Influenza Virus Non-Structural Protein 1 (NS1) Disrupts Interferon Signaling

Type I interferons (IFNs) function as the first line of defense against viral infections by modulating cell growth, establishing an antiviral state and influencing the activation of various immune cells. Viruses such as influenza have developed mechanisms to evade this defense mechanism and during infection with influenza A viruses, the non-structural protein 1 (NS1) encoded by the virus genome suppresses induction of IFNs-α/β. Here we show that expression of avian H5N1 NS1 in HeLa cells leads to a block in IFN signaling. H5N1 NS1 reduces IFN-inducible tyrosine phosphorylation of STAT1, STAT2 and STAT3 and inhibits the nuclear translocation of phospho-STAT2 and the formation of IFN-inducible STAT1:1-, STAT1:3- and STAT3:3- DNA complexes. Inhibition of IFN-inducible STAT signaling by NS1 in HeLa cells is, in part, a consequence of NS1-mediated inhibition of expression of the IFN receptor subunit, IFNAR1. In support of this NS1-mediated inhibition, we observed a reduction in expression of ifnar1 in ex vivo human non-tumor lung tissues infected with H5N1 and H1N1 viruses. Moreover, H1N1 and H5N1 virus infection of human monocyte-derived macrophages led to inhibition of both ifnar1 and ifnar2 expression. In addition, NS1 expression induces up-regulation of the JAK/STAT inhibitors, SOCS1 and SOCS3. By contrast, treatment of ex vivo human lung tissues with IFN-α results in the up-regulation of a number of IFN-stimulated genes and inhibits both H5N1 and H1N1 virus replication. The data suggest that NS1 can directly interfere with IFN signaling to enhance viral replication, but that treatment with IFN can nevertheless override these inhibitory effects to block H5N1 and H1N1 virus infections

The Role of CCR5 in Vaccinia virus Pathogenesis

Viral appropriation of chemokine receptors is an effective way to prevent a host immune response against the invading virus. Many viruses, including poxviruses, subvert the host immune response by encoding several chemokine receptor homologues, capable of binding to and thereby precluding chemokines from activating their cognate cell surface receptors. All poxviruses employ strategies to modulate chemokine activity, including virus-encoded chemokine-binding proteins, receptor homologues and ligand mimics. The potential for the involvement of certain chemokine receptors in poxviral infection was suggested in studies utilizing the rabbit poxvirus, myxoma. Specifically, CCR5 was implicated in mediating cell target susceptibility to infection. Our data suggest virus-CCR5 interactions may lead to the selective activation of distinct signaling pathways that are advantageous for the virus. VACV, a member of the poxvirus family, produces two structurally distinct forms of virions, the intracellular mature virus (IMV) and the extracellular enveloped virus (EEV), for which the immediate events following cell entry are ill-defined. Using confocal microscopy, we provided evidence that IMV and EEV enter both permissive and non-permissive cells, and that introduction of CCR5 into non-permissive cells – mouse fibroblasts and human PM1 T cells - renders them permissive for VACV replication. We showed that virus activation of CCR5 leads to the selective activation of distinct signaling pathways that are advantageous for the virus. We demonstrated that VACV infection in permissive cells is inhibited by siRNA knockdown of cell surface CCR5 expression and by the CCR5 antagonist, TAK-779. The importance of tyrosine phosphorylation of CCR5 was suggested by the observation that introduction of a CCR5 mutant, in which all the intracellular tyrosines are replaced by phenylalanines, effectively reduces VACV infection in permissive cells. Moreover, tyrosine-339 was implicated in CCR5 as the critical residue for mediating viral infection, since cells expressing CCR5.Y339F do not support viral replication. The cascade of events that leads to permissive phenotype of these cells includes phosphorylation activation of multiple signaling effectors: Jak-2, IRS-2, ERK1/ 2 and Grb2. These data were supported by findings that viral replication in permissive CCR5 expressing cells is blocked by Herbimycin A, and the Jak2 inhibitor, tyrophostin AG490, but not pertussis toxin. Viewed altogether, a critical role of post-entry events, specifically intracellular tyrosine phosphorylation events, was established in determining permissiveness of cells to VACV replication. Furthermore, evidence was provided that introduction of CCR5 in primary human T cells renders them permissive to VACV replication. Since permissive infection of T cells might represent a mechanism for VACV dissemination throughout the lymphatic system, we hypothesized that the absence of CCR5 may be protective against VACV infection in vivo. To test this hypothesis, wild-type and CCR5 null mice were challenged with VACV by intranasal inoculation. In time course studies we identified aggressive viral replication in the lungs and spleens of CCR5+/+ mice, with no evidence of infection in the CCR5-/- mice. Moreover, associated with VACV infection, we provided evidence for CD4+ and CD8+ T as well as CD11c+ and F4/80+ cell infiltration into the lungs of CCR5+/+ but not CCR5-/- mice, and showed that CCR5-expressing T cells harbor replicating virus. We showed that this CCR5-dependence is VACV-specific, since CCR5-/- mice were as susceptible to intranasal influenza (A/WSN/33) infection as CCR5+/+ mice. In a final series of experiments we provided evidence that adoptive transfer of CCR5+/+ bone marrow into CCR5-/- mice restored VACV permissiveness, with evidence of lung and spleen infection. Taken together, our data showed a critical and novel role for CCR5 in VACV infection and dissemination in vivo. Moreover, our confocal studies suggested a possible physical interaction between cellular proteins and the VACV in cytosole. Using mass spectrometry-based proteomics, glomulin was identified as a host cell protein that interacts with VACV. Knockdown of glomulin expression in human PM1.CCR5 T cells reduced VACV infection. We demonstrate that treatment of PM1.CCR5 T cells with a c-Met phosphorylation inhibitor led to a significant reduction in VACV infectivity. The data indicated that inhibition of c-Met phosphorylation, reduces the cytosolic availability of activated glomulin, thus leading to a decrease in VACV infectivity. These data identify glomulin as a permissivity factor for VACV infection, and as a potential therapeutic target for VACV.Ph

Role for CCR5 in Dissemination of Vaccinia Virus In Vivo▿

In an earlier report, we provided evidence that expression of CCR5 by primary human T cells renders them permissive for vaccinia virus (VACV) replication. This may represent a mechanism for dissemination throughout the lymphatic system. To test this hypothesis, wild-type CCR5+/+ and CCR5 null mice were challenged with VACV by intranasal inoculation. In time course studies using different infective doses of VACV, we identified viral replication in the lungs of both CCR5+/+ and CCR5−/− mice, yet there were diminished viral loads in the spleens and brains of CCR5−/− mice compared with CCR5+/+ mice. Moreover, in association with VACV infection, we provide evidence for CD4+ and CD8+ T-cell as well as CD11c+ and F4/80+ cell infiltration into the lungs of CCR5+/+ but not CCR5−/− mice, and we show that the CCR5-expressing T cells harbor virus. We demonstrate that this CCR5 dependence is VACV specific, since CCR5−/− mice are as susceptible to intranasal influenza virus (A/WSN/33) infection as CCR5+/+ mice. In a final series of experiments, we provide evidence that adoptive transfer of CCR5+/+ bone marrow leukocytes into CCR5−/− mice restores VACV permissiveness, with evidence of lung and spleen infection. Taken together, our data suggest a novel role for CCR5 in VACV dissemination in vivo

Vaccinia Virus Activation of CCR5 Invokes Tyrosine Phosphorylation Signaling Events That Support Virus Replication

Vaccinia virus, a poxvirus, produces structurally distinct forms of virions for which the immediate events following cell entry are ill-defined. We provide evidence that intracellular mature virus (IMV) enters both permissive and nonpermissive T-cell lines and that introduction of CCR5 into nonpermissive mouse fibroblasts or human primary T cells renders the cells permissive for vaccinia replication. Notably, T cells expressing CCR5 in which tyrosine 339 in the intracellular region is replaced by phenylalanine no longer support virus replication or virus-inducible activation of specific host cell signaling effectors IRS-2, Grb2, and Erk1/2. We show that following IMV entry into the cell, the intact but not the tyrosine-deficient CCR5 is rapidly internalized and colocalizes with virus. This colocalization precedes virus-inducible signaling and replication

Type I Interferon Protects Antiviral CD8(+) T Cells from NK Cell Cytotoxicity

Despite development of new antiviral drugs, viral infections are still a major health problem. The most potent antiviral defense mechanism is the innate production of type I interferon (IFN-I), which not only limits virus replication but also promotes antiviral T cell immunity through mechanisms, which remain insufficiently studied. Using the murine lymphocytic choriomeningitis virus model system, we show here that IFN-I signaling on T cells prevented their rapid elimination in vivo. Microarray analyses uncovered that IFN-I triggered the expression of selected inhibitory NK-cell-receptor ligands. Consequently, T cell immunity of IFN-I receptor (IFNAR)-deficient T cells could be restored by NK cell depletion or in NK-cell-deficient hosts (Nfil3(-/-)).The elimination of Ifnar1(-/-) T cells was dependent on NK-cell-mediated perforin expression. In summary, we identified IFN-I as a key player regulating the protection of T cells against regulatory NK cell function

H5N1 NS1 reduces surface IFNAR1 but not IFNAR2 expression.

<p><b>A</b>) HeLa cells were transfected with either GFP vector alone (green) or GFP vector containing HA-tagged NS1 (red), then 24 hr post-transfection, GFP-positive cells were FACS sorted and stained for IFNAR1 or IFNAR2 and analyzed by FACS. Data are representative of three independent experiments. <b>B</b>) HeLa cells were transfected with HA-tagged NS1 plasmid and 24 hr post-transfection were fixed and stained for HA (green) and either IFNAR1 (red; upper panel) or IFNAR2 (red; lower panel), and analyzed by confocal microscopy. Data are representative of two independent experiments. <b>C</b>) HeLa cells were transfected with either GFP vector alone () or GFP vector containing HA-tagged NS1 (▪). 24 hr post-transfection, GFP positive cells were FACS sorted, RNA extracted and cDNA synthesized. <i>Ifnar1</i>, <i>ifnar2</i> and <i>β</i>-actin gene expression were analyzed by RT-PCR. Gene expression was calculated relative to β-actin gene expression and normalized to cells transfected with GFP vector alone. Data are representative of two independent experiments. Significant differences (asterisk) were determined by Student's t-test (p<0.05).</p

Treatment with IFN alfacon-1 inhibits <i>pandemic</i> H1N1 2009 virus replication in human lung tissue.

<p>Three different human surgical lung tissue explants were infected with pandemic H1N1 2009 virus for 24 hr, then either left untreated (▪) or treated with IFN alfacon-1 (1.2×104 U/mL) () for a further 48 hr. At the indicated times <b>A</b>) Viral titers (TCID₅₀) and <b>B</b>) Influenza A <i>m</i> gene expression were measured; Significant differences were determined by Student t-test: * ∼ p<0.05; ** ∼ p<0.01. <b>C</b>) Thin sections of infected human lung explants, either untreated (i) or IFN-treated (ii) were stained for influenza A nucleoprotein (pink).</p

Influenza virus infection reduces <i>ifnar1</i> and <i>ifnar2</i> expression at 24 hours post-infection in human monocyte-derived macrophages and <i>ex vivo</i> lung tissues.

<p>Human monocyte-derived macrophages were infected with A/HK/483/97 H5N1 or A/HK/54/98 H1N1 virus (Multiplicity of Infection (MOI)  = 2). RNA was extracted from the cells at <b>A</b>) 3 hr, <b>B</b>) 6 hr, and <b>C</b>) 24 hr post-infection. Following cDNA synthesis, <i>ifnar1</i> (▪) and <i>ifnar2</i> () expression was assayed by real-time PCR. Data shown are fold induction of gene expression relative to mock-infected control after normalizing to β-actin in each sample. Representative data of duplicate experiments with means of triplicate assays are shown; <b>D</b>) Human lung explant tissue was either mock-infected (PBS) or infected with A/HK/483/97 H5N1 or A/HK/54/98 H1N1 influenza A viruses, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013927#s2" target="_blank">Materials & Methods</a>. 18 hr post-infection tissue was processed to extract RNA. cDNA was synthesized and expression of <i>ifnar1</i>, <i>ifnar2</i> and <i>β-actin</i> gene expression was measured by RT-PCR analysis. Gene expression was calculated relative to <i>β-actin</i> gene expression and normalized to mock infected tissues. Data are representative of two independent experiments. Significant differences (asterisk) were determined by Student's t-test (p<0.05).</p