The replication and life cycle of the influenza virus is governed by an intricate network of intracellular regulatory events during infection, including interactions with an even more complex system of biochemical interactions of the host cell. Computational modeling and systems biology have been successfully employed to further the understanding of various biological systems, however, computational studies of the complexity of intracellular interactions during influenza infection is lacking. In this work, we present the first large-scale dynamical model of the infection and replication cycle of influenza, as well as some of its interactions with the host's signaling machinery. Specifically, we focus on and visualize the dynamics of the internalization and endocytosis of the virus, replication and translation of its genomic components, as well as the assembly of progeny virions. Simulations and analyses of the models dynamics qualitatively reproduced numerous biological phenomena discovered in the laboratory. Finally, comparisons of the dynamics of existing and proposed drugs, our results suggest that a drug targeting PB1:PA would be more efficient than existing Amantadin/Rimantaine or Zanamivir/Oseltamivir. Helikar Tomas thelika@unmc.edu University of Nebraska - Lincoln 2012-03-19T00:16:44Z 2016-01-22T07:08:46Z 2015-12-19T22:02:17Z

<span style="font-size: 12px;">Methyl Beta Cyclodextrin (MCD) is a potent lipid microdomain disrupting agent and cholesterol depleting agent [1]. This is an external node and activation must be user specified (ie: amount of MCD added to cells).</span>

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<span style="font-size: 10pt; ">This is an external node and must be user specified. 'HA_SialicAcid_Affinity' represents the binding ability of cleaved hemagglutinin to its receptor Sialic Acid.</span>

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\r\n<p style="margin-bottom: 0in"><font size="3">Bisindolylmaleimide I,\r\nhighly specific PKC inhibitor of all isoforms. Prevented replication\r\nwhen added during infection, not affect after 2 hours. Virus entry\r\ninto cell, not receptor binding, inhibited.</font></p>\r\n

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The PB1:PA binding site can be blocked by using peptides that block the binding interface [1], [2].

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Viral_Dose represents the amount of intact influenza A virus particles being introduced into a population of epithelial cells.<br>

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null<br><br>null

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'RTK_Inhibitor' is a generic term for the many types of inhibitors of receptor tyrosine kinase signalling. This is an external node and activation must be user specified (ie: amount of inhibitor added to cells).

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<p><span style="font-size: 12px;">Wortmannin (PubChem ID 24278777) is a potent and specific PI3K inhibitor [1]. This is an external node and activation must be user specified (ie: amount of wortmannin added).</span></p>

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<p><span id="parentSpecies:viewSpeciesDescription" style="font-size: 12px;"><span style="font-size:11.0pt;line-height:115%;\r\nfont-family:"Calibri","sans-serif";mso-ascii-theme-font:minor-latin;mso-fareast-font-family:\r\nCalibri;mso-fareast-theme-font:minor-latin;mso-hansi-theme-font:minor-latin;\r\nmso-bidi-font-family:"Times New Roman";mso-bidi-theme-font:minor-bidi;\r\nmso-ansi-language:EN-US;mso-fareast-language:EN-US;mso-bidi-language:AR-SA">M1\r\nappears to play a role in the binding of progeny vRNA with NP in the nucleus to\r\nform nucleocapsids. Both M1 and NP bind to ssRNA (vRNA) but at different\r\nstoichiometric values: 1NP:20 nucleotides versus 1M1:200 nucleotides; M1:vRNA\r\ncomplexes are more stable. M1 may serve as a switch mechanism from replication\r\nto assembly – at low vRNA concentrations M1:M1 aggregation is favored, at high\r\nvRNA concentrations M1:vRNA is favored [1].</span></span></p>

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<span id="parentSpecies:viewSpeciesDescription" style="font-size: 12px;">Progeny\r\n vRNP are exported into the cytoplasm after being formed in the nucleus \r\n[1], [4]. This is mediated by NS2 and an NS2 cellular factor regulated \r\nby ERK [2].[3].</span>

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<font face="Arial, Verdana" size="2">HA mRNA localized in cytoplasm. Export of viral mRNA is thought to be mediated by hnRNP proteins that function as nuclear export signals NES. hnRNP NESs can be overridden by nuclear retention signals NRSs. Nuclear export of cellular mRNA but not viral mRNA is selectively inhibited thought the NS1 protein; not much is known about the mechanism of nuclear export of viral mRNA and hence not emphasized in the model [1].</font>

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A cellular factor for NS2 (NEP; also known as Nuclear Export Protein) is involved in the nuclear export of vRNPs regulated by ERK [1].

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<span style="font-size:11.0pt;line-height:115%;\r\nfont-family:"Calibri","sans-serif";mso-ascii-theme-font:minor-latin;mso-fareast-font-family:\r\nCalibri;mso-fareast-theme-font:minor-latin;mso-hansi-theme-font:minor-latin;\r\nmso-bidi-font-family:"Times New Roman";mso-bidi-theme-font:minor-bidi;\r\nmso-ansi-language:EN-US;mso-fareast-language:EN-US;mso-bidi-language:AR-SA">PB1\r\nand PA form a dimer in the cytoplasm and is imported into the nucleus\r\nseparately from PB2. Once in the nucleus the PB1/PA dimer associated with PB2\r\nto form the trimeric polymerase </span><span style="font-size:11.0pt;line-height:115%;font-family:"Calibri","sans-serif";\r\nmso-ascii-theme-font:minor-latin;mso-fareast-font-family:Calibri;mso-fareast-theme-font:\r\nminor-latin;mso-hansi-theme-font:minor-latin;mso-bidi-font-family:"Times New Roman";\r\nmso-bidi-theme-font:minor-bidi;mso-ansi-language:EN-US;mso-fareast-language:\r\nEN-US;mso-bidi-language:AR-SA">(Huet <i>et al.</i>, 2010)</span><span style="font-size:11.0pt;line-height:115%;font-family:"Calibri","sans-serif";\r\nmso-ascii-theme-font:minor-latin;mso-fareast-font-family:Calibri;mso-fareast-theme-font:\r\nminor-latin;mso-hansi-theme-font:minor-latin;mso-bidi-font-family:"Times New Roman";\r\nmso-bidi-theme-font:minor-bidi;mso-ansi-language:EN-US;mso-fareast-language:\r\nEN-US;mso-bidi-language:AR-SA">. </span><br><br>null

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<p><span id="parentSpecies:viewSpeciesDescription" style="font-size: 12px;">cRNP\r\n is the ribonucleoprotein encapsulating the complementary viral RNA. The\r\n ribonucleoprotein consists of the heterotrimeric polymerase subunit \r\nconsisting of PB1, PB2, and PA. </span></p><div><span style="font-size: 10pt; "><br></span></div><div><span style="font-size: 10pt; ">Stabilization of replication is stabilized by an intermediate cRNP [1].</span></div><div><span style="font-size: 10pt; "><br></span></div><div><span style="font-size: 10pt; ">The negative sense vRNA is copied to positive sense cRNA that serves as a template for progeny vRNA. </span><div>Unbound\r\n NP are required during cRNA synthesis for antitermination at the \r\npoly(A) site used during mRNA synthesis. Unbound NP is also required for\r\n copying cRNA into vRNA. Absence of NP causes termination at multiple \r\nsites [2].</div></div>

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<p><span id="parentSpecies:viewSpeciesDescription" style="font-size: 12px;">Progeny\r\n vRNP is formed in the nucleus. The progeny vRNP consists of NP, PB1, \r\nPB2, PA, and vRNA. The vRNP then leaves the nucleus [1].</span></p>

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<span style="font-size:11.0pt;line-height:115%;\r\nfont-family:"Calibri","sans-serif";mso-ascii-theme-font:minor-latin;mso-fareast-font-family:\r\nCalibri;mso-fareast-theme-font:minor-latin;mso-hansi-theme-font:minor-latin;\r\nmso-bidi-font-family:"Times New Roman";mso-bidi-theme-font:minor-bidi;\r\nmso-ansi-language:EN-US;mso-fareast-language:EN-US;mso-bidi-language:AR-SA">Once\r\nHemagglutinin is bound to its Sialic Acid receptor, endocytosis must occur in\r\norder to establish successful replication/transcription. Although various modes\r\nof virus uptake may take place, it is though clathrin-mediated endocytosis is\r\nmost common since the possibility exists that expression of dominant negative\r\nmutants leads to an up regulation of other endocytic processes that are not\r\nused by influenza under unperturbed cellular conditions </span><span style="font-size:11.0pt;line-height:115%;font-family:"Calibri","sans-serif";\r\nmso-ascii-theme-font:minor-latin;mso-fareast-font-family:Calibri;mso-fareast-theme-font:\r\nminor-latin;mso-hansi-theme-font:minor-latin;mso-bidi-font-family:"Times New Roman";\r\nmso-bidi-theme-font:minor-bidi;mso-ansi-language:EN-US;mso-fareast-language:\r\nEN-US;mso-bidi-language:AR-SA"><span style="mso-no-proof:yes">(Lakadamyali,\r\nRust, & Zhuang, 2004)</span></span><span style="font-size:11.0pt;line-height:115%;font-family:"Calibri","sans-serif";\r\nmso-ascii-theme-font:minor-latin;mso-fareast-font-family:Calibri;mso-fareast-theme-font:\r\nminor-latin;mso-hansi-theme-font:minor-latin;mso-bidi-font-family:"Times New Roman";\r\nmso-bidi-theme-font:minor-bidi;mso-ansi-language:EN-US;mso-fareast-language:\r\nEN-US;mso-bidi-language:AR-SA">. <br><br><span style="color:#00B050">Note: Filamentous\r\nvirions are too large to fit in CCP, thus filamentous IVA may enter through\r\nmacropinocytosis – filamentous Ebola virus enters via macropinocytosis </span></span><span style="font-size:11.0pt;line-height:115%;font-family:"Calibri","sans-serif";\r\nmso-ascii-theme-font:minor-latin;mso-fareast-font-family:Calibri;mso-fareast-theme-font:\r\nminor-latin;mso-hansi-theme-font:minor-latin;mso-bidi-font-family:"Times New Roman";\r\nmso-bidi-theme-font:minor-bidi;color:#00B050;mso-ansi-language:EN-US;\r\nmso-fareast-language:EN-US;mso-bidi-language:AR-SA"><span style="mso-no-proof:\r\nyes">(Nanbo et al., 2010)</span></span><span style="font-size:11.0pt;line-height:\r\n115%;font-family:"Calibri","sans-serif";mso-ascii-theme-font:minor-latin;\r\nmso-fareast-font-family:Calibri;mso-fareast-theme-font:minor-latin;mso-hansi-theme-font:\r\nminor-latin;mso-bidi-font-family:"Times New Roman";mso-bidi-theme-font:minor-bidi;\r\ncolor:#00B050;mso-ansi-language:EN-US;mso-fareast-language:EN-US;mso-bidi-language:\r\nAR-SA">; this may explain </span><span style="font-size:11.0pt;line-height:115%;font-family:"Calibri","sans-serif";\r\nmso-ascii-theme-font:minor-latin;mso-fareast-font-family:Calibri;mso-fareast-theme-font:\r\nminor-latin;mso-hansi-theme-font:minor-latin;mso-bidi-font-family:"Times New Roman";\r\nmso-bidi-theme-font:minor-bidi;color:#00B050;mso-ansi-language:EN-US;\r\nmso-fareast-language:EN-US;mso-bidi-language:AR-SA"><span style="mso-no-proof:\r\nyes">(Rust, Lakadamyali, Zhang, & Zhuang, 2004)</span></span><span style="font-size:11.0pt;line-height:\r\n115%;font-family:"Calibri","sans-serif";mso-ascii-theme-font:minor-latin;\r\nmso-fareast-font-family:Calibri;mso-fareast-theme-font:minor-latin;mso-hansi-theme-font:\r\nminor-latin;mso-bidi-font-family:"Times New Roman";mso-bidi-theme-font:minor-bidi;\r\ncolor:#00B050;mso-ansi-language:EN-US;mso-fareast-language:EN-US;mso-bidi-language:\r\nAR-SA"> claim of influenza using an unknown, clathrin and caveolin independent\r\nentry. </span><span style="font-size:11.0pt;line-height:115%;font-family:"Calibri","sans-serif";\r\nmso-ascii-theme-font:minor-latin;mso-fareast-font-family:Calibri;mso-fareast-theme-font:\r\nminor-latin;mso-hansi-theme-font:minor-latin;mso-bidi-font-family:"Times New Roman";\r\nmso-bidi-theme-font:minor-bidi;mso-ansi-language:EN-US;mso-fareast-language:\r\nEN-US;mso-bidi-language:AR-SA">Our modeling has focused exclusively on\r\nclathrin-mediated endocytosis. It has been revealed that influenza using\r\nclathrin-mediated endocytosis causes the formation of <i style="mso-bidi-font-style:\r\nnormal">de novo</i> formation of clathrin-coated pits (CCP) </span><span style="font-size:11.0pt;line-height:115%;font-family:"Calibri","sans-serif";\r\nmso-ascii-theme-font:minor-latin;mso-fareast-font-family:Calibri;mso-fareast-theme-font:\r\nminor-latin;mso-hansi-theme-font:minor-latin;mso-bidi-font-family:"Times New Roman";\r\nmso-bidi-theme-font:minor-bidi;mso-ansi-language:EN-US;mso-fareast-language:\r\nEN-US;mso-bidi-language:AR-SA"><span style="mso-no-proof:yes">(Rust et al.,\r\n2004)</span></span><span style="font-size:\r\n11.0pt;line-height:115%;font-family:"Calibri","sans-serif";mso-ascii-theme-font:\r\nminor-latin;mso-fareast-font-family:Calibri;mso-fareast-theme-font:minor-latin;\r\nmso-hansi-theme-font:minor-latin;mso-bidi-font-family:"Times New Roman";\r\nmso-bidi-theme-font:minor-bidi;mso-ansi-language:EN-US;mso-fareast-language:\r\nEN-US;mso-bidi-language:AR-SA">. It is currently hypothesized that the highly\r\nmultivalent binding of influenza to multiple sialic acid receptors may induce\r\nlocal curvature on the plasma membrane promoting the formation of CCP </span><span style="font-size:11.0pt;line-height:115%;font-family:"Calibri","sans-serif";\r\nmso-ascii-theme-font:minor-latin;mso-fareast-font-family:Calibri;mso-fareast-theme-font:\r\nminor-latin;mso-hansi-theme-font:minor-latin;mso-bidi-font-family:"Times New Roman";\r\nmso-bidi-theme-font:minor-bidi;mso-ansi-language:EN-US;mso-fareast-language:\r\nEN-US;mso-bidi-language:AR-SA"><span style="mso-no-proof:yes">(Rust et al.,\r\n2004)</span></span><span style="font-size:\r\n11.0pt;line-height:115%;font-family:"Calibri","sans-serif";mso-ascii-theme-font:\r\nminor-latin;mso-fareast-font-family:Calibri;mso-fareast-theme-font:minor-latin;\r\nmso-hansi-theme-font:minor-latin;mso-bidi-font-family:"Times New Roman";\r\nmso-bidi-theme-font:minor-bidi;mso-ansi-language:EN-US;mso-fareast-language:\r\nEN-US;mso-bidi-language:AR-SA">.</span><br><br>null

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<p><span id="species:viewSpeciesDescription" style="font-size: 12px;">mRNA is translated in the cytoplasm by host ribosomes. </span></p>

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<div><font face="Arial, Verdana" size="2">After endocytosis, viral ribonucleoprotein (vRNP) for each gene segment is released into the cytoplasm [1]. This vRNP is from the parental (incoming) influenza A virus.</font></div><div><font face="Arial, Verdana" size="2"><br></font></div><div><font face="Arial, Verdana" size="2">The influenza A genome consists of eight distinct RNA gene segments [2]. The three largest gene segments code for a heterotrimeric polymerase complex of basic (PB1 and PB2) and acidic (PA) subunits. Fourth, fifth, and sixth gene segments code for HA, NP, and NA, respectively.  The seventh segment codes for the collinear transcript M1 mRNA and M2 from spliced mRNA. The eight segments codes for NS1 and NS2 due to alternative splicing [3].</font></div><div><font face="Arial, Verdana" size="2"><br></font></div><div><font face="Arial, Verdana" size="2">Each gene segment is encapsulated by NP and bound to a polymerase complex. NP binds vRNA backbone without sequence specificity exposing the bases to the outside and hence melting all secondary structures. This allows the polymerase to transcribe mRNA as soon as vRNP enters the nucleus without requiring NP to dissociate or being stopped by a secondary RNA structure [4].  Taken together, each gene segment behaves as an independent replication and transcription unit [5] allowing double-infected cells to exhibit high frequency of gene reassortment [6]. </font></div>

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<p><span id="parentSpecies:viewSpeciesDescription" style="font-size: 12px;">After\r\n endocytosis, viral ribonucleoprotein (vRNP) for each gene segment is \r\nreleased into the cytoplasm [1]. This vRNP is from the parental \r\n(incoming) influenza A virus.</span></p><div><br></div><div><div>The \r\ninfluenza A genome consists of eight distinct RNA gene segments [2]. The\r\n three largest gene segments code for a heterotrimeric polymerase \r\ncomplex of basic (PB1 and PB2) and acidic (PA) subunits. Fourth, fifth, \r\nand sixth gene segments code for HA, NP, and NA, respectively.  The \r\nseventh segment codes for the collinear transcript M1 mRNA and M2 from \r\nspliced mRNA. The eight segments codes for NS1 and NS2 due to \r\nalternative splicing [3].</div><div><br></div><div>Each gene segment is \r\nencapsulated by NP and bound to a polymerase complex. NP binds vRNA \r\nbackbone without sequence specificity exposing the bases to the outside \r\nand hence melting all secondary structures. This allows the polymerase \r\nto transcribe mRNA as soon as vRNP enters the nucleus without requiring \r\nNP to dissociate or being stopped by a secondary RNA structure [4]. \r\n Taken together, each gene segment behaves as an independent replication\r\n and transcription unit [5] allowing double-infected cells to exhibit \r\nhigh frequency of gene reassortment [6].</div></div>

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<span id="parentSpecies:viewSpeciesDescription" style="font-size: 12px;">Once\r\n within the nucleus, the RNA dependent polymerase (temp_vRNP) begins \r\nboth transcription and replication trough mRNA and cRNA formation, \r\nrespectively. cRNA is quickly degraded by host proteases unless \r\nstabilized by the polymerase subunit and NP [1].</span>

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<div><font face="Arial, Verdana" size="2">Once within the nucleus, the RNA dependent polymerase begins both transcription and replication trough mRNA and cRNA formation, respectively. For further information see [1].</font></div><div><font face="Arial, Verdana" size="2"><br></font></div><div><font face="Arial, Verdana" size="2">Viral mRNA is capped and polyadenylated (contains methylated 5’ guanosine residue and polyadenylic acid at 3’ end, respectively).</font></div>

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<span id="parentSpecies:viewSpeciesDescription" style="font-size: 12px;">Template\r\n vRNP is responsible for transcription/replication. Incoming (as well as\r\n newly synthesized) vRNPs can serve as the template vRNP. Transcription \r\nand replication of RNA take place in the nucleus [4] at fixed sites [3].\r\n <br><br>Both transcription and replication is driven by the \r\nheterotrimeric RNA-dependent RNA polymerase complex of proteins PA, PB1,\r\n and PB2 [5]. Absence of any subunit were completely negative for mRNA \r\nor cRNA accumulation indicating a requirement of all three subunits for \r\nefficient transcription and replication [6]. PB1 is the catalytically \r\nactive polymerase that synthesis all RNA by adding nucleotides to the \r\ngrowing RNA chain [7]. PB1 is able to bind and recognize vRNA and cRNA \r\ntemplates [8]. The influenza polymerase does not have a proofreading \r\nmechanism which results in seasonal antigenic drift due to a gene \r\nmutation rate of approximately one error per replicated genome [9].</span>

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<span id="species:viewSpeciesDescription" style="font-size: 12px;"><span style="font-size:11.0pt;line-height:115%;\r\nfont-family:"Calibri","sans-serif";mso-fareast-font-family:"Droid Sans Fallback";\r\nmso-bidi-font-family:"Times New Roman";mso-bidi-theme-font:minor-bidi;\r\nmso-ansi-language:EN-US;mso-fareast-language:EN-US;mso-bidi-language:AR-SA">HA\r\nassociates with lipid rafts in the absence of other viral proteins whereas NA\r\nassociated within the cluster of HA-Lipid Rafts; M2 does not concentrate within\r\nlipid raft domains (Leser & Lamb, 2005). Cytoplasmic tails of HA and NA are\r\nresponsible for association with lipid raft domains; deletion of either HA or\r\nNA cytoplasmic tails resulted in decreased association (J. Zhang, Pekosz, &\r\nLamb, 2000). </span></span>

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<div style="font-size: 10pt;">mitogen-activated protein kinase <span style="font-size: 10pt;">kinase 1</span></div><div style="font-size: 10pt;"><span style="font-size: 10pt;"><br/></span></div><div style="font-size: 10pt;">MAP2K5:</div><div style="font-size: 10pt;">UniProt Accession ID: Q13163</div><div style="font-size: 10pt;"><br/></div><div style="font-size: 10pt;">MAP2K6:</div><div style="font-size: 10pt;">UniProt Accession ID: P52564 </div><div style="font-size: 10pt;"><br/></div><div style="font-size: 10pt;">MAP2K3:</div><div style="font-size: 10pt;">UniProt Accession ID: P46734 </div><div style="font-size: 10pt;"><br/></div><div style="font-size: 10pt;">MAP2K1:</div><div style="font-size: 10pt;">UniProt Accession ID: Q02750</div><div style="font-size: 10pt;"><br/></div><div><font size="2">MAP2K2:</font></div><div><font size="2">UniProt Accession ID: P36507</font></div>

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<font face="Arial, Verdana" size="2">M1 from the cytoplasm enter the nucleus. </font><span style="font-family: Calibri, sans-serif; font-size: 11pt; line-height: 115%; ">M1\r\nappears to play a role in the binding of progeny vRNA with NP in the nucleus to\r\nform nucleocapsids. Both M1 and NP bind to ssRNA (vRNA) but at different\r\nstoichiometric values: 1NP:20 nucleotides versus 1M1:200 nucleotides; M1:vRNA\r\ncomplexes are more stable. M1 may serve as a switch mechanism from replication\r\nto assembly – at low vRNA concentrations M1:M1 aggregation is favored, at high\r\nvRNA concentrations M1:vRNA is favored [1].</span>

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<span style="font-size: 12px;">Node representing late phase of viral life cycle.</span>

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<p><span id="parentSpecies:viewSpeciesDescription" style="font-size: 12px;"><font face="Arial, Verdana" size="2">Newly synthesized hemagglutinin in the cellular cytoplasm.</font></span></p>

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<div><font face="Georgia"><span style="line-height: 19.999780654907227px; background-color: rgb(255, 255, 255);">Zinc fingers and homeoboxes 2 [</span><em style="line-height: 19.999780654907227px; background-color: rgb(255, 255, 255);"><span class="highlight">Homo</span>sapiens</em><span style="line-height: 19.999780654907227px; background-color: rgb(255, 255, 255);"> (human)]</span></font></div><font face="Georgia">Uniprot ID: <a href="http://www.uniprot.org/uniprot/P04049">P04049</a></font><div><font face="Georgia">Gene Name: <a href="http://www.ncbi.nlm.nih.gov/gene/22882" ref="ordinalpos=1&ncbi_uid=22882&link_uid=22882" style="font-size: 10pt; line-height: 19.999780654907227px; white-space: nowrap; background-color: rgb(255, 255, 255);">ZHX2</a></font></div><div><font face="Georgia">Gene ID: <span style="background-color: rgb(255, 255, 255); font-size: 12.222222328186035px; line-height: 17.999801635742188px;">22882</span></font></div>

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Progeny vRNP are exported into the cytoplasm after being formed in the nucleus [1], [4]. This is mediated by NS2 and an NS2 cellular factor regulated by ERK [2].[3].<br>

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<span id="parentSpecies:viewSpeciesDescription" style="font-size: 12px;"><font face="Arial, Verdana" size="2">Newly synthesized matrix protein 1 in the cellular cytoplasm.</font></span>

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<span id="parentSpecies:viewSpeciesDescription" style="font-size: 12px;">HA\r\n mRNA localized in cytoplasm. Export of viral mRNA is thought to be \r\nmediated by hnRNP proteins that function as nuclear export signals NES. \r\nhnRNP NESs can be overridden by nuclear retention signals NRSs. Nuclear \r\nexport of cellular mRNA but not viral mRNA is selectively inhibited \r\nthought the NS1 protein; not much is known about the mechanism of \r\nnuclear export of viral mRNA and hence not emphasized in the model [1].</span>

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Influenza is a negative sense RNA virus and thus requires an intermediate positive sense complementary RNA strand (cRNA) before replicating its genome [1],

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cRNP is the ribonucleoprotein encapsulating the complementary viral RNA. The ribonucleoprotein consists of the heterotrimeric polymerase subunit consisting of PB1, PB2, and PA. <div><span style="font-size: 10pt; "><br></span></div><div><span style="font-size: 10pt; ">Stabilization of replication is stabilized by an intermediate cRNP [1].</span></div><div><span style="font-size: 10pt; "><br></span></div><div><span style="font-size: 10pt; ">The negative sense vRNA is copied to positive sense cRNA that serves as a template for progeny vRNA. </span><div>Unbound NP are required during cRNA synthesis for antitermination at the poly(A) site used during mRNA synthesis. Unbound NP is also required for copying cRNA into vRNA. Absence of NP causes termination at multiple sites [2].</div><div><br></div></div>

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<p><span id="parentSpecies:viewSpeciesDescription" style="font-size: 12px;">Once\r\n within the nucleus, the RNA dependent polymerase (temp_vRNP) begins \r\nboth transcription and replication trough mRNA and cRNA formation, \r\nrespectively. cRNA is quickly degraded by host proteases unless \r\nstabilized by the polymerase subunit and NP [1].</span></p>

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<font face="Arial, Verdana" size="2">Newly synthesized hemagglutinin in the cellular cytoplasm.</font>

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<span id="parentSpecies:viewSpeciesDescription" style="font-size: 12px;">Influenza\r\n is a negative sense RNA virus and thus requires an intermediate \r\npositive sense complementary RNA strand (cRNA) before replicating its \r\ngenome [1],</span>

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2015-12-19T22:02:17Z 2015-12-19T22:02:17Z

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<div><font face="Arial, Verdana" size="2">cRNP is the ribonucleoprotein encapsulating the complementary viral RNA. The ribonucleoprotein consists of the heterotrimeric polymerase subunit consisting of PB1, PB2, and PA. </font></div><div><font face="Arial, Verdana" size="2"><br></font></div><div><font face="Arial, Verdana" size="2">Stabilization of replication is stabilized by an intermediate cRNP [1].</font></div><div><font face="Arial, Verdana" size="2"><br></font></div><div><font face="Arial, Verdana" size="2">The negative sense vRNA is copied to positive sense cRNA that serves as a template for progeny vRNA. </font></div><div><font face="Arial, Verdana" size="2">Unbound NP are required during cRNA synthesis for antitermination at the poly(A) site used during mRNA synthesis. Unbound NP is also required for copying cRNA into vRNA. Absence of NP causes termination at multiple sites [2].</font></div>

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Hemagglutinin  (HA) is in despicable for the influenza life cycle as it mediates both receptor binding and membrane fusion [3,5]. Mature HA exists as a single-chain precursor HA<sub>0</sub> [3,5]. Precursor HA<sub>0</sub> which is not able to mediate membrane fusion must be cleaved into HA1 (receptor binding) and HA2 (membrane fusion) by host protease [1,2]. <br>

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<span class="highlight" style="background-color:">Influenza virus</span> <span class="highlight" style="background-color:">genome</span> <span class="highlight" style="background-color:">segment</span> 8 is transcribed into <span class="highlight" style="background-color:">two</span> separate mRNAs that code for <span class="highlight" style="background-color:">two</span> <span class="highlight" style="background-color:">polypeptides</span>, NS1 and NS2 [1],[2].<div><br><div><div>Once within the nucleus, the RNA dependent polymerase begins both \r\ntranscription and replication trough mRNA and cRNA formation, \r\nrespectively. For further information see [3].</div><div><br></div><div>Viral\r\n mRNA is capped and polyadenylated (contains methylated 5’ guanosine \r\nresidue and polyadenylic acid at 3’ end, respectively).</div></div></div>

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HA mRNA localized in cytoplasm. Export of viral mRNA is thought to be mediated by hnRNP proteins that function as nuclear export signals NES. hnRNP NESs can be overridden by nuclear retention signals NRSs. Nuclear export of cellular mRNA but not viral mRNA is selectively inhibited thought the NS1 protein; not much is known about the mechanism of nuclear export of viral mRNA and hence not emphasized in the model [1].

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The EE proceed to form an intermediate maturing endosome (ME) and subsequent late endosome (LE), Rab5/ESCRT and Rab5/Rab7/LAMP-1 respectively. <br>

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<p><p><font face="Arial, Verdana"><span style="font-size: 12px;">After endocytosis, viral ribonucleoprotein (vRNP) for each gene segment is released into the cytoplasm [1]. This vRNP is from the parental (incoming) influenza A virus.</span></font></p><p><font face="Arial, Verdana"><span style="font-size: 12px;"><br></span></font></p><p><font face="Arial, Verdana"><span style="font-size: 12px;">The influenza A genome consists of eight distinct RNA gene segments [2]. The three largest gene segments code for a heterotrimeric polymerase complex of basic (PB1 and PB2) and acidic (PA) subunits. Fourth, fifth, and sixth gene segments code for HA, NP, and NA, respectively.  The seventh segment codes for the collinear transcript M1 mRNA and M2 from spliced mRNA. The eight segments codes for NS1 and NS2 due to alternative splicing [3].</span></font></p><p><font face="Arial, Verdana"><span style="font-size: 12px;"><br></span></font></p><p><font face="Arial, Verdana"><span style="font-size: 12px;">Each gene segment is encapsulated by NP and bound to a polymerase complex. NP binds vRNA backbone without sequence specificity exposing the bases to the outside and hence melting all secondary structures. This allows the polymerase to transcribe mRNA as soon as vRNP enters the nucleus without requiring NP to dissociate or being stopped by a secondary RNA structure [4].  Taken together, each gene segment behaves as an independent replication and transcription unit [5] allowing double-infected cells to exhibit high frequency of gene reassortment [6].</span></font></p></p>

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<p><span id="species:viewSpeciesDescription" style="font-size: 12px;">mRNA is translated in the cytoplasm by host ribosomes. </span></p>

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<font face="Arial, Verdana" size="2" style="font-weight: normal; font-size: 10pt;">phosphoinositide-3-kinase</font><div><font size="2" style="font-weight: normal;"><br/></font><div style="font-weight: normal;"><font size="2">catalytic, alpha polypeptide (PIK3CA)</font></div><div style="font-weight: normal;"><font size="2">UniProt Accession ID: P42336</font></div><div style="font-weight: normal;"><font size="2"><br/></font></div><div><div style="font-weight: normal;"><font size="2">catalytic, beta polypeptide (PIK3CB)</font></div><div style="font-weight: normal;"><font size="2">UniProt Accession ID: Q6PJ60</font></div><div style="font-weight: normal;"><font size="2"><br/></font></div><div style="font-weight: normal;"><div><font size="2">catalytic, gamma polypeptide (PIK3CG)</font></div><div><font size="2">UniProt Accession ID: P48736</font></div></div><div style="font-weight: normal;"><font size="2"><br/></font></div><div><span style="font-family: sans-serif; font-size: small; line-height: 16px; background-color: rgb(255, 255, 255);">Phosphoinositide 3-kinase regulatory subunit 6</span></div><div style="font-weight: normal; font-size: 10pt;"><font face="Arial, Verdana" size="2">UniProt Accession ID: </font><a href="http://www.uniprot.org/uniprot/Q5UE93" style="font-size: small; text-decoration: none; font-family: sans-serif; background-color: rgb(255, 255, 255);">Q5UE93</a></div><div style="font-weight: normal; font-size: 10pt;"><br/></div><div style="font-size: 10pt;"><span style="font-family: sans-serif; font-size: small; line-height: 16px; background-color: rgb(255, 255, 255);">Phosphatidylinositol 4-phosphate 3-kinase C2 domain-containing subunit beta</span></div><div style="font-weight: normal; font-size: 10pt;">UniProt Accession ID: <a href="http://www.uniprot.org/uniprot/O00750" style="font-size: small; text-decoration: none; font-family: sans-serif; background-color: rgb(255, 255, 255);">O00750</a></div><div style="font-weight: normal; font-size: 10pt;"><br/></div><div style="font-size: 10pt;"><span style="font-family: sans-serif; font-size: small; line-height: 16px; background-color: rgb(255, 255, 255);">Phosphatidylinositol 4-phosphate 3-kinase C2 domain-containing subunit alpha</span></div><div style="font-weight: normal; font-size: 10pt;">UniProt Accession ID: <a href="http://www.uniprot.org/uniprot/O00443" style="font-size: small; text-decoration: none; font-family: sans-serif; background-color: rgb(255, 255, 255);">O00443</a></div></div></div>

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<span style="font-size:11.0pt;line-height:115%;\r\nfont-family:"Calibri","sans-serif";mso-ascii-theme-font:minor-latin;mso-fareast-font-family:\r\nCalibri;mso-fareast-theme-font:minor-latin;mso-hansi-theme-font:minor-latin;\r\nmso-bidi-font-family:"Times New Roman";mso-bidi-theme-font:minor-bidi;\r\nmso-ansi-language:EN-US;mso-fareast-language:EN-US;mso-bidi-language:AR-SA">CCP\r\nlead to the formation of Clathrin-coated vesicles (CCV) that pinch off the\r\nplasma membrane (for a review and list of cellular factors required see </span><span style="font-size:11.0pt;line-height:115%;font-family:"Calibri","sans-serif";\r\nmso-ascii-theme-font:minor-latin;mso-fareast-font-family:Calibri;mso-fareast-theme-font:\r\nminor-latin;mso-hansi-theme-font:minor-latin;mso-bidi-font-family:"Times New Roman";\r\nmso-bidi-theme-font:minor-bidi;mso-ansi-language:EN-US;mso-fareast-language:\r\nEN-US;mso-bidi-language:AR-SA"><span style="mso-no-proof:yes">(Mercer,\r\nSchelhaas, & Helenius, 2010)</span></span><span style="font-size:11.0pt;line-height:115%;font-family:"Calibri","sans-serif";\r\nmso-ascii-theme-font:minor-latin;mso-fareast-font-family:Calibri;mso-fareast-theme-font:\r\nminor-latin;mso-hansi-theme-font:minor-latin;mso-bidi-font-family:"Times New Roman";\r\nmso-bidi-theme-font:minor-bidi;mso-ansi-language:EN-US;mso-fareast-language:\r\nEN-US;mso-bidi-language:AR-SA">. Our model assumes all components are present. <br></span>

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<span style="font-size: 11pt; line-height: 115%; font-family: " calibri","sans-serif";"="">The\r\nvRNP is released into the cytoplasm after reaching the LE due to the intrinsic\r\nion channel activity of M2(Pinto, 1992; Takeda, 2002). This is due to the\r\nrelative low pH of the LE and the shuttling of hydrogen ions to disrupt M1-vRNP\r\ninteractions (Bui, 1996).<br><br></span><span style="font-size:11.0pt;line-height:115%;\r\nfont-family:" calibri","sans-serif";mso-fareast-font-family:"droid="" sans="" fallback";="" mso-bidi-font-family:"times="" new="" roman";mso-bidi-theme-font:minor-bidi;="" mso-ansi-language:en-us;mso-fareast-language:en-us;mso-bidi-language:ar-sa"="">Popular\r\ndrugs Amantadine/Rimantadine target the M2 ion channel preventing nuclear\r\nimport due to the large size of M1-vRNP (Bui 1996). Popular drugs\r\nAmantadine/Rimantadine block nuclear import vicariously by blocking the ion\r\nchannel activity of M2 (Hay,1985). </span><br>

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<p><span id="species:viewSpeciesDescription" style="font-size: 12px;">mRNA is translated in the cytoplasm by host ribosomes. </span></p>

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<span style="color: rgb(34, 34, 34); font-family: Calibri, sans-serif; font-size: 14px; font-style: normal; font-variant: normal; font-weight: normal; letter-spacing: normal; line-height: 16px; orphans: 3; text-align: left; text-indent: 0px; text-transform: none; white-space: normal; widows: 3; word-spacing: 0px; -webkit-text-size-adjust: auto; -webkit-text-stroke-width: 0px; background-color: rgb(255, 255, 255); display: inline !important; float: none; ">Of the 11 proteins encoded by influenza’s 8 gene segments, 8 are assembled into progeny virions (Lamb & Krug, 2001). The viral envelope is made up of HA, NA, and the M2 ion channel protein. Within the envelope M1 provides the frame of the virus and connects the vRNP to the viral envelope. vRNP is composed of PB1, PB2, PA as well as NP that binds and packages influenza’s genome (influenza virus contains only one copy of the genome (Noda<span class="Apple-converted-space"> </span></span><i style="color: rgb(34, 34, 34); font-family: Calibri, sans-serif; font-size: 14px; font-variant: normal; font-weight: normal; letter-spacing: normal; line-height: 16px; orphans: 3; text-align: left; text-indent: 0px; text-transform: none; white-space: normal; widows: 3; word-spacing: 0px; -webkit-text-size-adjust: auto; -webkit-text-stroke-width: 0px; background-color: rgb(255, 255, 255); ">et al.</i><span style="color: rgb(34, 34, 34); font-family: Calibri, sans-serif; font-size: 14px; font-style: normal; font-variant: normal; font-weight: normal; letter-spacing: normal; line-height: 16px; orphans: 3; text-align: left; text-indent: 0px; text-transform: none; white-space: normal; widows: 3; word-spacing: 0px; -webkit-text-size-adjust: auto; -webkit-text-stroke-width: 0px; background-color: rgb(255, 255, 255); display: inline !important; float: none; ">, 2006). Nonstructural protein NS1 is primary for immune system evasion, NS2 is for vRNP nuclear export, and finally some strains express PB1-F2 involved in cellular apoptosis</span>

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Once within the nucleus, the RNA dependent polymerase (temp_vRNP) begins both transcription and replication trough mRNA and cRNA formation, respectively. cRNA is quickly degraded by host proteases unless stabilized by the polymerase subunit and NP [1].

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Once cleaved, influenza binds to cell surface receptors sialic acid with alpha-(2,3) or alpha-(2,6) linkages, avian and human respectively [1]. IVA_SialicAcid represents the virus bound to the host cell receptor.

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<div><font face="Arial, Verdana" size="2">Template vRNP is responsible for transcription/replication. Incoming (as well as newly synthesized) vRNPs can serve as the template vRNP. Transcription and replication of RNA take place in the nucleus [4] at fixed sites [3].</font></div><div><font face="Arial, Verdana" size="2"><br></font></div><div><font face="Arial, Verdana" size="2">Both transcription and replication is driven by the heterotrimeric RNA-dependent RNA polymerase complex of proteins PA, PB1, and PB2 [5]. Absence of any subunit were completely negative for mRNA or cRNA accumulation indicating a requirement of all three subunits for efficient transcription and replication [6]. PB1 is the catalytically active polymerase that synthesis all RNA by adding nucleotides to the growing RNA chain [7]. PB1 is able to bind and recognize vRNA and cRNA templates [8]. The influenza polymerase does not have a proofreading mechanism which results in seasonal antigenic drift due to a gene mutation rate of approximately one error per replicated genome [9].</font></div>

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<div>Once within the nucleus, the RNA dependent polymerase begins both \r\ntranscription and replication trough mRNA and cRNA formation, \r\nrespectively. For further information see [1].</div><div><br></div><div>Viral\r\n mRNA is capped and polyadenylated (contains methylated 5’ guanosine \r\nresidue and polyadenylic acid at 3’ end, respectively).</div>

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<p><span id="parentSpecies:viewSpeciesDescription" style="font-size: 12px;">Influenza\r\n is a negative sense RNA virus and thus requires an intermediate \r\npositive sense complementary RNA strand (cRNA) before replicating its \r\ngenome [1],</span></p>

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<span style="font-size: 12px;">NP is required to form RNP within the nucleus as well as stabilize cRNA [1].</span>

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<p><span id="species:viewSpeciesDescription" style="font-size: 12px;">mRNA is translated in the cytoplasm by host ribosomes. </span></p>

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Influenza virus genome segment 8 is transcribed into two separate mRNAs that code for two polypeptides, NS1 and NS2 [1],[2].<br><br>Once within the nucleus, the RNA dependent polymerase begins both transcription and replication trough mRNA and cRNA formation, respectively. For further information see [3].<br><br>Viral mRNA is capped and polyadenylated (contains methylated 5’ guanosine residue and polyadenylic acid at 3’ end, respectively).

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Receptor tyrosine kinases (RTKs) are cell surface receptors involved in diverse signal transduction mechanisms. These receptors are coupled to signal transduction networks that regulate many biological responses. Reviewed in [1].

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Template vRNP is responsible for transcription/replication. Incoming (as well as newly synthesized) vRNPs can serve as the template vRNP. Transcription and replication of RNA take place in the nucleus [4] at fixed sites [3]. <br><br>Both transcription and replication is driven by the heterotrimeric RNA-dependent RNA polymerase complex of proteins PA, PB1, and PB2 [5]. Absence of any subunit were completely negative for mRNA or cRNA accumulation indicating a requirement of all three subunits for efficient transcription and replication [6]. PB1 is the catalytically active polymerase that synthesis all RNA by adding nucleotides to the growing RNA chain [7]. PB1 is able to bind and recognize vRNA and cRNA templates [8]. The influenza polymerase does not have a proofreading mechanism which results in seasonal antigenic drift due to a gene mutation rate of approximately one error per replicated genome [9].<br>

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<p><span id="parentSpecies:viewSpeciesDescription" style="font-size: 12px;"><font face="Arial, Verdana" size="2">Newly synthesized hemagglutinin in the cellular cytoplasm.</font></span></p>

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Progeny vRNP is formed in the nucleus. The progeny vRNP consists of NP, PB1, PB2, PA, and vRNA. The vRNP then leaves the nucleus [1].

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PB2 forms one of the subunits of the heterotrimeric polymerase. PB2 is responsible for RNA transcription and posses cap binding [1]. N-terminal of PB2 plays a role in viral RNA replication as well [2],

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M2 mRNA is translated into M2 protein in the cytoplasm by host ribosomes.

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<div><font face="Georgia" size="2">The protein kinase c family consists of 15 isozymes in humans [49]. </font></div><div><font face="Georgia" size="2"><br/></font></div><div><font face="Georgia" size="2">Protein Kinase C alpha</font></div><div><font face="Georgia" size="2">Uniprot ID: <a href="http://www.uniprot.org/uniprot/P17252">P17252</a></font></div><div><font face="Georgia" size="2">Gene Name: PRKCA</font></div><div><font face="Georgia" size="2">Gene ID:<span style="background-color: rgb(255, 255, 255); line-height: 16.78819465637207px; white-space: nowrap;">5578</span></font></div><div><div><font face="Georgia" size="2">Protein Kinase C beta</font></div><div><font face="Georgia" size="2">Uniprot ID: <a href="http://www.uniprot.org/uniprot/P05771" style="text-decoration: none; background-color: rgb(255, 255, 255);">P05771</a></font></div><div><font face="Georgia" size="2">Gene Name: PRKCB</font></div><div><font face="Georgia" size="2">Gene ID:<span style="background-color: rgb(255, 255, 255); line-height: 16.78819465637207px; white-space: nowrap;">5579</span></font></div></div><div><div><div><font face="Georgia" size="2">Protein Kinase C gamma</font></div><div><font face="Georgia" size="2">Uniprot ID: <a href="http://www.uniprot.org/uniprot/P05129" style="text-decoration: none; background-color: rgb(255, 255, 255);">P05129</a></font></div><div><font face="Georgia" size="2">Gene Name: PRKCG</font></div><div><font face="Georgia" size="2">Gene ID:<span style="background-color: rgb(255, 255, 255); line-height: 16.78819465637207px; white-space: nowrap;">5582</span></font></div></div><div><div><font face="Georgia" size="2">Protein Kinase C delta</font></div><div><font face="Georgia" size="2">Uniprot ID: <a href="http://www.uniprot.org/uniprot/Q05655" style="text-decoration: none; background-color: rgb(255, 255, 255);">Q05655</a></font></div><div><font face="Georgia" size="2">Gene Name: PRKCD</font></div><div><font face="Georgia" size="2">Gene ID:<span style="background-color: rgb(255, 255, 255); line-height: 16.78819465637207px; white-space: nowrap;">5580</span></font></div></div><div><div><font face="Georgia" size="2">Protein Kinase C epsilon</font></div><div><font face="Georgia" size="2">Uniprot ID: <a href="http://www.uniprot.org/uniprot/Q02156" style="text-decoration: none; background-color: rgb(255, 255, 255);">Q02156</a></font></div><div><font face="Georgia" size="2">Gene Name: PRKCE</font></div><div><font face="Georgia" size="2">Gene ID:<span style="background-color: rgb(255, 255, 255); line-height: 16.78819465637207px; white-space: nowrap;">5581</span></font></div></div><div><div><font face="Georgia" size="2">Protein Kinase C eta</font></div><div><font face="Georgia" size="2">Uniprot ID: <a href="http://www.uniprot.org/uniprot/P24723" style="text-decoration: none; background-color: rgb(255, 255, 255);">P24723</a></font></div><div><font face="Georgia" size="2">Gene Name: PRKCH</font></div><div><font face="Georgia" size="2">Gene ID:<span style="background-color: rgb(255, 255, 255); line-height: 16.78819465637207px; white-space: nowrap;">5583</span></font></div></div><div><div><font face="Georgia" size="2">Protein Kinase C theta</font></div><div><font face="Georgia" size="2">Uniprot ID: <a href="http://www.uniprot.org/uniprot/Q04759" style="text-decoration: none; background-color: rgb(255, 255, 255);">Q04759</a></font></div><div><font face="Georgia" size="2">Gene Name: PRKCQ</font></div><div><font face="Georgia" size="2">Gene ID:<span style="background-color: rgb(255, 255, 255); line-height: 16.78819465637207px; white-space: nowrap;">5588</span></font></div></div><div><div><font face="Georgia" size="2">Protein Kinase C iota</font></div><div><font face="Georgia" size="2">Uniprot ID: <a href="http://www.uniprot.org/uniprot/P41743" style="text-decoration: none; background-color: rgb(255, 255, 255);">P41743</a></font></div><div><font face="Georgia" size="2">Gene Name: PRKCI</font></div><div><font face="Georgia" size="2">Gene ID:<span style="background-color: rgb(255, 255, 255); line-height: 16.78819465637207px; white-space: nowrap;">5584</span></font></div></div><div><div><font face="Georgia" size="2">Protein Kinase C zeta</font></div><div><font face="Georgia" size="2">Uniprot ID: <a href="http://www.uniprot.org/uniprot/Q05513">Q05513</a></font></div><div><font face="Georgia" size="2">Gene Name: PRKCZ</font></div><div><font face="Georgia" size="2">Gene ID:<span style="background-color: rgb(255, 255, 255); line-height: 16.78819465637207px; white-space: nowrap;">5590</span></font></div></div></div>

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<span style="font-size:11.0pt;line-height:115%;\r\nfont-family:"Calibri","sans-serif";mso-fareast-font-family:"Droid Sans Fallback";\r\nmso-bidi-font-family:"Times New Roman";mso-bidi-theme-font:minor-bidi;\r\nmso-ansi-language:EN-US;mso-fareast-language:EN-US;mso-bidi-language:AR-SA">M1\r\nappears to play a role in the binding of progeny vRNA with NP in the nucleus to\r\nform nucleocapsids. Both M1 and NP bind to ssRNA (vRNA) but at different\r\nstoichiometric values: 1NP:20 nucleotides versus 1M1:200 nucleotides; M1:vRNA\r\ncomplexes are more stable. M1 may serve as a switch mechanism from replication\r\nto assembly – at low vRNA concentrations M1:M1 aggregation is favored, at high\r\nvRNA concentrations M1:vRNA is favored (Wakefield & Brownlee, 1989).</span>

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<p><span id="parentSpecies:viewSpeciesDescription" style="font-size: 12px;"><font face="Arial, Verdana" size="2">Newly synthesized hemagglutinin in the cellular cytoplasm.</font></span></p>

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<div>Once within the nucleus, the RNA dependent polymerase begins both transcription and replication trough mRNA and cRNA formation, respectively. For further information see [1].</div><div><br></div><div>Viral mRNA is capped and polyadenylated (contains methylated 5’ guanosine residue and polyadenylic acid at 3’ end, respectively).</div>

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<font face="Arial, Verdana" size="2">mitogen-activated protein kinase</font><div><span style="font-family: sans-serif; font-size: small; line-height: 16px; background-color: rgb(255, 255, 255);">Mitogen-activated protein kinase 1</span></div><div><span style="font-family: sans-serif; font-size: small; line-height: 16px; background-color: rgb(255, 255, 255);">UniProt ID: P28482</span></div><div><span style="font-family: sans-serif; font-size: small; line-height: 16px; background-color: rgb(255, 255, 255);"><br/></span></div><div><span style="font-family: sans-serif; font-size: small; line-height: 16px; background-color: rgb(255, 255, 255);">Mitogen-activated protein kinase 3</span></div><div><span style="font-family: sans-serif; font-size: small; line-height: 16px; background-color: rgb(255, 255, 255);">UniProt Accession ID: P27361</span></div><div><br/></div><div>Extracellular signal-regulated kinases</div><div><span style="font-family: sans-serif; font-size: 16px; background-color: rgb(255, 255, 255);"><br/></span></div>

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After endocytosis, viral ribonucleoprotein (vRNP) for each gene segment is released into the cytoplasm [1]. This vRNP is from the parental (incoming) influenza A virus.<div><br></div><div><div>The influenza A genome consists of eight distinct RNA gene segments [2]. The three largest gene segments code for a heterotrimeric polymerase complex of basic (PB1 and PB2) and acidic (PA) subunits. Fourth, fifth, and sixth gene segments code for HA, NP, and NA, respectively.  The seventh segment codes for the collinear transcript M1 mRNA and M2 from spliced mRNA. The eight segments codes for NS1 and NS2 due to alternative splicing [3].</div><div><br></div><div>Each gene segment is encapsulated by NP and bound to a polymerase complex. NP binds vRNA backbone without sequence specificity exposing the bases to the outside and hence melting all secondary structures. This allows the polymerase to transcribe mRNA as soon as vRNP enters the nucleus without requiring NP to dissociate or being stopped by a secondary RNA structure [4].  Taken together, each gene segment behaves as an independent replication and transcription unit [5] allowing double-infected cells to exhibit high frequency of gene reassortment [6]. </div></div>

2015-12-19T22:02:17Z

<span style="font-size:11.0pt;line-height:115%;\r\nfont-family:"Calibri","sans-serif";mso-ascii-theme-font:minor-latin;mso-fareast-font-family:\r\nCalibri;mso-fareast-theme-font:minor-latin;mso-hansi-theme-font:minor-latin;\r\nmso-bidi-font-family:"Times New Roman";mso-bidi-theme-font:minor-bidi;\r\nmso-ansi-language:EN-US;mso-fareast-language:EN-US;mso-bidi-language:AR-SA">The\r\nCCV then enters the early endosome (EE) and can be recycled to the plasma\r\nmembrane directly or through the recycling endosome (RE), Rab4 and Rab22/Rab11\r\nrespectively. Our model assumes once within the EE, sialic acids is recycled\r\nback into the plasma membrane through RE and that no other virus can infect the\r\ncell (see Dr. Donze for reference). </span>

2015-12-19T22:02:17Z

<span id="parentSpecies:viewSpeciesDescription" style="font-size: 12px;"><span style="font-size:11.0pt;line-height:115%;\r\nfont-family:"Calibri","sans-serif";mso-ascii-theme-font:minor-latin;mso-fareast-font-family:\r\nCalibri;mso-fareast-theme-font:minor-latin;mso-hansi-theme-font:minor-latin;\r\nmso-bidi-font-family:"Times New Roman";mso-bidi-theme-font:minor-bidi;\r\nmso-ansi-language:EN-US;mso-fareast-language:EN-US;mso-bidi-language:AR-SA">M1\r\nappears to play a role in the binding of progeny vRNA with NP in the nucleus to\r\nform nucleocapsids. Both M1 and NP bind to ssRNA (vRNA) but at different\r\nstoichiometric values: 1NP:20 nucleotides versus 1M1:200 nucleotides; M1:vRNA\r\ncomplexes are more stable. M1 may serve as a switch mechanism from replication\r\nto assembly – at low vRNA concentrations M1:M1 aggregation is favored, at high\r\nvRNA concentrations M1:vRNA is favored [1].</span></span>

2015-12-19T22:02:17Z

<span id="species:viewSpeciesDescription" style="font-size: 12px;">The vRNA:M1 complex requires both M1 and vRNA localized in the nucleus.</span><h1 id="firstHeading" class="firstHeading">\r\n </h1>\r\n

<p><span style="font-size: 12px;">vRNA is bound to M1.</span></p>

<p><span style="font-size: 12px;">M1 binds vRNA.</span></p>

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<font face="Arial, Verdana" size="2">progeny vRNP are exported from the nucleus into the cytoplasm mediated by NS2 and its cognate cellular factor. </font>

<font face="Arial, Verdana" size="2">Progeny vRNPs bound to M1 are exported into the cytoplasm. M1 association to vRNP promotes nuclear export of the M1-vRNP complex; this movement is bidirectional as M1 dissociation from vRNP is required for nuclear import [3]. </font>

<p><span style="font-size: 12px;">A cellular\r\n factor for NS2 (NEP; also known as Nuclear Export Protein) is involved \r\nin the nuclear export of vRNPs regulated by ERK [1]. </span></p>

<font face="Arial, Verdana" size="2">A functional domain of NS2 with nuclear export signal characteristics was mapped. This domain gives rise to a model by which NS2 acts as a protein adaptor molecule bridging vRNP with the nuclear pore complex and subsequent nuclear export [2]. </font>

S_10 1 S_4 1 S_78 1

<font face="Arial, Verdana" size="2">mRNA_HA_nuc activates mRNA_HA_cyto.</font>

<font face="Arial, Verdana" size="2">mRNA_HA_nuc is exported via host's nuclear export machinery to the cytoplasm; hence mRNA_HA_nuc activates mRNA_HA_cyto [1].</font>

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ERK activates an NS2 Cellular Factor.

<span style="font-family: Verdana, Arial, sans-serif; font-size: 12px; line-height: 24px; orphans: 3; widows: 3; background-color: rgb(255, 255, 255); ">ERK regulates a cellular factor required for NS2 mediated nuclear export of vRNPs [1].</span>

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PA and PB1 is required for PB1:PA complex formation. Blocking their binding interface is a possible drug target.<br>

<span style="color: rgb(0, 0, 0); font-family: Arial, Verdana; font-style: normal; font-variant: normal; font-weight: normal; letter-spacing: normal; line-height: 24px; orphans: 3; text-align: -webkit-auto; text-indent: 0px; text-transform: none; white-space: normal; widows: 3; word-spacing: 0px; -webkit-text-size-adjust: auto; -webkit-text-stroke-width: 0px; background-color: rgb(255, 255, 255); font-size: small; display: inline !important; float: none; ">Sequence analysis has shown that the PA-binding domain of PB1 is highly conserved as multiple offsetting mutations in both PA and PB1 would be required. Use of binding peptides at the PB1:PA interface blocked viral polymerase activity and inhibited viral replication (Ghanem et al., 2007; Wunderlich et al., 2009) similar to the use of L-741,001. This interface has thus been proposed as possibly druggable, and a 25-residue PB1-derived peptide has been shown to inhibit polymerase assembly and virus replication (Boivin, Cusack, Ruigrok, & Hart, 2010).</span>

S_120 1 S_12 1 S_41 1

<font face="Arial, Verdana" size="2">progeny vRNP are exported from the nucleus into the cytoplasm mediated by NS2 and its cognate cellular factor. </font>

<font face="Arial, Verdana" size="2">A cellular factor for NS2 (NEP; also known as Nuclear Export Protein) is involved in the nuclear export of vRNPs regulated by ERK [1].  </font>

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<span id="species:viewSpeciesDescription" style="font-size: 12px;">cRNP formation requires cRNA, NP, PB1, PA, and PB2 [1].</span>

<div style="font-size: 12px; font-family: Verdana, Arial, sans-serif; background-color: rgb(255, 255, 255); ">RNA transcription – cap binding [5].</div><div style="font-size: 12px; font-family: Verdana, Arial, sans-serif; background-color: rgb(255, 255, 255); ">N-terminal region of PB2 plays a role in viral RNA replication [6].</div>

<div style="font-size: 12px; font-family: Verdana, Arial, sans-serif; background-color: rgb(255, 255, 255); ">All\r\n RNA synthesis is by PB1; catalytically active polymerase – adds \r\nnucleotides to growing RNA chain [2]. PB1 is able to bind and recognize \r\nvRNA and cRNA templates [3].</div><div style="font-size: 12px; font-family: Verdana, Arial, sans-serif; background-color: rgb(255, 255, 255); "><br></div><div style="font-size: 12px; font-family: Verdana, Arial, sans-serif; background-color: rgb(255, 255, 255); ">PA is implicated in RNA replication with endonuclease activity [4].</div>

<p><span style="font-size: 12px;"><span style="font-family: Verdana, Arial, sans-serif; line-height: 24px; orphans: 3; widows: 3; background-color: rgb(255, 255, 255); ">cRNA is synthesized early in infection but is quickly degraded by host proteases unless stabilized by RNA polymerase and NP [1].</span></span></p>

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<p><span id="species:viewSpeciesDescription" style="font-size: 12px;">progeny vRNP are exported from the nucleus into the cytoplasm mediated by NS2 and its cognate cellular factor.</span></p>

<p><span style="font-size: 12px;">Progeny vRNPs bound to M1 are exported into the cytoplasm. <span style="font-size:11.0pt;line-height:115%;\r\nfont-family:" calibri","sans-serif";mso-ascii-theme-font:minor-latin;mso-fareast-font-family:="" calibri;mso-fareast-theme-font:minor-latin;mso-hansi-theme-font:minor-latin;="" mso-bidi-font-family:"times="" new="" roman";mso-bidi-theme-font:minor-bidi;="" mso-ansi-language:en-us;mso-fareast-language:en-us;mso-bidi-language:ar-sa"="">M1\r\nassociation to vRNP promotes nuclear export of the M1-vRNP complex; this\r\nmovement is bidirectional as M1 dissociation from vRNP is required for nuclear\r\nimport [3].</span></span></p>

<p><span style="font-size: 12px;">A cellular\r\n factor for NS2 (NEP; also known as Nuclear Export Protein) is involved \r\nin the nuclear export of vRNPs regulated by ERK [1]. </span></p>

<p><span style="font-size: 12px;"><span style="font-size:11.0pt;line-height:115%;\r\nfont-family:" calibri","sans-serif";mso-ascii-theme-font:minor-latin;mso-fareast-font-family:="" calibri;mso-fareast-theme-font:minor-latin;mso-hansi-theme-font:minor-latin;="" mso-bidi-font-family:"times="" new="" roman";mso-bidi-theme-font:minor-bidi;="" mso-ansi-language:en-us;mso-fareast-language:en-us;mso-bidi-language:ar-sa"="">A\r\nfunctional domain of NS2 with nuclear export signal characteristics was mapped.\r\nThis domain gives rise to a model by which NS2 acts as a protein adaptor\r\nmolecule bridging vRNP with the nuclear pore complex and subsequent nuclear\r\nexport [2].</span></span></p>

S_98 1 S_4 1 S_78 1

<span id="species:viewSpeciesDescription" style="font-size: 12px;">vRNP \r\nformation requires all three polymerase subunits and vRNA bound to NP. \r\nAs more vRNPs enter the cytoplasm, less vRNPs are localized in the \r\nnucleus.</span>

<p><span style="font-size: 12px;"><span style="font-family: Verdana, Arial, sans-serif; line-height: 24px; orphans: 3; widows: 3; background-color: rgb(255, 255, 255); ">The\r\n vRNP leaving the nucleus is a mutually exclusive event and thus this is\r\n a negative regulator (ie: less vRNPs in the nucleus as more vRNPs enter\r\n the cytoplasm).</span></span></p>

S_51 1 S_112 1 S_97 1 S_93 1 S_2 1

Receptor bound influenza A viruse ('IVA_SialicAcid_CCP') lead to endocytosis via clathrin coated pits.

Influenza undergoes receptor bound endocytosis mainly via clathrin coated pits [1].

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<p><span id="species:viewSpeciesDescription" style="font-size: 12px;">mRNA is translated in the cytoplasm by host ribosomes. </span></p>

<p><span style="font-size: 12px;">mRNA is translated into its protein.</span></p>

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<font face="Arial, Verdana" size="2">After entering the late endosome, vRNPs are released into the cytoplasm. </font>

<div style="orphans: 3; text-align: left; widows: 3;"><font color="#222222" face="Verdana, Arial, sans-serif"><span style="font-size: 14px; line-height: 16px;">The vRNP is released into the cytoplasm after reaching the late endosome (LE) due to the intrinsic ion channel activity of M2 [1],[2]. </span></font></div>

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<font face="Arial, Verdana" size="2">mRNA_HA_nuc activates mRNA_HA_cyto. </font>

<font face="Arial, Verdana" size="2">mRNA_HA_nuc is exported via host's nuclear export machinery to the cytoplasm; hence mRNA_HA_nuc activates mRNA_HA_cyto [1].</font>

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<p><span id="species:viewSpeciesDescription" style="font-size: 12px;">After entering the late endosome, vRNPs are released into the cytoplasm. </span></p>

The vRNP is released into the cytoplasm after reaching the late endosome (LE) due to the intrinsic ion channel activity of M2 [1],[2]. 

S_75 1

<span id="species:viewSpeciesDescription" style="font-size: 12px;">Incoming vRNP are newly formed vRNPs are able to stabilize cRNA synthesis.</span>

<span style="font-size: 12px;">Incoming or newly formed vRNP <span style="font-size:11.0pt;line-height:115%;\r\nfont-family:"Calibri","sans-serif";mso-fareast-font-family:"Droid Sans Fallback";\r\nmso-bidi-font-family:"Times New Roman";mso-bidi-theme-font:minor-bidi;\r\nmso-ansi-language:EN-US;mso-fareast-language:EN-US;mso-bidi-language:AR-SA">begins\r\nboth transcription and replication trough mRNA and cRNA formation,\r\nrespectively [1].</span></span>

<span style="font-size: 12px;">An intermediate is required for cRNA stabilization [1].</span>

<span style="font-size: 12px;">All polymerase subunits are required for effective replication [1].</span>

<span style="font-size: 12px;">NP is required for NP stabilization [1].</span>

S_83 1 S_112 1 S_97 1 S_112 1 S_97 1 S_93 1 S_53 1 S_112 1 S_97 1 S_93 1 S_112 1 S_97 1

<font face="Arial, Verdana" size="2">vRNA transcribes mRNA. </font>

<font face="Arial, Verdana" size="2">vRNP initiates both transcription and replication [1]. </font>

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<p><span style="font-size: 12px;">The incoming vRNP contains the complete polymerase subunits and RNA encapsulated by NP [1].</span></p>

<p><span style="font-size: 12px;">Template vRNP requires RNA for replication and transcription [3].</span></p>

<p><span style="font-size: 12px;">All three subunits are required for efficient transcription and replication [4].</span></p>

<p><span style="font-size: 12px;">Each gene segment is encapsulated by NP \r\nand bound to a polymerase complex. NP binds vRNA backbone without \r\nsequence specificity exposing the bases to the outside and hence melting\r\n all secondary structures. This allows the polymerase to transcribe mRNA\r\n as soon as vRNP enters the nucleus without requiring NP to dissociate \r\nor being stopped by a secondary RNA structure [2]</span></p>

S_89 1 S_112 1 S_97 1 S_93 1 S_40 1

<span style="font-size:11.0pt;line-height:115%;\r\nfont-family:"Calibri","sans-serif";mso-fareast-font-family:"Droid Sans Fallback";\r\nmso-bidi-font-family:"Times New Roman";mso-bidi-theme-font:minor-bidi;\r\nmso-ansi-language:EN-US;mso-fareast-language:EN-US;mso-bidi-language:AR-SA">HA\r\nassociates with lipid rafts in the absence of other viral proteins whereas NA\r\nassociated within the cluster of HA-Lipid Rafts; M2 does not concentrate within\r\nlipid raft domains (Leser & Lamb, 2005). Cytoplasmic tails of HA and NA are\r\nresponsible for association with lipid raft domains; deletion of either HA or\r\nNA cytoplasmic tails resulted in decreased association (J. Zhang, Pekosz, &\r\nLamb, 2000). </span>

<p><span id="parentSpecies:viewSpeciesDescription" style="font-size: 12px;"><span calibri","sans-serif";mso-ascii-theme-font:minor-latin;mso-fareast-font-family:="" calibri;mso-fareast-theme-font:minor-latin;mso-hansi-theme-font:minor-latin;="" mso-bidi-theme-font:minor-latin;mso-ansi-language:en-us;mso-fareast-language:="" en-us;mso-bidi-language:ar-sa"="">A currently approved drug that targets the viral surface protein neuraminidase (Zanamivir/Oseltamivir). </span>Budding\r\n of the progeny virions (last step of the influenza life cycle) is \r\nmediated by neuraminidase. Thus inhibition of NA prevents virus release \r\nfrom infected cells (Gubareva, Kaiser, & Hayden, 2000). (Gong, Xu, \r\n& Zhang, 2007; Skehel, 2009).</span></p>

<span style="color: rgb(34, 34, 34); font-family: Calibri, sans-serif; font-size: 14px; line-height: 16px; orphans: 3; text-align: left; widows: 3; background-color: rgb(255, 255, 255); ">HA associates with lipid rafts in the absence of other viral proteins whereas NA associated within the cluster of HA-Lipid Rafts; M2 does not concentrate within lipid raft domains (Leser & Lamb, 2005).</span>

<p><span id="parentSpecies:viewSpeciesDescription" style="font-size: 12px;">Methyl\r\n Beta Cyclodextrin (MCD) is a potent lipid microdomain disrupting agent \r\nand cholesterol depleting agent [3]. This is an external node and \r\nactivation must be user specified (ie: amount of MCD added to cells).</span></p>

NA associates with lipid rafts.

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<font face="Arial, Verdana" size="2">progeny vRNP are exported from the nucleus into the cytoplasm mediated by NS2 and its cognate cellular factor. </font>

<font face="Arial, Verdana" size="2">A cellular factor for NS2 (NEP; also known as Nuclear Export Protein) is involved in the nuclear export of vRNPs regulated by ERK [1].  </font>

S_64 1 S_4 1 S_78 1

<font face="Arial, Verdana" size="2">mRNA_HA_nuc activates mRNA_HA_cyto.</font>

<font face="Arial, Verdana" size="2">mRNA_HA_nuc is exported via host's nuclear export machinery to the cytoplasm; hence mRNA_HA_nuc activates mRNA_HA_cyto [1].</font>

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The vRNA:M1 complex requires both M1 and vRNA localized in the nucleus.<br>

vRNA is bound to M1.

M1 binds vRNA.

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Raf activated MEK which can be inhibited by U0126.

Raf phosphorylates and activates MEK [1],

CAS no 1173097-76-1. U0126 blocks the Raf/MEK/ERK cascade by inhibiting MEK. Inhibition of MEK resulted in nuclear retention of vRNPs thus the Raf/MEK/ERK cascade is responsible for nuclear export of vRNPs. This results in reduced progeny virus titers [2].

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<span style="color: rgb(34, 34, 34); font-family: Verdana, Arial, sans-serif; font-size: 12px; font-style: normal; font-variant: normal; font-weight: normal; letter-spacing: normal; line-height: 17px; orphans: 3; text-align: left; text-indent: 0px; text-transform: none; white-space: normal; widows: 3; word-spacing: 0px; -webkit-text-size-adjust: auto; -webkit-text-stroke-width: 0px; background-color: rgb(255, 255, 255); display: inline !important; float: none; ">M1 from the cytoplasm enter the nucleus [1].</span>

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Initiation of virus binding and establishing the replication cycle is necessary for the cell to become successfully infected.<br>

Initiation of the viral life cycle.

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<p><span style="font-size: 12px;"><span style="font-family: Verdana, Arial, sans-serif; line-height: 24px; orphans: 3; widows: 3; background-color: rgb(255, 255, 255); ">The\r\n vRNP leaving the nucleus is a mutually exclusive event and thus this is\r\n a negative regulator (ie: less vRNPs in the nucleus as more vRNPs enter\r\n the cytoplasm).</span></span></p>

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<span id="species:viewSpeciesDescription" style="font-size: 12px;">cRNP formation requires cRNA, NP, PB1, PA, and PB2 [1].</span>

<p><span style="font-size: 12px;"><span style="font-family: Verdana, Arial, sans-serif; line-height: 24px; orphans: 3; widows: 3; background-color: rgb(255, 255, 255); ">cRNA is synthesized early in infection but is quickly degraded by host proteases unless stabilized by RNA polymerase and NP [1].</span></span></p>

<span style="font-size: 12px;"><span style="font-family: Verdana, Arial, sans-serif; line-height: 24px; orphans: 3; widows: 3; background-color: rgb(255, 255, 255); ">NP is required to form RNP as well as stabilize cRNA [1].</span></span>

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<p><span id="species:viewSpeciesDescription" style="font-size: 12px;">mRNA is translated in the cytoplasm by host ribosomes. </span></p>

<p><span style="font-size: 12px;">mRNA is translated into its protein.</span></p>

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<font face="Arial, Verdana" size="2">mRNA_HA_nuc activates mRNA_HA_cyto.</font>

<font face="Arial, Verdana" size="2">mRNA_HA_nuc is exported via host's nuclear export machinery to the cytoplasm; hence mRNA_HA_nuc activates mRNA_HA_cyto [1].</font>

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Hemagglutinin accumulation on cellular membrane is an important requirement for MAPK activation via PKC.  This is a late phase activation possibly representing a mechanism that coordinated vRNP export when all viral components are ready [1]. For this reason a node representing late phase infection 'Infected' was included as a prerequisite for PKC mediated activation of Raf.

Node representing late phase of viral life cycle.

<div><span style="font-size: 10pt; ">* PKC can directly phosphorylate and activate Raf {[12]-p(4)} </span></div><div>* PKC can directly phosphorylate and regulate activity of Raf, however mutation of PKC sites in Raf doesn't affect its regulations by other agents {[75]-p(4),[55]-p(2)} </div><div>* PKC activates Raf {[76]-p(8)} </div><div>* Activation of Raf by PKC is Ras-dependent {[69]-p(371), [26]-p(2)} </div><div>* PKC directly phosphorylates Raf1; activation requires Ras (broken cells) {[82]-p(63)} </div><div>* PKA phosphorylates Rap1, which not only blocks c-Raf activation, but also binds b-Raf, resulting in b-Raf activation. {[85]-p(2)} </div><div>* PKC? activates raf-1 {[79]-p(293), table 1} </div><div>* PKC phosphorylates (activates) Raf {[81]-p(1)} </div><div>* PKC phosphorylation of Raf is associated with T491 and S494, but the significance of this phosphorylation is not clear. {[77]-p(5)} Therefore, leaving out this connection.</div>

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progeny vRNP are exported from the nucleus into the cytoplasm mediated by NS2 and its cognate cellular factor.

<font face="Arial, Verdana">A cellular factor for NS2 (NEP; also known as Nuclear Export Protein) is involved in the nuclear export of vRNPs regulated by ERK [1]. </font>

Progeny vRNPs bound to M1 are exported into the cytoplasm. <span style="font-size:11.0pt;line-height:115%;\r\nfont-family:" calibri","sans-serif";mso-ascii-theme-font:minor-latin;mso-fareast-font-family:="" calibri;mso-fareast-theme-font:minor-latin;mso-hansi-theme-font:minor-latin;="" mso-bidi-font-family:"times="" new="" roman";mso-bidi-theme-font:minor-bidi;="" mso-ansi-language:en-us;mso-fareast-language:en-us;mso-bidi-language:ar-sa"="">M1\r\nassociation to vRNP promotes nuclear export of the M1-vRNP complex; this\r\nmovement is bidirectional as M1 dissociation from vRNP is required for nuclear\r\nimport [3].</span>

<span style="font-size:11.0pt;line-height:115%;\r\nfont-family:" calibri","sans-serif";mso-ascii-theme-font:minor-latin;mso-fareast-font-family:="" calibri;mso-fareast-theme-font:minor-latin;mso-hansi-theme-font:minor-latin;="" mso-bidi-font-family:"times="" new="" roman";mso-bidi-theme-font:minor-bidi;="" mso-ansi-language:en-us;mso-fareast-language:en-us;mso-bidi-language:ar-sa"="">A\r\nfunctional domain of NS2 with nuclear export signal characteristics was mapped.\r\nThis domain gives rise to a model by which NS2 acts as a protein adaptor\r\nmolecule bridging vRNP with the nuclear pore complex and subsequent nuclear\r\nexport [2].</span>

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Electron micrograph experiments by Rulgrok et.al. demonstrated that M1 is not membrane inserted but is membrane associated through electrostatic interactions (Ruigrok et al., 2000). Furthermore, detergent treatment experiments by Ali et.al. have indicated M1 interacts with HA and NA (Ali, Avalos, Ponimaskin, & Nayak, 2000). M1 is able to oligomerize and associate with vRNP via NP (Noton et al., 2007; Ye, Liu, Offringa, McInnis, & Levandowski, 1999). All enveloped proteins (HA,NA,M2) are transported to the apical surface of polarized cells (Whittaker, 2001). Taken together, these observations indicate M1 bridges the vRNP to the enveloped proteins.<br>

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<font face="Arial, Verdana" size="2">mRNA_HA_nuc activates mRNA_HA_cyto.</font>

<font face="Arial, Verdana" size="2">mRNA_HA_nuc is exported via host's nuclear export machinery to the cytoplasm; hence mRNA_HA_nuc activates mRNA_HA_cyto [1].</font>

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<span style="font-family: Verdana, Arial, sans-serif; font-size: 12px; line-height: 24px; orphans: 3; widows: 3; background-color: rgb(255, 255, 255); ">cRNA leads to vRNA replication [1],</span>

S_81 1

cRNP formation requires cRNA, NP, PB1, PA, and PB2 [1].<br>

<div style="font-size: 12px; color: rgb(0, 0, 0); font-family: Verdana, Arial, sans-serif; font-style: normal; font-variant: normal; font-weight: normal; letter-spacing: normal; line-height: normal; orphans: 2; text-align: -webkit-auto; text-indent: 0px; text-transform: none; white-space: normal; widows: 2; word-spacing: 0px; -webkit-text-size-adjust: auto; -webkit-text-stroke-width: 0px; background-color: rgb(255, 255, 255); ">All RNA synthesis is by PB1; catalytically active polymerase – adds nucleotides to growing RNA chain [2]. PB1 is able to bind and recognize vRNA and cRNA templates [3].</div><div style="font-size: 12px; color: rgb(0, 0, 0); font-family: Verdana, Arial, sans-serif; font-style: normal; font-variant: normal; font-weight: normal; letter-spacing: normal; line-height: normal; orphans: 2; text-align: -webkit-auto; text-indent: 0px; text-transform: none; white-space: normal; widows: 2; word-spacing: 0px; -webkit-text-size-adjust: auto; -webkit-text-stroke-width: 0px; background-color: rgb(255, 255, 255); "><br></div><div style="font-size: 12px; color: rgb(0, 0, 0); font-family: Verdana, Arial, sans-serif; font-style: normal; font-variant: normal; font-weight: normal; letter-spacing: normal; line-height: normal; orphans: 2; text-align: -webkit-auto; text-indent: 0px; text-transform: none; white-space: normal; widows: 2; word-spacing: 0px; -webkit-text-size-adjust: auto; -webkit-text-stroke-width: 0px; background-color: rgb(255, 255, 255); ">PA is implicated in RNA replication with endonuclease activity [4].</div>

S_81 1 S_112 1 S_97 1 S_93 1

<span id="species:viewSpeciesDescription" style="font-size: 12px;">Incoming vRNP are newly formed vRNPs are able to stabilize cRNA synthesis.</span>

<p><span style="font-size: 12px;"><span style="font-family: Verdana, Arial, sans-serif; line-height: 24px; orphans: 3; widows: 3; background-color: rgb(255, 255, 255); ">Incoming or newly formed vRNP </span><span style="orphans: 3; widows: 3; background-color: rgb(255, 255, 255); font-size: 11pt; line-height: 16px; font-family: Calibri, sans-serif; ">begins both transcription and replication trough mRNA and cRNA formation, respectively [1].</span></span></p>

S_29 1 S_112 1 S_97 1 S_93 1 S_112 1 S_97 1 S_19 1 S_112 1 S_97 1 S_93 1 S_112 1 S_97 1

<font face="Arial, Verdana" size="2">After entering the late endosome, vRNPs are released into the cytoplasm. </font>

<span style="color: rgb(34, 34, 34); font-family: Verdana, Arial, sans-serif; font-size: 14px; font-style: normal; font-variant: normal; font-weight: normal; letter-spacing: normal; line-height: 16px; orphans: 3; text-align: left; text-indent: 0px; text-transform: none; white-space: normal; widows: 3; word-spacing: 0px; -webkit-text-size-adjust: auto; -webkit-text-stroke-width: 0px; background-color: rgb(255, 255, 255); display: inline !important; float: none; ">The vRNP is released into the cytoplasm after reaching the late endosome (LE) due to the intrinsic ion channel activity of M2 [1],[2]. </span>

S_75 1

mRNA is translated in the cytoplasm by host ribosomes. 

mRNA is translated into its protein.

S_65 1

<p><span style="font-size: 12px;">Template vRNP requires RNA for replication and transcription [3].</span></p>

<p><span style="font-size: 12px;">All three subunits are required for efficient transcription and replication [4].</span></p>

<font face="Arial, Verdana" size="2">Each gene segment is encapsulated by NP and bound to a polymerase complex. NP binds vRNA backbone without sequence specificity exposing the bases to the outside and hence melting all secondary structures. This allows the polymerase to transcribe mRNA as soon as vRNP enters the nucleus without requiring NP to dissociate or being stopped by a secondary RNA structure [2] </font>

<p><span style="font-size: 12px;">The incoming vRNP contains the complete polymerase subunits and RNA encapsulated by NP [1].</span></p>

S_49 1 S_112 1 S_97 1 S_93 1 S_13 1

<font face="Arial, Verdana" size="2">vRNA transcribes mRNA. </font>

<font face="Arial, Verdana" size="2">vRNP initiates both transcription and replication [1]. </font>

S_102 1

<p><span id="species:viewSpeciesDescription" style="font-size: 12px;"><span style="font-family: Verdana, Arial, sans-serif; line-height: 24px; orphans: 3; widows: 3; background-color: rgb(255, 255, 255); ">cRNA leads to vRNA replication [1],</span></span></p>

S_91 1

<span id="species:viewSpeciesDescription" style="font-size: 12px;">Incoming vRNP are newly formed vRNPs are able to stabilize cRNA synthesis.</span>

<p><span style="font-size: 12px;"><span style="font-family: Verdana, Arial, sans-serif; line-height: 24px; orphans: 3; widows: 3; background-color: rgb(255, 255, 255); ">Incoming or newly formed vRNP </span><span style="orphans: 3; widows: 3; background-color: rgb(255, 255, 255); font-size: 11pt; line-height: 16px; font-family: Calibri, sans-serif; ">begins both transcription and replication trough mRNA and cRNA formation, respectively [1].</span></span></p>

S_102 1 S_112 1 S_97 1 S_112 1 S_97 1 S_93 1 S_50 1 S_112 1 S_97 1 S_112 1 S_97 1 S_93 1

S_118 1

<p><span style="font-size: 12px;">cRNA leads to vRNA replication [1],</span></p>

S_60 1

<span id="species:viewSpeciesDescription" style="font-size: 12px;">cRNP formation requires cRNA, NP, PB1, PA, and PB2 [1].</span>

<p><span style="font-size: 12px;"><span style="font-family: Verdana, Arial, sans-serif; line-height: 24px; orphans: 3; widows: 3; background-color: rgb(255, 255, 255); ">cRNA is synthesized early in infection but is quickly degraded by host proteases unless stabilized by RNA polymerase and NP [1].</span></span></p>

S_47 1 S_112 1 S_97 1 S_93 1

<p><span style="font-size: 12px;">vRNA is bound to M1.</span></p>

<p><span style="font-size: 12px;">M1 binds vRNA.</span></p>

S_89 1 S_26 1

<p><span style="font-size: 12px;"><span style="font-family: Verdana, Arial, sans-serif; line-height: 24px; orphans: 3; widows: 3; background-color: rgb(255, 255, 255); ">The\r\n vRNP leaving the nucleus is a mutually exclusive event and thus this is\r\n a negative regulator (ie: less vRNPs in the nucleus as more vRNPs enter\r\n the cytoplasm).</span></span></p>

S_1 1 S_112 1 S_97 1 S_93 1 S_127 1

<div>RNA transcription – cap binding [6].</div><div>N-terminal region of PB2 plays a role in viral RNA replication [7].</div>

<span style="font-size: 12px;">cRNA is synthesized early in infection but is quickly degraded by host proteases unless stabilized by RNA polymerase and NP [1].</span>

<div>All RNA synthesis is by PB1; catalytically active polymerase – adds\r\n nucleotides to growing RNA chain [3]. PB1 is able to bind and recognize\r\n vRNA and cRNA templates [4].</div><div><br></div><div>PA is implicated in RNA replication with endonuclease activity [5].</div>

<span style="font-size: 12px;">NP is required to form RNP as well as stabilize cRNA [1].</span>

S_16 1 S_112 1 S_97 1 S_93 1

S_16 1

<p><span id="species:viewSpeciesDescription" style="font-size: 12px;">mRNA_HA_nuc activates mRNA_HA_cyto.</span></p>

<font face="Arial, Verdana" size="2">mRNA_HA_nuc is exported via host's nuclear export machinery to the cytoplasm; hence mRNA_HA_nuc activates mRNA_HA_cyto [1].</font>

S_77 1

<p><span style="font-size: 12px;"><span style="font-family: Verdana, Arial, sans-serif; line-height: 24px; orphans: 3; widows: 3; background-color: rgb(255, 255, 255); ">The\r\n vRNP leaving the nucleus is a mutually exclusive event and thus this is\r\n a negative regulator (ie: less vRNPs in the nucleus as more vRNPs enter\r\n the cytoplasm).</span></span></p>

S_119 1 S_112 1 S_97 1 S_93 1 S_70 1

<span id="species:viewSpeciesDescription" style="font-size: 12px;">Incoming vRNP are newly formed vRNPs are able to stabilize cRNA synthesis.</span>

<p><span style="font-size: 12px;"><span style="font-family: Verdana, Arial, sans-serif; line-height: 24px; orphans: 3; widows: 3; background-color: rgb(255, 255, 255); ">Incoming or newly formed vRNP </span><span style="orphans: 3; widows: 3; background-color: rgb(255, 255, 255); font-size: 11pt; line-height: 16px; font-family: Calibri, sans-serif; ">begins both transcription and replication trough mRNA and cRNA formation, respectively [1].</span></span></p>

S_68 1 S_112 1 S_97 1 S_112 1 S_97 1 S_93 1 S_76 1 S_112 1 S_97 1 S_112 1 S_97 1 S_93 1

<font face="Arial, Verdana" size="2">In this version of the influeza A model, proteases listed in the previous page are assumed to be present to successfully cleave HA. Future versions of the model will incorporate host proteases specific to HA cleavage.</font>

This model assumes proteases are present and are able to cleave all incoming influenza A particles depicted by 'Viral_Dose'

S_42 1

<p><span id="species:viewSpeciesDescription" style="font-size: 12px;">vRNA transcribes mRNA.</span></p>

S_103 1

<span id="species:viewSpeciesDescription" style="font-size: 12px;">Incoming vRNP are newly formed vRNPs are able to stabilize cRNA synthesis.</span>

<p><span style="font-size: 12px;"><span style="font-family: Verdana, Arial, sans-serif; line-height: 24px; orphans: 3; widows: 3; background-color: rgb(255, 255, 255); ">Incoming or newly formed vRNP </span><span style="orphans: 3; widows: 3; background-color: rgb(255, 255, 255); font-size: 11pt; line-height: 16px; font-family: Calibri, sans-serif; ">begins both transcription and replication trough mRNA and cRNA formation, respectively [1].</span></span></p>

S_7 1 S_112 1 S_97 1 S_112 1 S_97 1 S_93 1 S_44 1 S_112 1 S_97 1 S_93 1 S_112 1 S_97 1

HA binds to lipid rafts unless MCD is active.

<span style="font-size:11.0pt;line-height:115%;\r\nfont-family:"Calibri","sans-serif";mso-fareast-font-family:"Droid Sans Fallback";\r\nmso-bidi-font-family:"Times New Roman";mso-bidi-theme-font:minor-bidi;\r\nmso-ansi-language:EN-US;mso-fareast-language:EN-US;mso-bidi-language:AR-SA">Influenza\r\nutilizes lipid raft domains for both assembly and budding. Raft-associated\r\nproteins such as HA form a viral ‘budozone’ (Schmitt & Lamb, 2005).</span>

S_43 1 S_18 1

<span style="font-size: 12px;">cRNA is synthesized early in infection but is quickly degraded by host proteases unless stabilized by RNA polymerase and NP [1].</span>

<div>RNA transcription – cap binding [6].</div><div>N-terminal region of PB2 plays a role in viral RNA replication [7].</div>

<span style="font-size: 12px;">NP is required to form RNP as well as stabilize cRNA [1].</span>

S_110 1 S_112 1 S_97 1 S_93 1

<p><span style="font-size: 12px;"><span style="font-family: Verdana, Arial, sans-serif; line-height: 24px; orphans: 3; widows: 3; background-color: rgb(255, 255, 255); ">The\r\n vRNP leaving the nucleus is a mutually exclusive event and thus this is\r\n a negative regulator (ie: less vRNPs in the nucleus as more vRNPs enter\r\n the cytoplasm).</span></span></p>

<p style="orphans: 3; widows: 3; "><p><span style="font-size: 12px;"><span style="font-family: Verdana, Arial, sans-serif; line-height: 24px; background-color: rgb(255, 255, 255); ">NP is required to form RNP [1].</span></span></p></p>

S_92 1 S_112 1 S_97 1 S_93 1 S_21 1

<span style="font-size: 12px;">mRNA_HA_nuc activates mRNA_HA_cyto.</span>

mRNA_HA_nuc is exported via host's nuclear export machinery to the cytoplasm; hence mRNA_HA_nuc activates mRNA_HA_cyto [1].

S_121 1

Early endosome leads to an intermediate endosomal compartment called maturing endomsome (represented as 'IVA_ME'; this represents the endocytosed influenza virus within the maturing endosome).

<div>The EE proceed to form an intermediate maturing endosome (ME) and \r\nsubsequent late endosome (LE), Rab5/ESCRT and Rab5/Rab7/LAMP-1 \r\nrespectively [1].</div>

S_130 1

<font face="Arial, Verdana" size="2">After entering the late endosome, vRNPs are released into the cytoplasm. </font>

S_75 1

<span id="species:viewSpeciesDescription" style="font-size: 12px;">cRNP formation requires cRNA, NP, PB1, PA, and PB2 [1].</span>

<p><span style="font-size: 12px;"><span style="font-family: Verdana, Arial, sans-serif; line-height: 24px; orphans: 3; widows: 3; background-color: rgb(255, 255, 255); ">cRNA is synthesized early in infection but is quickly degraded by host proteases unless stabilized by RNA polymerase and NP [1].</span></span></p>

S_57 1 S_112 1 S_97 1 S_93 1

<p><span id="species:viewSpeciesDescription" style="font-size: 12px;">mRNA_HA_nuc activates mRNA_HA_cyto.</span></p>

<span style="font-size: 12px;">mRNA_HA_nuc is exported via host's nuclear export machinery to the cytoplasm; hence mRNA_HA_nuc activates mRNA_HA_cyto [1].</span>

S_17 1

<p><span id="species:viewSpeciesDescription" style="font-size: 12px;">progeny vRNP are exported from the nucleus into the cytoplasm mediated by NS2 and its cognate cellular factor.</span></p>

<p><span style="font-size: 12px;">A cellular\r\n factor for NS2 (NEP; also known as Nuclear Export Protein) is involved \r\nin the nuclear export of vRNPs regulated by ERK [1]. </span></p>

S_56 1 S_4 1 S_78 1

<p><span id="species:viewSpeciesDescription" style="font-size: 12px;">mRNA is translated in the cytoplasm by host ribosomes. </span></p>

<p><span style="font-size: 12px;">mRNA is translated into its protein.</span></p>

S_31 1

<font face="Arial, Verdana" size="2">RTKs are able to activate PI3K signaling which is required for viral entry. Nonstructural Protein 1 (NS1) of influenza is able to bind and activate PI3K signaling. Wortmannin, a potent PI3K inhibitor, is dominant over any positive regulation.</font>

PI3K is a downstream effector of Gab1 via RTK (EGFR) signaling. Tyr1068 at EGFR is phosphorylated which generates binding site for p85 [2<font color="rgb(0, 0, 0)"><font face="Arial,Verdana">].</font></font><br>

NS1 binds the regulatory subunit p85beta – this is strain specific [1]. This is late phase activation.<br>

Wortmannin (PubChem ID 24278777) is a potent and specific PI3K inhibitor [3].

S_30 1 S_116 1 S_107 1 S_116 1

Influenza viruses bound to sialic acid within clathrin coated vesicles (represented by 'IVA_SialicAcid_CCP') lead to the formation of clathrin coated vesicles. All prerequisite proteins (ie: adaptors) are assumed to be present.

<font face="Arial, Verdana" size="2">Clathrin coated vesicles lead to the formation of clathrin coated vesicles [1].</font>

S_11 1

<p><span style="font-size: 12px;">vRNA is bound to M1.</span></p>

<p><span style="font-size: 12px;">M1 binds vRNA.</span></p>

S_49 1 S_26 1

Maturing endosome ('IVA_ME') leads to late endosome formation ('IVA_LE') both containing the incoming influenza A virus. Late endosome formation requires PKC and thus the "AND" relationship between 'IVA_ME' and 'PKC'. M2 inhibitors ('M2_inhibitor) such as Amantadine/Rimantadine block the ion channel activity of M2 and prevent vRNP entry into the cytoplasm (instead vRNP is bound to M1). This inhibition is not related to endosome formation and thus is independent (as well as dominant) over all positive regulators. <br>

<p style="margin-bottom: 0in;">\r\n</p><p style="margin-bottom: 0in"><font size="3">Influenza down-regulates\r\namiloride-sensitive sodium ion channels in repiratory epithelia (\r\ndisturbance of fluid balance in respiratory tract associated with\r\nrespiratory pathogens such as influenza ). This is caused by HA to\r\ncell-surface receptor which then activated phospholipase C and PKC. \r\nThis does not require endocytosis or entry in cytoplasm. [4]</font>\r\n</p>\r\n<p style="margin-bottom: 0in"><font size="3">PKC isoform is PKCbetaII.\r\nInfluenza requires pH of approximately 5.5 for membrane fusion such\r\nas in the late endosome [</font><font size="3"><b>2,1</b></font><font size="3"><b></b></font><font size="3">]. In\r\ncells lacking PKCBeteII, influenza (and EGFR) accumulates in late\r\nendosomes. Likely that late endosome is not fully functional in\r\nabsence of PKCBetaII [3].<br></font></p>\r\n\r\n

<span style="color: rgb(34, 34, 34); font-family: Verdana, Arial, sans-serif; font-size: 14px; font-style: normal; font-variant: normal; font-weight: normal; letter-spacing: normal; line-height: 16px; orphans: 3; text-align: left; text-indent: 0px; text-transform: none; white-space: normal; widows: 3; word-spacing: 0px; -webkit-text-size-adjust: auto; -webkit-text-stroke-width: 0px; background-color: rgb(255, 255, 255); display: inline !important; float: none; ">M2_inhibitors block nuclear import due to the large size of M1-vRNP [6]. Popular drugs Amantadine/Rimantadine block nuclear import vicariously by blocking the ion channel activity of M2 that pumps hydrogen ions within the viral envelope to break the bonds between M1 and vRNP [7].</span>

S_66 1 S_115 1 S_95 1

<p><span id="species:viewSpeciesDescription" style="font-size: 12px;"><span style="font-size: 14px; line-height: 16px;">Incoming vRNPs or newly synthesized vRNPs are able to act as a template for transcription/translation. Newly </span><span style="font-size: 14px; line-height: 16px; ">synthesized </span><span style="font-size: 14px; line-height: 16px;">vRNPs require NP, PB1, PA, PB2, and RNA.</span></span></p>

<p><span style="font-size: 12px;">Template vRNP requires RNA for replication and transcription [3].</span></p>

<p><span style="font-size: 12px;">All three subunits are required for efficient transcription and replication [4].</span></p>

<p><span style="font-size: 12px;">The incoming vRNP contains the complete polymerase subunits and RNA encapsulated by NP [1].</span></p>

S_85 1 S_106 1 S_112 1 S_97 1 S_93 1

<p><span id="species:viewSpeciesDescription" style="font-size: 12px;">vRNA transcribes mRNA.</span></p>

S_76 1

A functional domain of NS2 with nuclear export signal characteristics was mapped. This domain gives rise to a model by which NS2 acts as a protein adaptor molecule bridging vRNP with the nuclear pore complex and subsequent nuclear export (O’Neill, Talon, & Palese, 1998).<br>

<p><span style="font-size: 12px;">mRNA is translated into its protein.</span></p>

S_87 1

<p><span id="species:viewSpeciesDescription" style="font-size: 12px;">progeny vRNP are exported from the nucleus into the cytoplasm mediated by NS2 and its cognate cellular factor.</span></p>

<p><span style="font-size: 12px;">A cellular\r\n factor for NS2 (NEP; also known as Nuclear Export Protein) is involved \r\nin the nuclear export of vRNPs regulated by ERK [1]. </span></p>

S_28 1 S_4 1 S_78 1

<span style="font-size:11.0pt;line-height:115%;\r\nfont-family:"Calibri","sans-serif";mso-fareast-font-family:"Droid Sans Fallback";\r\nmso-bidi-font-family:"Times New Roman";mso-bidi-theme-font:minor-bidi;\r\nmso-ansi-language:EN-US;mso-fareast-language:EN-US;mso-bidi-language:AR-SA">Of\r\nthe 11 proteins encoded by influenza’s 8 gene segments, 8 are assembled into\r\nprogeny virions (Lamb & Krug, 2001). The viral envelope is made up of HA,\r\nNA, and the M2 ion channel protein. Within the envelope M1 provides the frame\r\nof the virus and connects the vRNP to the viral envelope. vRNP is composed of\r\nPB1, PB2, PA as well as NP that binds and packages influenza’s genome\r\n(influenza virus contains only one copy of the genome (Noda <i>et al.</i>, 2006). Nonstructural protein NS1\r\nis primary for immune system evasion, NS2 is for vRNP nuclear export, and\r\nfinally some strains express PB1-F2 involved in cellular apoptosis</span>

S_20 1 S_33 1 S_6 1 S_21 1 S_79 1 S_8 1 S_127 1 S_61 1 S_70 1 S_114 1 S_2 1

Incoming vRNP are newly formed vRNPs are able to stabilize cRNA synthesis.<br>

<span style="color: rgb(0, 0, 0); font-family: Verdana, Arial, sans-serif; font-size: 12px; font-style: normal; font-variant: normal; font-weight: normal; letter-spacing: normal; line-height: 24px; orphans: 3; text-align: -webkit-auto; text-indent: 0px; text-transform: none; white-space: normal; widows: 3; word-spacing: 0px; -webkit-text-size-adjust: auto; -webkit-text-stroke-width: 0px; background-color: rgb(255, 255, 255); display: inline !important; float: none; ">Incoming or newly formed vRNP </span><span style="color: rgb(0, 0, 0); font-style: normal; font-variant: normal; font-weight: normal; letter-spacing: normal; orphans: 3; text-align: -webkit-auto; text-indent: 0px; text-transform: none; white-space: normal; widows: 3; word-spacing: 0px; -webkit-text-size-adjust: auto; -webkit-text-stroke-width: 0px; background-color: rgb(255, 255, 255); font-size: 11pt; line-height: 16px; font-family: Calibri, sans-serif; ">begins both transcription and replication trough mRNA and cRNA formation, respectively [1].</span>

S_38 1 S_112 1 S_97 1 S_93 1 S_112 1 S_97 1 S_108 1 S_112 1 S_97 1 S_112 1 S_97 1 S_93 1

'IVA_SialicAcid' represents intact influenza A virus particles (with cleaved HA) bound to Sialic Acid. Sucesfully receptor binding requires binding affinity ('HA_SialicAcid_Affinity') between cleaved HA ('IVA_HA1_HA2') [2]. Thus these Upstream Regulators are in an "AND" relationship. 

<font face="Arial, Verdana" size="2">Hemagglutinin (ligand) affinity and specificity to Sialic Acid (receptor) is an essential factor in establishing infection [1], [2].</font>

Only influenza viruses with cleaved hemagglutinin are able to mediate receptor binding with Sialic Acid [3], [4]. [5], [6].

S_58 1 S_23 1

<p><span style="font-size: 12px;">The incoming vRNP contains the complete polymerase subunits and RNA encapsulated by NP [1].</span></p>

<p><span style="font-size: 12px;">All three subunits are required for efficient transcription and replication [4].</span></p>

<p><p><span style="font-size: 12px;">Template vRNP requires RNA for replication and transcription [3].</span></p></p>

<p><span style="font-size: 12px;">All three subunits are required for efficient transcription and replication [4].</span></p>

S_15 1 S_54 1 S_112 1 S_97 1 S_93 1

<p><span style="font-size: 12px;"><span style="font-family: Verdana, Arial, sans-serif; line-height: 24px; orphans: 3; widows: 3; background-color: rgb(255, 255, 255); ">The\r\n vRNP leaving the nucleus is a mutually exclusive event and thus this is\r\n a negative regulator (ie: less vRNPs in the nucleus as more vRNPs enter\r\n the cytoplasm).</span></span></p>

S_74 1 S_112 1 S_97 1 S_93 1 S_6 1

<font face="Arial, Verdana" size="2">After entering the late endosome, vRNPs are released into the cytoplasm. </font>

S_75 1

<font face="Arial, Verdana" size="2">vRNA transcribes mRNA. </font>

<font face="Arial, Verdana" size="2">vRNP initiates both transcription and replication [1]. </font>

S_100 1

<font face="Arial, Verdana" size="2">mRNA_HA_nuc activates mRNA_HA_cyto. </font>

<font face="Arial, Verdana" size="2">mRNA_HA_nuc is exported via host's nuclear export machinery to the cytoplasm; hence mRNA_HA_nuc activates mRNA_HA_cyto [1].</font>

S_104 1

<font face="Arial, Verdana" size="2">vRNA transcribes mRNA. </font>

<font face="Arial, Verdana" size="2">vRNP initiates both transcription and replication [1]. </font>

S_19 1

<p><span style="font-size: 12px;">cRNA leads to vRNA replication [1],</span></p>

S_39 1

<span id="species:viewSpeciesDescription" style="font-size: 12px;">cRNP formation requires cRNA, NP, PB1, PA, and PB2 [1].</span>

<span style="font-size: 12px;"><span style="font-family: Verdana, Arial, sans-serif; line-height: 24px; orphans: 3; widows: 3; background-color: rgb(255, 255, 255); ">cRNA is synthesized early in infection but is quickly degraded by host proteases unless stabilized by RNA polymerase and NP [1].</span></span>

S_91 1 S_112 1 S_97 1 S_93 1

<span id="species:viewSpeciesDescription" style="font-size: 12px;">Incoming vRNP are newly formed vRNPs are able to stabilize cRNA synthesis.</span>

<p><span style="font-size: 12px;"><span style="font-family: Verdana, Arial, sans-serif; line-height: 24px; orphans: 3; widows: 3; background-color: rgb(255, 255, 255); ">Incoming or newly formed vRNP </span><span style="orphans: 3; widows: 3; background-color: rgb(255, 255, 255); font-size: 11pt; line-height: 16px; font-family: Calibri, sans-serif; ">begins both transcription and replication trough mRNA and cRNA formation, respectively [1].</span></span></p>

<span style="font-size: 12px;"><span style="font-family: Verdana, Arial, sans-serif; line-height: 24px; orphans: 3; widows: 3; background-color: rgb(255, 255, 255); ">An intermediate is required for cRNA stabilization [1].</span></span>

<span style="font-size: 12px;"><span style="font-family: Verdana, Arial, sans-serif; line-height: 24px; orphans: 3; widows: 3; background-color: rgb(255, 255, 255); ">All polymerase subunits are required for effective replication [1].</span></span>

S_90 1 S_112 1 S_97 1 S_112 1 S_97 1 S_93 1 S_103 1 S_112 1 S_97 1 S_93 1 S_112 1 S_97 1

<font face="Arial, Verdana" size="2">The vRNA:M1 complex requires both M1 and vRNA localized in the nucleus.</font><h1 id="firstHeading" class="firstHeading" style="font-family: Arial, Verdana; font-size: 10pt; font-style: normal; font-variant: normal; font-weight: normal; line-height: normal; ">\r\n </h1>\r\n

<p><span style="font-size: 12px;">vRNA is bound to M1.</span></p>

<p><span style="font-size: 12px;">M1 binds vRNA.</span></p>

S_46 1 S_26 1

NP from cytoplasm enter the nucleus. 

<span style="font-family: Verdana, Arial, sans-serif; font-size: 12px; line-height: 24px; orphans: 3; widows: 3; background-color: rgb(255, 255, 255); ">Newly translated NP enter the nucleus to form the RNP [1].</span>

S_96 1

S_110 1

<p><span id="species:viewSpeciesDescription" style="font-size: 12px;">mRNA is translated in the cytoplasm by host ribosomes. </span></p>

<p><span style="font-size: 12px;">mRNA is translated into its protein.</span></p>

S_55 1

<span style="font-family: Verdana, Arial, sans-serif; font-size: 12px; line-height: 24px; orphans: 3; widows: 3; background-color: rgb(255, 255, 255); ">PB1:PA complex enters the nucleus [1],</span>

S_5 1

<p><span style="font-size: 12px;"><span style="font-family: Verdana, Arial, sans-serif; line-height: 24px; orphans: 3; widows: 3; background-color: rgb(255, 255, 255); ">The\r\n vRNP leaving the nucleus is a mutually exclusive event and thus this is\r\n a negative regulator (ie: less vRNPs in the nucleus as more vRNPs enter\r\n the cytoplasm).</span></span></p>

S_131 1 S_112 1 S_97 1 S_93 1 S_8 1

<font face="Arial, Verdana" size="2">mRNA_HA_nuc activates mRNA_HA_cyto. </font>

<font face="Arial, Verdana" size="2">mRNA_HA_nuc is exported via host's nuclear export machinery to the cytoplasm; hence mRNA_HA_nuc activates mRNA_HA_cyto [1].</font>

S_59 1

<font face="Arial, Verdana" size="2">Incoming vRNPs or newly synthesized vRNPs are able to act as a template for transcription/translation. Newly synthesized vRNPs require NP, PB1, PA, PB2, and RNA. </font><h1 id="firstHeading" class="firstHeading" style="font-family: Arial, Verdana; font-size: 10pt; font-style: normal; font-variant: normal; font-weight: normal; line-height: normal; ">\r\n </h1>

<p><span style="font-size: 12px;">The incoming vRNP contains the complete polymerase subunits and RNA encapsulated by NP [1].</span></p>

<font face="Arial, Verdana" size="2">All three subunits are required for efficient transcription and replication [4].</font>

<font face="Arial, Verdana" size="2">Template vRNP requires RNA for replication and transcription [3].</font>

S_128 1 S_94 1 S_112 1 S_97 1 S_93 1

<p><span style="font-size: 12px;">The incoming vRNP contains the complete polymerase subunits and RNA encapsulated by NP [1].</span></p>

<p><span style="font-size: 12px;">Template vRNP requires RNA for replication and transcription [3].</span></p>

<p><span style="font-size: 12px;">All three subunits are required for efficient transcription and replication [4].</span></p>

S_67 1 S_122 1 S_112 1 S_97 1 S_93 1

<p><span style="font-size: 12px;">Template vRNP requires RNA for replication and transcription [3].</span></p>

<p><span style="font-size: 12px;">All three subunits are required for efficient transcription and replication [4].</span></p>

<p><span style="font-size: 12px;">The incoming vRNP contains the complete polymerase subunits and RNA encapsulated by NP [1].</span></p>

<p><span style="font-size: 12px;">All three subunits are required for efficient transcription and replication [4].</span></p>

S_46 1 S_112 1 S_97 1 S_93 1 S_105 1

vRNA transcribes mRNA.

vRNP initiates both transcription and replication [1].

S_103 1

<font face="Arial, Verdana" size="2">After entering the late endosome, vRNPs are released into the cytoplasm. </font>

S_75 1

<p><span style="font-size: 12px;">cRNA leads to vRNA replication [1],</span></p>

S_57 1

Multivalent binding of influenza A to Sialic Acid ('IVA_SialicAcid') is sufficient for receptor tyrosine kinase (RTK) activation. Disruption of lipid raft domains, the signaling platform for RTK activation, by Methyl Beta Cyclodextrin or direct inhibition of RTK by RTK inhibitors such as genistein causes significantly reduced virus uptake. Negative regulators of RTK (the inhibitors) are dominant over positive regulators (Influenza binding sialic acid) because treatment of cells with inhibitors before infection caused reduced RTK activation.<br>

<span style="color: rgb(0, 0, 0); font-family: Verdana, Arial, sans-serif; font-size: 12px; font-style: normal; font-variant: normal; font-weight: normal; letter-spacing: normal; line-height: 24px; orphans: 3; text-align: -webkit-auto; text-indent: 0px; text-transform: none; white-space: normal; widows: 3; word-spacing: 0px; -webkit-text-size-adjust: auto; -webkit-text-stroke-width: 0px; background-color: rgb(255, 255, 255); display: inline !important; float: none; ">Treatment of cell prior to infection with a broad-range tyrosine kinase inhibitor such as genistein (PubChem Substance ID 24278036), represented by 'RTK_Inhibitor'  caused virus particles to aggregate on the cell surface with subsequent reduced levels of viral protein levels and progeny virus titers [2].</span>

<span style="color: rgb(0, 0, 0); font-family: Verdana, Arial, sans-serif; font-size: 12px; font-style: normal; font-variant: normal; font-weight: normal; letter-spacing: normal; line-height: 24px; orphans: 3; text-align: -webkit-auto; text-indent: 0px; text-transform: none; white-space: normal; widows: 3; word-spacing: 0px; -webkit-text-size-adjust: auto; -webkit-text-stroke-width: 0px; background-color: rgb(255, 255, 255); display: inline !important; float: none; ">Influenza A binds multivalently to its receptor Sialic Acid (represented by 'IVA_SialicAcid'). Multivalent binding to the cell surface results in formation of raft signaling domains that are able to activate receptor tyrosine kinases such as EGF by bringing receptor monomers in close proximity [2].</span>

S_82 1 S_101 1 S_18 1

<span style="font-size: 14px; line-height: 16px;">Incoming vRNPs or newly synthesized vRNPs are able to act as a template for transcription/translation. Newly </span><span style="font-size: 14px; line-height: 16px; ">synthesized </span><span style="font-size: 14px; line-height: 16px;">vRNPs require NP, PB1, PA, PB2, and RNA.</span>

<p><span style="font-size: 12px;">Template vRNP requires RNA for replication and transcription [3].</span></p>

<p><span style="font-size: 12px;">All three subunits are required for efficient transcription and replication [4].</span></p>

<span style="font-size: 12px;">Each gene segment is encapsulated by NP \r\nand bound to a polymerase complex. NP binds vRNA backbone without \r\nsequence specificity exposing the bases to the outside and hence melting\r\n all secondary structures. This allows the polymerase to transcribe mRNA\r\n as soon as vRNP enters the nucleus without requiring NP to dissociate \r\nor being stopped by a secondary RNA structure [2]</span>

<span style="font-size: 12px;">The incoming vRNP contains the complete polymerase subunits and RNA encapsulated by NP [1].</span>

S_125 1 S_36 1 S_112 1 S_97 1 S_93 1

<p><span id="species:viewSpeciesDescription" style="font-size: 12px;">mRNA is translated in the cytoplasm by host ribosomes. </span></p>

<p><span style="font-size: 12px;">mRNA is translated into its protein.</span></p>

S_35 1

<span id="species:viewSpeciesDescription" style="font-size: 12px;">Incoming vRNP are newly formed vRNPs are able to stabilize cRNA synthesis.</span>

<span style="font-size: 12px;"><span style="font-family: Verdana, Arial, sans-serif; line-height: 24px; orphans: 3; widows: 3; background-color: rgb(255, 255, 255); ">All polymerase subunits are required for effective replication [1].</span></span>

<span style="font-size: 12px;"><span style="font-family: Verdana, Arial, sans-serif; line-height: 24px; orphans: 3; widows: 3; background-color: rgb(255, 255, 255); ">An intermediate is required for cRNA stabilization [1].</span></span>

<p><span style="font-size: 12px;"><span style="font-family: Verdana, Arial, sans-serif; line-height: 24px; orphans: 3; widows: 3; background-color: rgb(255, 255, 255); ">Incoming or newly formed vRNP </span><span style="orphans: 3; widows: 3; background-color: rgb(255, 255, 255); font-size: 11pt; line-height: 16px; font-family: Calibri, sans-serif; ">begins both transcription and replication trough mRNA and cRNA formation, respectively [1].</span></span></p>

S_62 1 S_112 1 S_97 1 S_112 1 S_97 1 S_93 1 S_100 1 S_112 1 S_97 1 S_93 1 S_112 1 S_97 1

vRNP formation requires all three polymerase subunits and vRNA bound to NP. As more vRNPs enter the cytoplasm, less vRNPs are localized in the nucleus.<br>

S_24 1 S_112 1 S_97 1 S_93 1 S_33 1

PB2 is imported into the nucleus.<br>

S_71 1

<p><span style="font-size: 12px;">M1 binds vRNA.</span></p>

<p><span style="font-size: 12px;">vRNA is bound to M1.</span></p>

S_122 1 S_26 1

Deletion of M2 or mutation of M2 cytoplasmic tail inhibits virus budding (Iwatsuki-Horimoto et al., 2006). This may be due to the fact that M2 stabilizes M1 shell structure via direct cytoplasmic domain interactions. On the other hand, M2 has been copurified with NP indicating M2-NP association which would lead to M2-vRNP association (Bron, Kendal, Klenk, & Wilschut, 1993)<br>

M2 mRNA enters cytoplasm to be translated.

S_14 1

PKC can be activated by PI3K or HA_LipidRafts. Bisindolylmaleimide I is a potent inhibitor of PKC.

Bisindolylmaleimide I is a highly specific PKC inhibitor of all PKC isoforms. Addition of Bisindolylmaleimide I prevented replication when added to infected cells [2].

<font face="Arial, Verdana" size="2">Hemagglutinin attachment to cell surface lipid raft domains causes activation of PKC. This causes down-regulation of amiloride-sensitive sodium ion channels in respiratory epithelia. This is consistent with the clinical observation of fluid balance disruption during respiratory pathogens such as influenza [1].</font>

PI3K is able to induce PKC activation [3].

S_61 1 S_37 1 S_72 1 S_37 1

M1 forms dimers if vRNA concentrations are not high enough for vRNA:M1 complexes.

S_34 1 S_51 1 S_24 1 S_1 1 S_92 1 S_113 1 S_74 1 S_119 1 S_131 1

S_72 1

<p><span style="font-size: 12px;">M1 binds vRNA.</span></p>

<p><span style="font-size: 12px;">vRNA is bound to M1.</span></p>

S_94 1 S_26 1

<p><span id="species:viewSpeciesDescription" style="font-size: 12px;">mRNA is translated in the cytoplasm by host ribosomes. </span></p>

<p><span style="font-size: 12px;">mRNA is translated into its protein.</span></p>

<font face="Arial, Verdana" size="2">Theoretical drug target.</font>

S_22 1 S_9 1

vRNA transcribes mRNA.

<span style="font-family: Verdana, Arial, sans-serif; font-size: 12px; line-height: 24px; orphans: 3; widows: 3; background-color: rgb(255, 255, 255); ">vRNP initiates both transcription and replication [1].</span>

S_108 1

<p><span style="font-size: 12px;">cRNA leads to vRNA replication [1],</span></p>

S_47 1

<font face="Arial, Verdana" size="2">vRNA transcribes mRNA. </font>

<font face="Arial, Verdana" size="2">vRNP initiates both transcription and replication [1]. </font>

S_44 1

MEK phosphorylates and activated ERK.

<font face="Arial, Verdana" size="2">Raf phosphorylates and activates MEK [1], </font>

S_25 1

After entering the late endosome, vRNPs are released into the cytoplasm.

S_75 1

<p><span id="species:viewSpeciesDescription" style="font-size: 12px;">progeny vRNP are exported from the nucleus into the cytoplasm mediated by NS2 and its cognate cellular factor.</span></p>

<p><span style="font-size: 12px;">A cellular\r\n factor for NS2 (NEP; also known as Nuclear Export Protein) is involved \r\nin the nuclear export of vRNPs regulated by ERK [1]. </span></p>

S_52 1 S_4 1 S_78 1

<font face="Arial, Verdana" size="2">After entering the late endosome, vRNPs are released into the cytoplasm. </font>

<div style="orphans: 3; text-align: left; widows: 3;">The vRNP is released into the cytoplasm after reaching the late endosome (LE) due to the intrinsic ion channel activity of M2 [1],[2]. </div>

S_75 1

<p><span id="species:viewSpeciesDescription" style="font-size: 12px;">vRNA transcribes mRNA.</span></p>

S_83 1

For influenza to enter the early endosome, PIP3 and entry (and formation of vesicle formation) via clathrin is required. This is an "AND" relationship between 'IVA_SialicAcid_CCV' and 'PIP3'.<br>

\r\n\r\n<p class="MsoNormal" style="margin-bottom:0in;margin-bottom:.0001pt;line-height:\r\nnormal">\r\n\r\n</p><p class="MsoNormal" style="margin-bottom:0in;margin-bottom:.0001pt;line-height:\r\nnormal"><span style="font-size:8.0pt">PI3K has a bivalent role in influenza.\r\nEarly in infection PI3K is involved in virus entry [1]. According to [1] this\r\nis independent of PI3K effector Akt (or EEA1 and PKCbetaII) but is regulated by\r\nPIP3 in early and late endosomal fusion (Model places PIP3 condition only on\r\nEE); exact molecular mechanism is unknown.</span></p>\r\n\r\n\r\n\r\n \r\n\r\n\r\n\r\n

<span style="color: rgb(0, 0, 0); font-family: Arial, Verdana; font-size: 13px; font-style: normal; font-variant: normal; font-weight: normal; letter-spacing: normal; line-height: normal; orphans: 2; text-align: -webkit-auto; text-indent: 0px; text-transform: none; white-space: normal; widows: 2; word-spacing: 0px; -webkit-text-size-adjust: auto; -webkit-text-stroke-width: 0px; display: inline !important; float: none; ">Influenza A viruses bound to Sialic Acid within clathrin coated vesicles (represented by 'IVA_SialicAcid_CCV') enter the early endosome [1].</span>

S_73 1 S_48 1

<p><span style="font-size: 12px;">M1 binds vRNA.</span></p>

<p><span style="font-size: 12px;">vRNA is bound to M1.</span></p>

S_54 1 S_26 1