The Human STAT2 Coiled-Coil Domain Contains a Degron for Zika Virus Interferon Evasion.

The ability of viruses to evade the host antiviral immune system determines their level of replication fitness, species specificity, and pathogenic potential. Flaviviruses rely on the subversion of innate immune barriers, including the type I and type III interferon (IFN) antiviral systems. Zika virus infection induces the degradation of STAT2, an essential component of the IFN-stimulated gene transcription factor ISGF3. The mechanisms that lead to STAT2 degradation by Zika virus are poorly understood, but it is known to be mediated by the viral NS5 protein that binds to STAT2 and targets it for proteasome-mediated destruction. To better understand how NS5 engages and degrades STAT2, functional analysis of the protein interactions that lead to Zika virus and NS5-dependent STAT2 proteolysis were investigated. Data implicate the STAT2 coiled-coil domain as necessary and sufficient for NS5 interaction and proteasome degradation after Zika virus infection. Molecular dissection reveals that the first two α-helices of the STAT2 coiled-coil domain contain a specific targeting region for IFN antagonism. These functional interactions provide a more complete understanding of the essential protein-protein interactions needed for Zika virus evasion of the host antiviral response and identify new targets for antiviral therapeutic approaches. IMPORTANCE Zika virus infection can cause mild fever, rash, and muscle pain and in rare cases can lead to brain or nervous system diseases, including Guillain-Barré syndrome. Infections in pregnant women can increase the risk of miscarriage or serious birth defects, including brain anomalies and microcephaly. There are no drugs or vaccines for Zika disease. Zika virus is known to break down the host antiviral immune response, and this research project reveals how the virus suppresses interferon signaling, and may reveal therapeutic vulnerabilities.

RNA sensor LGP2 inhibits TRAF ubiquitin ligase to negatively regulate innate immune signaling.

The production of type I interferon (IFN) is essential for cellular barrier functions and innate and adaptive antiviral immunity. In response to virus infections, RNA receptors RIG-I and MDA5 stimulate a mitochondria-localized signaling apparatus that uses TRAF family ubiquitin ligase proteins to activate master transcription regulators IRF3 and NFκB, driving IFN and antiviral target gene expression. Data indicate that a third RNA receptor, LGP2, acts as a negative regulator of antiviral signaling by interfering with TRAF family proteins. Disruption of LGP2 expression in cells results in earlier and overactive transcriptional responses to virus or dsRNA LGP2 associates with the C-terminus of TRAF2, TRAF3, TRAF5, and TRAF6 and interferes with TRAF ubiquitin ligase activity. TRAF interference is independent of LGP2 ATP hydrolysis, RNA binding, or its C-terminal domain, and LGP2 can regulate TRAF-mediated signaling pathways in trans, including IL-1ß, TNFα, and cGAMP These findings provide a unique mechanism for LGP2 negative regulation through TRAF suppression and extend the potential impact of LGP2 negative regulation beyond the IFN antiviral response.

Immune regulator LGP2 targets Ubc13/UBE2N to mediate widespread interference with K63 polyubiquitination and NF-κB activation.

Lysine 63-linked polyubiquitin (K63-Ub) chains activate a range of cellular immune and inflammatory signaling pathways, including the mammalian antiviral response. Interferon and antiviral genes are triggered by TRAF family ubiquitin ligases that form K63-Ub chains. LGP2 is a feedback inhibitor of TRAF-mediated K63-Ub that can interfere with diverse immune signaling pathways. Our results demonstrate that LGP2 inhibits K63-Ub by association with and sequestration of the K63-Ub-conjugating enzyme, Ubc13/UBE2N. The LGP2 helicase subdomain, Hel2i, mediates protein interaction that engages and inhibits Ubc13/UBE2N, affecting control over a range of K63-Ub ligase proteins, including TRAF6, TRIM25, and RNF125, all of which are inactivated by LGP2. These findings establish a unifying mechanism for LGP2-mediated negative regulation that can modulate a variety of K63-Ub signaling pathways.

A shared interface mediates paramyxovirus interference with antiviral RNA helicases MDA5 and LGP2.

Diverse members of the Paramyxovirus family of negative-strand RNA viruses effectively suppress host innate immune responses through the actions of their V proteins. The V protein mediates interference with the interferon regulatory RNA helicase MDA5 to avoid cellular antiviral responses. Analysis of the interaction interface revealed the MDA5 helicase C domain as necessary and sufficient for association with V proteins from human parainfluenza virus type 2, parainfluenza virus type 5, measles virus, mumps virus, Hendra virus, and Nipah virus. The identified approximately 130-residue region is highly homologous between MDA5 and the related antiviral helicase LGP2, but not RIG-I. Results indicate that the paramyxovirus V proteins can also associate with LGP2. The V protein interaction was found to disrupt ATP hydrolysis mediated by both MDA5 and LGP2. These findings provide a potential mechanistic basis for V protein-mediated helicase interference and identify LGP2 as a second cellular RNA helicase targeted by paramyxovirus V proteins.

STAT2 is a primary target for measles virus V protein-mediated alpha/beta interferon signaling inhibition.

Measles virus, a member of the Morbillivirus family, infects millions of people each year despite the availability of effective vaccines. The V protein of measles virus is an important virulence factor that can interfere with host innate immunity by inactivating alpha/beta interferon (IFN-alpha/beta) and IFN-gamma signaling through protein interactions with signal transducer and activator of transcription proteins STAT1 and STAT2. Here we demonstrate that although STAT1 interference results from protein interactions within a V protein N-terminal region encompassed by amino acids 110 to 130, detection of STAT1 interaction and IFN-gamma signaling inhibition requires the presence of cellular STAT2. Cell-specific variability in STAT1 interference was observed to correlate with V protein expression level. A more direct target for measles virus V protein-mediated IFN-alpha/beta evasion is STAT2. Results indicate that the widely conserved C-terminal zinc finger domain of measles virus V protein is both necessary and sufficient to bind STAT2 and disrupt IFN-alpha/beta signal transduction. Mutagenesis and molecular modeling define a contact surface for STAT2 association that includes aspartic acid residue 248 as critical for STAT2 interference and IFN antiviral immune suppression. These findings clearly define the molecular determinants for measles virus IFN evasion and validate specific targets as candidates for therapeutic intervention.

Detection of activated STAT proteins.

STAT proteins are activated by diverse cellular stimuli including cytokine and growth factor receptor signaling, proto-oncogene and oncogene expression, and cellular stress mediators. In most cases, canonical STAT activation by a particular treatment or cellular condition results in STAT protein phosphorylation on an activating tyrosine residue near the C terminus. This phosphotyrosine is recognized by SH2 domains in partner STATs, resulting in homo- or hetero-dimerization. The STAT dimers attain the ability to bind specific DNA response element sequences present in the promoters of target genes. Two methods are described for the detection of activated STAT proteins based on (1) acquisition of tyrosine phosphorylation and (2) acquisition of DNA binding ability.

Measles virus V protein inhibits p53 family member p73.

Paramyxovirus V proteins function as host interference factors that inactivate antiviral responses, including interferon. Characterization of cellular proteins that copurify with ectopically expressed measles virus V protein has revealed interactions with DNA binding domains of p53 family proteins, p53 and p73. Specific transcriptional assays reveal that expression of measles virus V cDNA inhibits p73, but not p53. Expression of measles virus V cDNA can delay cell death induced by genotoxic stress and also can decrease the abundance of the proapoptotic factor PUMA, a p73 target. Recombinant measles virus with an engineered deficiency in V protein is capable of inducing more severe cytopathic effects than the wild type, implicating measles virus V protein as an inhibitor of cell death. These findings also suggest that p73-PUMA signaling may be a previously unrecognized arm of cellular innate antiviral immunity.

Composition and assembly of STAT-targeting ubiquitin ligase complexes: paramyxovirus V protein carboxyl terminus is an oligomerization domain.

Transcription regulators STAT1 and STAT2 are key components of the interferon signaling system leading to innate antiviral immunity. The related STAT3 protein is a regulator of interleukin-6-type cytokine signals and can contribute to both cell growth and death important for cancer gene regulation and tumor survival. These three STAT proteins are targeted for proteasome-mediated degradation by RNA viruses in the Rubulavirus genus of the Paramyxoviridae. A single viral protein, the V protein, assembles STAT-specific ubiquitin ligase complexes from cellular components. Simian virus 5 (SV5) targets STAT1, human parainfluenza virus 2 targets STAT2, and mumps virus targets both STAT1 and STAT3. Analysis of the V-dependent degradation complex (VDC) composition and assembly revealed several features contributing to targeting specificity. SV5 and mumps V proteins require STAT2 to recruit the STAT1 target, yet mumps V protein binds STAT3 independent of STAT1 and STAT2. All Rubulavirus V proteins tested require cellular DDB1 to target STATs for degradation but differ in the use of Roc1, which is essential for mumps V STAT3 targeting. Protein interaction analysis reveals that paramyxovirus V proteins can homo- and heterooligomerize and that the conserved cysteine-rich zinc-binding C-terminal domain is necessary and sufficient for oligomerization. Purified SV5 V protein spontaneously assembles into spherical macromolecular particles, and similar particles constitute SV5 and mumps VDC preparations.

Nipah virus V protein evades alpha and gamma interferons by preventing STAT1 and STAT2 activation and nuclear accumulation.

Characterization of recent outbreaks of fatal encephalitis in southeast Asia identified the causative agent to be a previously unrecognized enveloped negative-strand RNA virus of the Paramyxoviridae family, Nipah virus. One feature linking Nipah virus to this family is a conserved cysteine-rich domain that is the hallmark of paramyxovirus V proteins. The V proteins of other paramyxovirus species have been linked with evasion of host cell interferon (IFN) signal transduction and subsequent antiviral responses by inducing proteasomal degradation of the IFN-responsive transcription factors, STAT1 or STAT2. Here we demonstrate that Nipah virus V protein escapes IFN by a distinct mechanism involving direct inhibition of STAT protein function. Nipah virus V protein differs from other paramyxovirus V proteins in its subcellular distribution but not in its ability to inhibit cellular IFN responses. Nipah virus V protein does not induce STAT degradation but instead inhibits IFN responses by forming high-molecular-weight complexes with both STAT1 and STAT2. We demonstrate that Nipah virus V protein accumulates in the cytoplasm by a Crm1-dependent mechanism, alters the STAT protein subcellular distribution in the steady state, and prevents IFN-stimulated STAT redistribution. Consistent with the formation of complexes, STAT protein tyrosine phosphorylation is inhibited in cells expressing the Nipah virus V protein. As a result, Nipah virus V protein efficiently prevents STAT1 and STAT2 nuclear translocation in response to IFN, inhibiting cellular responses to both IFN-alpha and IFN-gamma.

STAT2 acts as a host range determinant for species-specific paramyxovirus interferon antagonism and simian virus 5 replication.

The antiviral state induced by alpha/beta interferon (IFN-alpha/beta) is a powerful selective pressure for virus evolution of evasive strategies. The paramyxoviruses simian virus 5 (SV5) and human parainfluenza virus 2 (HPIV2) overcome IFN-alpha/beta responses through the actions of their V proteins, which induce proteasomal degradation of cellular IFN-alpha/beta-activated signal transducers and activators of transcription STAT1 and STAT2. SV5 infection induces STAT1 degradation and IFN-alpha/beta inhibition efficiently in human cells but not in mouse cells, effectively restricting SV5 host range. Here, the cellular basis for this species specificity is demonstrated to result from differences between human and murine STAT2. Expression in mouse cells of full-length or truncated human STAT2 cDNA is sufficient to permit antagonism of endogenous murine IFN-alpha/beta signaling by SV5 and HPIV2 V proteins. Furthermore, virus-induced STAT protein degradation is observed in mouse cells only in the presence of ectopically expressed human STAT2. The results indicate that STAT2 acts as an intracellular determinant of paramyxovirus host range restriction, which contributes to the species specificity of virus replication, and that human STAT2 can confer a growth advantage for SV5 in the murine host.

A hybrid IRF9-STAT2 protein recapitulates interferon-stimulated gene expression and antiviral response.

Type I interferon (IFN) signaling induces the heterotrimeric transcription complex, IFN-stimulated gene factor (ISGF) 3, which contains STAT1, STAT2, and the DNA binding subunit, interferon regulatory factor (IRF) 9. Because IRF9 is targeted to the nucleus in the absence of IFN stimulation, the potential of IRF9 protein for gene regulation was examined using a GAL4 DNA binding domain fusion system. GAL4-IRF9 was transcriptionally active in reporter gene assays but not in the absence of cellular STAT1 and STAT2. However, the inert IRF9 protein was readily converted to a constitutively active ISGF3-like activator by fusion with the C-terminal transcriptional activation domain of STAT2 or the acidic activation domain of herpesvirus VP16. The IRF9 hybrids are targeted to endogenous ISGF3 target loci and can activate their transcription. Moreover, expression of the IRF9-STAT2 fusion can recapitulate the type I IFN biological response, producing a cellular antiviral state that protects cells from virus-induced cytopathic effects and inhibits virus replication. The antiviral state generated by regulated IRF9-STAT2 hybrid protein expression is independent of autocrine IFN signaling and inhibits both RNA and DNA viruses.

STAT3 ubiquitylation and degradation by mumps virus suppress cytokine and oncogene signaling.

Mumps virus is a common infectious agent of humans, causing parotitis, meningitis, encephalitis, and orchitis. Like other paramyxoviruses in the genus Rubulavirus, mumps virus catalyzes the proteasomal degradation of cellular STAT1 protein, a means for escaping antiviral responses initiated by alpha/beta and gamma interferons. We demonstrate that mumps virus also eliminates cellular STAT3, a protein that mediates transcriptional responses to cytokines, growth factors, nonreceptor tyrosine kinases, and a variety of oncogenic stimuli. STAT1 and STAT3 are independently targeted by a single mumps virus protein, called V, that assembles STAT-directed ubiquitylation complexes from cellular components, including STAT1, STAT2, STAT3, DDB1, and Cullin4A. Consequently, mumps virus V protein prevents responses to interleukin-6 and v-Src signals and can induce apoptosis in STAT3-dependent multiple myeloma cells and transformed murine fibroblasts. These findings demonstrate a unique cytokine and oncogene evasion property of mumps virus that provides a molecular basis for its observed oncolytic properties.

STAT protein interference and suppression of cytokine signal transduction by measles virus V protein.

Measles virus, a paramyxovirus of the Morbillivirus genus, is responsible for an acute childhood illness that infects over 40 million people and leads to the deaths of more than 1 million people annually (C. J. Murray and A. D. Lopez, Lancet 349:1269-1276, 1997). Measles virus infection is characterized by virus-induced immune suppression that creates susceptibility to opportunistic infections. Here we demonstrate that measles virus can inhibit cytokine responses by direct interference with host STAT protein-dependent signaling systems. Expression of the measles V protein prevents alpha, beta, and gamma interferon-induced transcriptional responses. Furthermore, it can interfere with signaling by interleukin-6 and the non-receptor tyrosine kinase, v-Src. Affinity purification demonstrates that the measles V protein associates with cellular STAT1, STAT2, STAT3, and IRF9, as well as several unidentified partners. Mechanistic studies indicate that while the measles V protein does not interfere with STAT1 or STAT2 tyrosine phosphorylation, it causes a defect in IFN-induced STAT nuclear accumulation. The defective STAT nuclear redistribution is also observed in measles virus-infected cells, where some of the STAT protein is detected in cytoplasmic bodies that contain viral nucleocapsid protein and nucleic acids. Interference with STAT-inducible transcription may provide a novel intracellular mechanism for measles virus-induced cytokine inhibition that links innate immune evasion to adaptive immune suppression.

Selective STAT protein degradation induced by paramyxoviruses requires both STAT1 and STAT2 but is independent of alpha/beta interferon signal transduction.

The alpha/beta interferon (IFN-alpha/beta)-induced STAT signal transduction pathway leading to activation of the ISGF3 transcription complex and subsequent antiviral responses is the target of viral pathogenesis strategies. Members of the Rubulavirus genus of the Paramyxovirus family of RNA viruses have acquired the ability to specifically target either STAT1 or STAT2 for proteolytic degradation as a countermeasure for evading IFN responses. While type II human parainfluenza virus induces STAT2 degradation, simian virus 5 induces STAT1 degradation. The components of the IFN signaling system that are required for STAT protein degradation by these paramyxoviruses have been investigated in a series of human somatic cell lines deficient in IFN signaling proteins. Results indicate that neither the IFN-alpha/beta receptor, the tyrosine kinases Jak1 or Tyk2, nor the ISGF3 DNA-binding subunit, IFN regulatory factor 9 (IRF9), is required for STAT protein degradation induced by either virus. Nonetheless, both STAT1 and STAT2 are strictly required in the host cell to establish a degradation-permissive environment enabling both viruses to target their respective STAT protein. Complementation studies reveal that STAT protein-activating tyrosine phosphorylation and functional src homology 2 (SH2) domains are dispensable for creating a permissive STAT degradation environment in degradation-incompetent cells, but the N terminus of the missing STAT protein is essential. Protein-protein interaction analysis indicates that V and STAT proteins interact physically in vitro and in vivo. These results constitute genetic and biochemical evidence supporting a virus-induced, IFN-independent STAT protein degradation complex that contains at least STAT1 and STAT2.

Effects of influenza A virus NS1 protein on protein expression: the NS1 protein enhances translation and is not required for shutoff of host protein synthesis.

The influenza A virus NS1 protein, a virus-encoded alpha/beta interferon (IFN-alpha/beta) antagonist, appears to be a key regulator of protein expression in infected cells. We now show that NS1 protein expression results in enhancement of reporter gene activity from transfected plasmids. This effect appears to be mediated at the translational level, and it is reminiscent of the activity of the adenoviral virus-associated I (VAI) RNA, a known inhibitor of the antiviral, IFN-induced, PKR protein. To study the effects of the NS1 protein on viral and cellular protein synthesis during influenza A virus infection, we used recombinant influenza viruses lacking the NS1 gene (delNS1) or expressing truncated NS1 proteins. Our results demonstrate that the NS1 protein is required for efficient viral protein synthesis in COS-7 cells. This activity maps to the amino-terminal domain of the NS1 protein, since cells infected with wild-type virus or with a mutant virus expressing a truncated NS1 protein-lacking approximately half of its carboxy-terminal end-showed similar kinetics of viral and cellular protein expression. Interestingly, no major differences in host cell protein synthesis shutoff or in viral protein expression were found among NS1 mutant viruses in Vero cells. Thus, another viral component(s) different from the NS1 protein is responsible for the inhibition of host protein synthesis during viral infection. In contrast to the earlier proposal suggesting that the NS1 protein regulates the levels of spliced M2 mRNA, no effects on M2 protein accumulation were seen in Vero cells infected with delNS1 virus.