Influenza virus differentially activates mTORC1 and mTORC2 signaling to maximize late stage replication

<div><p>Influenza A virus usurps host signaling factors to regulate its replication. One example is mTOR, a cellular regulator of protein synthesis, growth and motility. While the role of mTORC1 in viral infection has been studied, the mechanisms that induce mTORC1 activation and the substrates regulated by mTORC1 during influenza virus infection have not been established. In addition, the role of mTORC2 during influenza virus infection remains unknown. Here we show that mTORC2 and PDPK1 differentially phosphorylate AKT upon influenza virus infection. PDPK1-mediated phoshorylation of AKT at a distinct site is required for mTORC1 activation by influenza virus. On the other hand, the viral NS1 protein promotes phosphorylation of AKT at a different site via mTORC2, which is an activity dispensable for mTORC1 stimulation but known to regulate apoptosis. Influenza virus HA protein and down-regulation of the mTORC1 inhibitor REDD1 by the virus M2 protein promote mTORC1 activity. Systematic phosphoproteomics analysis performed in cells lacking the mTORC2 component Rictor in the absence or presence of Torin, an inhibitor of both mTORC1 and mTORC2, revealed mTORC1-dependent substrates regulated during infection. Members of pathways that regulate mTORC1 or are regulated by mTORC1 were identified, including constituents of the translation machinery that once activated can promote translation. mTORC1 activation supports viral protein expression and replication. As mTORC1 activation is optimal midway through the virus life cycle, the observed effects on viral protein expression likely support the late stages of influenza virus replication when infected cells undergo significant stress.</p></div

DNA Tumor Virus Regulation of Host DNA Methylation and Its Implications for Immune Evasion and Oncogenesis

Viruses have evolved various mechanisms to evade host immunity and ensure efficient viral replication and persistence. Several DNA tumor viruses modulate host DNA methyltransferases for epigenetic dysregulation of immune-related gene expression in host cells. The host immune responses suppressed by virus-induced aberrant DNA methylation are also frequently involved in antitumor immune responses. Here, we describe viral mechanisms and virus–host interactions by which DNA tumor viruses regulate host DNA methylation to evade antiviral immunity, which may contribute to the generation of an immunosuppressive microenvironment during cancer development. Recent trials of immunotherapies have shown promising results to treat multiple cancers; however, a significant number of non-responders necessitate identifying additional targets for cancer immunotherapies. Thus, understanding immune evasion mechanisms of cancer-causing viruses may provide great insights for reversing immune suppression to prevent and treat associated cancers

mTORC1 activation promotes viral infection.

<p>(<b>A</b>) A549 cells were infected with WSN at MOI of 2 PFU/cell for 1 h and then treated with 250 nM Torin1 or DMSO for an additional 9 h followed by immunoblot analysis. (<b>B</b>) A549 cells were infected at MOI of 0.01 PFU/cell with WSN for 16 h in the presence of the depicted concentrations of Torin1 or DMSO. Viral titers were measured by plaque assay to yield plaque-forming units per mL (PFU/ml, plotted as % infection relative to DMSO control). Cell survival was also assessed at the depicted concentrations using the MTT assay. The following parameters were calculated: CC₅₀>500 nM; IC₅₀ = 0.46nM; and SI>1087. (<b>C</b>) Experiment was performed as in <b>B</b> except that cells were treated with 13.7 μM rapamycin or DMSO as control. Mean and standard error of the mean (SEM) are shown. <i>n</i> = 3, **p<0.01. (<b>D</b>) <i>Rictor</i>^<i>+/+</i> or <i>Rictor</i>^<i>-/-</i> MEFs were infected with WSN at MOI of 2 PFU/cell for 6 h and treated with 250 nM Torin1 or DMSO at 1 h post-infection. Immunoblot analyses were performed to detect viral proteins (HA, NP, PA, PB1, PB2, NS1, M1, M2 and NA) and host proteins (β-actin, Rictor, total and phosphorylated S6K and 4E-BP1). β-actin and S6K serve as loading controls. The upper band in the S6K/p-S6K blots is p85 S6K whereas the lower band is p70 S6K. (<b>E</b>) <i>Rictor</i>^<i>+/+</i> or <i>Rictor</i>^<i>-/-</i> MEFs were infected with WSN at MOI of 0.01 for 24 h and 48 h. Viral titers were measured by plaque assay to yield plaque-forming units per ml (PFU/ml). Mean and standard deviation (SD) are shown, <i>n</i> = 3, **p<0.01, *p<0.05.</p

The viral protein HA promotes mTORC1 activation.

<p>(<b>A</b>) A549 cells were transfected with siRNAs (pool of three each) targeting viral mRNAs and then infected for 6 h at MOI of 2 PFU/cell. Immunoblot analysis was performed against the depicted proteins. (<b>B</b>) The activity of reconstituted polymerase complexes was measured by transfecting cells with a control plasmid or plasmids encoding NP, the polymerase subunits (PA, PB1 and PB2) and luciferase reporter genes to measure influenza virus promoter activity (mini-genome assay). Luciferase assay and immunoblot analysis were performed with antibodies against the depicted proteins. (<b>C</b>) MDCK cells were transfected with control plasmids or with plasmids encoding HA or NA. MDCK cells were also mock infected or infected with WSN at MOI of 2 PFU/cell for 6h. Immunoblot analysis were performed with antibodies against the depicted proteins. Data are representative of three independent experiments.</p

Influenza virus down-regulates REDD1 to promote mTORC1 activity.

<p>(<b>A-C</b>) A549 cells were infected with WSN at MOI of 2 PFU/cell for the indicated times. Cell extracts were subjected to (<b>A</b>) western blot analysis to detect the depicted proteins and quantified as shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006635#ppat.1006635.s005" target="_blank">S5 Fig</a> or (<b>B</b>) RNA was purified for qRT-PCR to determine REDD1 mRNA levels. Mean and standard deviation are shown for qRT-PCR, <i>n</i> = 4 independent experiments done in triplicates. **<i>p</i><0.000004, Student's <i>t</i>-test. (<b>C</b>) A549 cells were transfected with siRNAs (pool of three each) targeting viral mRNAs and then infected for 7 h at MOI of 2 PFU/cell. Immunoblot analysis was performed to detect the depicted proteins, <i>n</i> = 3. (<b>D</b>) A549 cells were transfected with plasmids encoding the indicated virus proteins. At 48 h post-transfection, total RNA was purified and REDD1 mRNA levels were determined by qRT-PCR as in <b>B</b>. The bottom panel in <b>D</b> shows viral polymerase activity upon tranfection of the depicted viral proteins and/or minigenome as control. Minigenome mRNA was measured by qRT-PCR. In cells transfected with the complete set of plasmids that encode the viral polymerase we detect the minigenome RNA transcribed by pol I directly from the plasmid in addition to the minigenme RNA amplified by the influenza proteins, indicating protein activity. In cells transfected with the same plasmids except for PA, we only detect the minigenome RNA transcribed by pol I directly from the plasmid, and the average values was set to 1. The minigenome RNA level is higher when all plasmids were transfected (<i>n</i> = 3). (<b>E, F</b>) MDCK cells were transfected with control plasmid of plasmid enconding the M2 protein. In <b>E,</b> RNA was purified for qRT-PCR to determine REDD1 mRNA levels as in <b>B</b>, <i>n</i> = 3, ***p<0.001. In <b>F,</b> cell extracts were subjected to western blot analysis to detect the depicted proteins (<i>n</i> = 3). (<b>G</b>) U2OS-REDD1 cells were treated with vehicle or 1μg/ml tetracycline for 2 h prior to and during infection to induce REDD1 expression. Cells were infected at MOI of 2 PFU/cell for 6 h. Immunoblot analyses were performed to detect the depicted proteins. Total S6K serves as the loading control. The upper band in the S6K/p-S6K blots is p85 S6K, whereas the lower band is p70 S6K (<i>n</i> = 3).</p

Schematic representation of the model for mTOR activation by influenza virus midway through the virus life cycle.

<p>The viral protein HA and virus replication promote mTORC1 activation through PDPK1-mediated phosphorylation of AKT at T308. In addition, down-regulation of REDD1 by the viral M2 protein amplifies or support mTORC1 activation downstream of AKT. NS1 promotes AKT phosphorylation at S473 via mTORC2 and this process is known to regulate apoptosis. Differential AKT phosphorylation dictates downstream effects.</p

Viral replication is required for influenza virus to activate mTORC1 independently of NS1.

<p>(<b>A</b>, <b>B</b>) Vero cells were infected with (<b>A</b>) WSN or (<b>B</b>) PR8 wild-type or mutant viruses lacking NS1 at MOI of 2 PFU/cell for 6 h. (<b>C</b>,<b>D</b>) HCT116 <i>Akt1/2</i>^<i>+/+</i> and <i>Akt1/2</i>^<i>-/-</i> were infected with WSNΔNS1 or WSN at MOI of 5 PFU/cell for 10h. (<b>E</b>) A549 cells were infected with WSN or UV-inactivated WSN at MOI of 2 PFU/cell for 6h. (<b>F</b>) <i>Mavs</i>^<i>+/+</i> and <i>Mavs</i>^<i>-/-</i> or (<b>G</b>) <i>Ifitm3</i>^<i>+/+</i> and <i>Ifitm3</i>^<i>-/-</i> MEFs were infected with WSN at an MOI of 2 PFU/cell for 6 h. Immunoblot analyses were performed for detection of viral proteins (NS1, HA, NP and M1) and host proteins (β-actin, MAVS, IFITM3 as well as total and phosphorylated S6K, AKT and 4E-BP1). β-actin and total S6K serve as loading controls. The upper band in the S6K/p-S6K blots is p85 S6K whereas the lower band is p70 S6K. Data are representative of three independent experiments.</p

PDPK1 phosphorylates AKT to promote mTORC1 activation during influenza virus infection.

<p>(<b>A</b>) Schematic representation of the PI3K/AKT pathway and mTORC1 activation. Filled red lines represent regulation of the signaling pathway by influenza virus. Dotted lines depict gaps of knowledge in these pathways, and boxes with question marks indicate unknown regulatory factors. (<b>B</b>) HCT116 <i>Akt1/2</i>^<i>+/+</i> and <i>Akt1/2</i>^<i>-/-</i> were infected with WSN at MOI of 2 PFU/cell for 6h. Cell lysates were subjected to immunoblot analysis with antibodies against the depicted proteins. (<b>C</b>) <i>Rictor</i>^<i>+/+</i> and <i>Rictor</i>^<i>-/</i>- MEFs were processed as in <b>B</b> except that infection was performed for 10h. (<b>D</b>) <i>Tbk1</i>^<i>+/+</i> and <i>Tbk1</i>^<i>-/-</i> MEFs +/- 1 μM BX795 were processed as in <b>B</b>. (<b>E</b>) HCT116 <i>Pdpk1</i>^<i>+/+</i> and <i>Pdpk1</i>^<i>-/-</i> were processed as in <b>B</b>. Data are representative of three independent experiments. Dotted line in <b>C</b> indicates omission of unnecessary lanes from the same immunoblot membrane. M2, M1 and NS1: viral proteins; host proteins: β-actin, Rictor, TBK1, PPDPK1, total and phosphorylated S6K, AKT and 4E-BP1. Total S6K, β-actin and total AKT serve as loading controls.</p