Human parainfluenza virus type 4 is incapable of evading the interferon-induced antiviral effect

The V proteins of some paramyxoviruses have developed the ability to efficiently inactivate STAT protein function as a countermeasure for evading interferon (IFN) responses. Human parainfluenza virus type 4 (hPIV4) is one of the rubulaviruses, which are members of the family Paramyxoviridae, and has a V protein with a highly conserved cysteine-rich domain that is the hallmark of paramyxovirus V proteins. In order to study the function of the hPIV4 V protein, we established HeLa cells expressing the hPIV4A V protein (HeLa/FlagPIV4V). The hPIV4 V protein had no ability to reduce the level of STAT1 or STAT2, although it associated with STAT1, STAT2, DDB1, and Cul4A. It interfered with neither STAT1 and STAT2 tyrosine phosphorylation nor IFN-induced STAT nuclear accumulation. In addition, HeLa/FlagPIV4V cells are fully sensitive to both beta interferon (IFN-beta) and IFN-gamma, indicating that the hPIV4 V protein has no ability to block IFN-induced signaling. We further established HeLa cells expressing various chimeric proteins between the hPIV2 and hPIV4A V proteins. The lack of IFN-antagonistic activity of the hPIV4 V protein is caused by both the P/V common and V-specific domains. At least two regions (amino acids [aa] 32 to 45 and aa 143 to 164) of hPIV4 V in the P/V common domain and one region (aa 200 to 212) of the C terminus are involved in the inability to evade the IFN-induced signaling. Moreover, we established HeLa cells persistently infected with hPIV4 to make sure of the inability to escape IFN and confirmed that hPIV4 is the only paramyxovirus analyzed to date that can't evade the IFN-induced antiviral responses.     

Naturally occurring substitutions in the P/V gene convert the noncytopathic paramyxovirus simian virus 5 into a virus that induces alpha/beta interferon synthesis and cell death

The V protein of the paramyxovirus simian virus 5 (SV5) is responsible for targeted degradation of STAT1 and the block in alpha/beta interferon (IFN-alpha/beta) signaling that occurs after SV5 infection of human cells. We have analyzed the growth properties of a recombinant SV5 that was engineered to be defective in targeting STAT1 degradation. A recombinant SV5 (rSV5-P/V-CPI-) was engineered to contain six naturally occurring P/V protein mutations, three of which have been shown in previous transfection experiments to disrupt the V-mediated block in IFN-alpha/beta signaling. In contrast to wild-type (WT) SV5, human cells infected with rSV5-P/V-CPI- had STAT1 levels similar to those in mock-infected cells. Cells infected with rSV5-P/V-CPI- were found to express higher-than-WT levels of viral proteins and mRNA, suggesting that the P/V mutations had disrupted the regulation of viral RNA synthesis. Despite the inability to target STAT1 for degradation, single-step growth assays showed that the rSV5-P/V-CPI- mutant virus grew better than WT SV5 in all cell lines tested. Unexpectedly, cells infected with rSV5-P/V-CPI- but not WT SV5 showed an activation of a reporter gene that was under control of the IFN-beta promoter. The secretion of IFN from cells infected with rSV5-P/V-CPI- but not WT SV5 was confirmed by a bioassay for IFN. The rSV5-P/V-CPI- mutant grew to higher titers than did WT rSV5 at early times in multistep growth assays. However, rSV5-P/V-CPI- growth quickly reached a final plateau while WT rSV5 continued to grow and produced a final titer higher than that of rSV5-P/V-CPI- by late times postinfection. In contrast to WT rSV5, infection of a variety of cell lines with rSV5-P/V-CPI- induced cell death pathways with characteristics of apoptosis. Our results confirm a role for the SV5 V protein in blocking IFN signaling but also suggest new roles for the P/V gene products in controlling viral gene expression, the induction of IFN-alpha/beta synthesis, and virus-induced apoptosis.     

Single amino acid substitution in the V protein of simian virus 5 differentiates its ability to block interferon signaling in human and murine cells

Previous work has demonstrated that the V protein of simian virus 5 (SV5) targets STAT1 for proteasome-mediated degradation (thereby blocking interferon [IFN] signaling) in human but not in murine cells. In murine BF cells, SV5 establishes a low-grade persistent infection in which the virus fluxes between active and repressed states in response to local production of IFN. Upon passage of persistently infected BF cells, virus mutants were selected that were better able to replicate in murine cells than the parental W3 strain of SV5 (wild type [wt]). Viruses with mutations in the Pk region of the N-terminal domain of the V protein came to predominate the population of viruses carried in the persistently infected cell cultures. One of these mutant viruses, termed SV5 mci-2, was isolated. Sequence analysis of the V/P gene of SV5 mci-2 revealed two nucleotide differences compared to wt SV5, only one of which resulted in an amino acid substitution (asparagine [N], residue 100, to aspartic acid [D]) in V. Unlike the protein of wt SV5, the V protein of SV5 mci-2 blocked IFN signaling in murine cells. Since the SV5 mci-2 virus had additional mutations in genes other than the V/P gene, a recombinant virus (termed rSV5-V/P N(100)D) was constructed that contained this substitution alone within the wt SV5 backbone to evaluate what effect the asparagine-to-aspartic-acid substitution in V had on the virus phenotype. In contrast to wt SV5, rSV5-V/P N(100)D blocked IFN signaling in murine cells. Furthermore, rSV5-V/P N(100)D virus protein synthesis in BF cells continued for significantly longer periods than that for wt SV5. However, even in cells infected with rSV5-V/P N(100)D, there was a late, but significant, inhibition in virus protein synthesis. Nevertheless, there was an increase in virus yield from BF cells infected with rSV5-V/P N(100)D compared to wt SV5, demonstrating a clear selective advantage to SV5 in being able to block IFN signaling in these cells.     

The p127 subunit (DDB1) of the UV-DNA damage repair binding protein is essential for the targeted degradation of STAT1 by the V protein of the paramyxovirus simian virus 5

The V protein of simian virus 5 (SV5) blocks interferon signaling by targeting STAT1 for proteasome-mediated degradation. Here we present three main pieces of evidence which demonstrate that the p127 subunit (DDB1) of the UV damage-specific DNA binding protein (DDB) plays a central role in this degradation process. First, the V protein of an SV5 mutant which fails to target STAT1 for degradation does not bind DDB1. Second, mutations in the N and C termini of V which abolish the binding of V to DDB1 also prevent V from blocking interferon (IFN) signaling. Third, treatment of HeLa/SV5-V cells, which constitutively express the V protein of SV5 and thus lack STAT1, with short interfering RNAs specific for DDB1 resulted in a reduction in DDB1 levels with a concomitant increase in STAT1 levels and a restoration of IFN signaling. Furthermore, STAT1 is degraded in GM02415 (2RO) cells, which have a mutation in DDB2 (the p48 subunit of DDB) which abolishes its ability to interact with DDB1, thereby demonstrating that the role of DDB1 in STAT1 degradation is independent of its association with DDB2. Evidence is also presented which demonstrates that STAT2 is required for the degradation of STAT1 by SV5. These results suggest that DDB1, STAT1, STAT2, and V may form part of a large multiprotein complex which leads to the targeted degradation of STAT1 by the proteasome.     

Inhibition of interferon signaling by the New York 99 strain and Kunjin subtype of West Nile virus involves blockage of STAT1 and STAT2 activation by nonstructural proteins

The interferon (IFN) response is the first line of defense against viral infections, and the majority of viruses have developed different strategies to counteract IFN responses in order to ensure their survival in an infected host. In this study, the abilities to inhibit IFN signaling of two closely related West Nile viruses, the New York 99 strain (NY99) and Kunjin virus (KUN), strain MRM61C, were analyzed using reporter plasmid assays, as well as immunofluorescence and Western blot analyses. We have demonstrated that infections with both NY99 and KUN, as well as transient or stable transfections with their replicon RNAs, inhibited the signaling of both alpha/beta IFN (IFN-alpha/beta) and gamma IFN (IFN-gamma) by blocking the phosphorylation of STAT1 and its translocation to the nucleus. In addition, the phosphorylation of STAT2 and its translocation to the nucleus were also blocked by KUN, NY99, and their replicons in response to treatment with IFN-alpha. IFN-alpha signaling and STAT2 translocation to the nucleus was inhibited when the KUN nonstructural proteins NS2A, NS2B, NS3, NS4A, and NS4B, but not NS1 and NS5, were expressed individually from the pcDNA3 vector. The results clearly demonstrate that both NY99 and KUN inhibit IFN signaling by preventing STAT1 and STAT2 phosphorylation and identify nonstructural proteins responsible for this inhibition.     

Ebola virus VP24 binds karyopherin alpha1 and blocks STAT1 nuclear accumulation

Ebola virus (EBOV) infection blocks cellular production of alpha/beta interferon (IFN-alpha/beta) and the ability of cells to respond to IFN-alpha/beta or IFN-gamma. The EBOV VP35 protein has previously been identified as an EBOV-encoded inhibitor of IFN-alpha/beta production. However, the mechanism by which EBOV infection inhibits responses to IFNs has not previously been defined. Here we demonstrate that the EBOV VP24 protein functions as an inhibitor of IFN-alpha/beta and IFN-gamma signaling. Expression of VP24 results in an inhibition of IFN-induced gene expression and an inability of IFNs to induce an antiviral state. The VP24-mediated inhibition of cellular responses to IFNs correlates with the impaired nuclear accumulation of tyrosine-phosphorylated STAT1 (PY-STAT1), a key step in both IFN-alpha/beta and IFN-gamma signaling. Consistent with this proposed function for VP24, infection of cells with EBOV also confers a block to the IFN-induced nuclear accumulation of PY-STAT1. Further, VP24 is found to specifically interact with karyopherin alpha1, the nuclear localization signal receptor for PY-STAT1, but not with karyopherin alpha2, alpha3, or alpha4. Overexpression of VP24 results in a loss of karyopherin alpha1-PY-STAT1 interaction, indicating that the VP24-karyopherin alpha1 interaction contributes to the block to IFN signaling. These data suggest that VP24 is likely to be an important virulence determinant that allows EBOV to evade the antiviral effects of IFNs.     

Identification of paramyxovirus V protein residues essential for STAT protein degradation and promotion of virus replication

Some paramyxovirus V proteins induce STAT protein degradation, and the amino acids essential for this process in the human parainfluenza virus type 2 (hPIV2) V protein have been studied. Various recombinant hPIV2s and cell lines constitutively expressing various mutant V proteins were generated. We found that V proteins with replacement of Cys residues of the Cys cluster were still able to bind STATs but were unable to induce their degradation. The hPIV2 V protein binds STATs via a W-(X)3-W-(X)9-W Trp motif located just upstream of the Cys cluster. Replacements of two or more Trp residues in this motif resulted in a failure to form a V/STAT2 complex. We have also identified two Phe residues of the hPIV2 V protein that are essential for STAT degradation, namely, Phe207, lying within the Cys cluster, and Phe143, in the P/V common region of the protein. Interestingly, infection of BHK cells with hPIV2 led to the specific degradation of STAT1 and not STAT2. Other evidence for the cell species specificity of hPIV2-induced STAT degradation is presented. Finally, a V-minus hPIV2, which can express only the P protein from its P gene, was generated and partially characterized. In contrast to V-minus viruses of other paramyxovirus genera, this V-minus rubulavirus was highly debilitated, and its growth even in Vero cells was very limited. The structural rubulavirus V proteins, as expected, are thus clearly important in promoting virus growth, independent of their anti-interferon (IFN) activity. Interestingly, many of the residues that are essential for anti-IFN activity, e.g., the Cys of this cluster and Phe207 within this cluster, as well as the Trp of this motif, are also essential for promoting virus growth.     

Sendai virus and simian virus 5 block activation of interferon-responsive genes: importance for virus pathogenesis

Sendai virus (SeV) is highly pathogenic for mice. In contrast, mice (including SCID mice) infected with simian virus 5 (SV5) showed no overt signs of disease. Evidence is presented that a major factor which prevented SV5 from productively infecting mice was its inability to circumvent the interferon (IFN) response in mice. Thus, in murine cells that produce and respond to IFN, SV5 protein synthesis was rapidly switched off. In marked contrast, once SeV protein synthesis began, it continued, even if the culture medium was supplemented with alpha/beta IFN (IFN-alpha/beta). However, in human cells, IFN-alpha/beta did not inhibit the replication of either SV5 or SeV once virus protein synthesis was established. To begin to address the molecular basis for these observations, the effects of SeV and SV5 infections on the activation of an IFN-alpha/beta-responsive promoter and on that of the IFN-beta promoter were examined in transient transfection experiments. The results demonstrated that (i) SeV, but not SV5, inhibited an IFN-alpha/beta-responsive promoter in murine cells; (ii) both SV5 and SeV inhibited the activation of an IFN-alpha/beta-responsive promoter in human cells; and (iii) in both human and murine cells, SeV was a strong inducer of the IFN-beta promoter, whereas SV5 was a poor inducer. The ability of SeV and SV5 to inhibit the activation of IFN-responsive genes in human cells was confirmed by RNase protection experiments. The importance of these results in terms of paramyxovirus pathogenesis is discussed.     

Nipah virus V and W proteins have a common STAT1-binding domain yet inhibit STAT1 activation from the cytoplasmic and nuclear compartments, respectively

In previous reports it was demonstrated that the Nipah virus V and W proteins have interferon (IFN) antagonist activity due to their ability to block signaling from the IFN-alpha/beta receptor (J. J. Rodriguez, J. P. Parisien, and C. M. Horvath, J. Virol. 76:11476-11483, 2002; M. S. Park et al., J. Virol. 77:1501-1511, 2003). The V, W, and P proteins are all encoded by the same viral gene and share an identical 407-amino-acid N-terminal region but have distinct C-terminal sequences. We now show that the P protein also has anti-IFN function, confirming that the common N-terminal domain is responsible for the antagonist activity. Truncation of this N-terminal domain revealed that amino acids 50 to 150 retain the ability to block IFN and to bind STAT1, a key component of the IFN signaling pathway. Subcellular localization studies demonstrate that the V and P proteins are predominantly cytoplasmic whereas the W protein is localized to the nucleus. In all cases, STAT1 colocalizes with the corresponding Nipah virus protein. These interactions are sufficient to inhibit STAT1 activation, as demonstrated by the lack of STAT1 phosphorylation on tyrosine 701 in IFN-stimulated cells expressing P, V, or W. Therefore, despite their common STAT1-binding domain, the Nipah virus V and P proteins act by retaining STAT1 in the cytoplasm while the W protein sequesters STAT1 in the nucleus, creating both a cytoplasmic and a nuclear block for STAT1. We also show that the IFN antagonist activity of the P protein is not as strong as that of V or W, perhaps explaining why Nipah virus has evolved to express these two edited products.     

Hepatitis B virus X protein and simian virus 5 V protein exhibit similar UV-DDB1 binding properties to mediate distinct activities

The UV-damaged DNA-binding activity protein (UV-DDB) consists of two subunits, DDB1 and DDB2, and functions in DNA repair and cell cycle regulation. The DDB1 subunit is a target for the hepatitis B virus X protein (HBx). Binding of HBx to DDB1 interferes with cell growth and viability in culture and has been implicated in the establishment of viral infection. DDB1 also interacts with the V proteins encoded by several paramyxoviruses including simian virus 5 (SV5), which prevent interferon signaling by targeting either STAT1 or STAT2 proteins for proteolysis. The role of V binding to DDB1, however, remains unclear. Here we show that the V protein of SV5 (SV5-V) and HBx exhibit strikingly similar DDB1 binding properties. Thus, SV5-V and HBx bind to DDB1 in a mutually exclusive manner, and SV5-V shares with HBx the ability to enhance the steady-state levels of DDB1 and to inhibit its association with DDB2. Yet only HBx induces cell death, and SV5-V can prevent HBx from doing so by blocking its interaction with DDB1. Binding of SV5-V to DDB1 may serve another function, since SV5-V shows a decreased ability to induce STAT1 degradation in cells expressing reduced amounts of DDB1. These findings demonstrate that HBx performs a unique function through its association with DDB1 for which SV5-V cannot substitute and suggest that SV5-V and HBx have evolved to bind DDB1 to achieve distinct functions, both by a mechanism that does not involve DDB2.     

The nucleocytoplasmic rabies virus P protein counteracts interferon signaling by inhibiting both nuclear accumulation and DNA binding of STAT1

Rabies virus P protein inhibits alpha interferon (IFN-alpha)- and IFN-gamma-stimulated Jak-STAT signaling by retaining phosphorylated STAT1 in the cytoplasm. Here, we show that P also blocks an intranuclear step that is the STAT1 binding to the DNA promoter of IFN-responsive genes. As P is a nucleocytoplasmic shuttling protein, we first investigated the effect of the cellular distribution of P on the localization of STAT1 and consequently on IFN signaling. We show that the localization of STAT1 is correlated with the localization of P: in cells expressing a nuclear form of P (the short P3 isoform or the complete P in the presence of the export inhibitor leptomycin B), STAT1 is nuclear, whereas in cells expressing a cytoplasmic form of P, STAT1 is cytoplasmic. However, the expression of nuclear forms of P inhibits the signaling of both IFN-gamma and IFN-alpha, demonstrating that the retention of STAT1 in the cytoplasm is not the only mechanism involved in the inhibition of IFN signaling. Electrophoretic mobility shift analysis indicates that P expression in the cell extracts of infected cells or in stable cell lines prevents IFN-induced DNA binding of STAT1. The loss of the DNA binding of STAT1 and ISGF3 was also observed when purified recombinant P or P3 was added to the extracts of IFN-gamma- or IFN-alpha-treated cells, indicating that P directly affects the DNA binding activity of STAT1. Then products of the rabies virus P gene are able to counteract IFN signaling by creating both cytoplasmic and nuclear blocks for STAT1.