Hendra virus V protein inhibits interferon signaling by preventing STAT1 and STAT2 nuclear accumulation

The V protein of the recently emerged paramyxovirus, Nipah virus, has been shown to inhibit interferon (IFN) signal transduction through cytoplasmic sequestration of cellular STAT1 and STAT2 in high-molecular-weight complexes. Here we demonstrate that the closely related Hendra virus V protein also inhibits cellular responses to IFN through binding and cytoplasmic sequestration of both STAT1 and STAT2, but not STAT3. These findings demonstrate a V protein-mediated IFN signal evasion mechanism that is a general property of the known Henipavirus species.     

A novel nuclear trafficking module regulates the nucleocytoplasmic localization of the rabies virus interferon antagonist, p protein

Regulated nucleocytoplasmic transport of proteins is central to cellular function and dysfunction during processes such as viral infection. Active protein trafficking into and out of the nucleus is dependent on the presence within cargo proteins of intrinsic specific modular signals for nuclear import (nuclear localization signals, NLSs) and export (nuclear export signals, NESs). Rabies virus (RabV) phospho (P) protein, which is largely responsible for antagonising the host anti-viral response, is expressed as five isoforms (P1-P5). The subcellular trafficking of these isoforms is thought to depend on a balance between the activities of a dominant N-terminal NES (N-NES) and a distinct C-terminal NLS (C-NLS). Specifically, the N-NES-containing isoforms P1 and P2 are cytoplasmic, whereas the shorter P3-P5 isoforms, which lack the N-NES, are believed to be nuclear through the activity of the C-NLS. Here, we show for the first time that RabV P contains an additional strong NLS in the N-terminal region (N-NLS), which, intriguingly, overlaps with the N-NES. This arrangement represents a novel nuclear trafficking module where the N-NLS is inactive in P1 but becomes activated in P3, concomitant with truncation of the N-NES, to become the principal targeting signal conferring nuclear accumulation. Understanding this unique switch arrangement of overlapping, co-regulated NES/NLS sequences is vital to delineating the critical role of RabV P protein in viral infection.     

Deactivation of the antiviral state by rabies virus through targeting and accumulation of persistently phosphorylated STAT1

Antagonism of the interferon (IFN)-mediated antiviral state is critical to infection by rabies virus (RABV) and other viruses, and involves interference in the IFN induction and signaling pathways in infected cells, as well as deactivation of the antiviral state in cells previously activated by IFN. The latter is required for viral spread in the host, but the precise mechanisms involved and roles in RABV pathogenesis are poorly defined. Here, we examined the capacity of attenuated and pathogenic strains of RABV that differ only in the IFN-antagonist P protein to overcome an established antiviral state. Importantly, P protein selectively targets IFN-activated phosphorylated STAT1 (pY-STAT1), providing a molecular tool to elucidate specific roles of pY-STAT1. We find that the extended antiviral state is dependent on a low level of pY-STAT1 that appears to persist at a steady state through ongoing phosphorylation/dephosphorylation cycles, following an initial IFN-induced peak. P protein of pathogenic RABV binds and progressively accumulates pY-STAT1 in inactive cytoplasmic complexes, enabling recovery of efficient viral replication over time. Thus, P protein-pY-STAT1 interaction contributes to ‘disarming’ of the antiviral state. P protein of the attenuated RABV is defective in this respect, such that replication remains suppressed over extended periods in cells pre-activated by IFN. These data provide new insights into the nature of the antiviral state, indicating key roles for residual pY-STAT1 signaling. They also elucidate mechanisms of viral deactivation of antiviral responses, including specialized functions of P protein in selective targeting and accumulation of pY-STAT1.     

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.     

Mumps virus V protein antagonizes interferon without the complete degradation of STAT1

Mumps virus (MuV) has been shown to antagonize the antiviral effects of interferon (IFN) through proteasome-mediated complete degradation of STAT1 by using the viral V protein (T. Kubota et al., Biochem. Biophys. Res. Commun. 283:255-259, 2001). However, we found that MuV could inhibit IFN signaling and the generation of a subsequent antiviral state long before the complete degradation of cellular STAT1 in infected cells. In MuV-infected cells, nuclear translocation and phosphorylation of STAT1 and STAT2 tyrosine residue (Y) at 701 and 689, respectively, by IFN-beta were significantly inhibited but the phosphorylation of Jak1 and Tyk2 was not inhibited. The transiently expressed MuV V protein also inhibited IFN-beta-induced Y701-STAT1 and Y689-STAT2 phosphorylation, suggesting that the V protein could block IFN-beta-induced signal transduction without the aid of other viral components. Finally, a substitution of an alanine residue in place of a cysteine residue in the C-terminal V-unique region known to be required for STAT1 degradation and inhibition of anti-IFN signaling resulted in the loss of V protein function to inhibit the Y701-STAT1 and Y689-STAT2 phosphorylation.     

Mechanism of inhibiting type I interferon induction by hepatitis B virus X protein

Hepatitis B virus (HBV) is regarded as a stealth virus, invading and replicating efficiently in human liver undetected by host innate antiviral immunity. Here, we show that type I interferon (IFN) induction but not its downstream signaling is blocked by HBV replication in HepG2.2.15 cells. This effect may be partially due to HBV X protein (HBx), which impairs IFNβ promoter activation by both Sendai virus (SeV) and components implicated in signaling by viral sensors. As a deubiquitinating enzyme (DUB), HBx cleaves Lys63-linked polyubiquitin chains from many proteins except TANK-binding kinase 1 (TBK1). It binds and deconjugates retinoic acid-inducible gene I (RIG I) and TNF receptor-associated factor 3 (TRAF3), causing their dissociation from the downstream adaptor CARDIF or TBK1 kinase. In addition to RIG I and TRAF3, HBx also interacts with CARDIF, TRIF, NEMO, TBK1, inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase epsilon (IKKi) and interferon regulatory factor 3 (IRF3). Our data indicate that multiple points of signaling pathways can be targeted by HBx to negatively regulate production of type I IFN.     

DDB2 induces nuclear accumulation of the hepatitis B virus X protein independently of binding to DDB1

The hepatitis B virus (HBV) X protein (HBx) is critical for the life cycle of the virus. HBx associates with several host cell proteins including the DDB1 subunit of the damaged-DNA binding protein DDB. Recent studies on the X protein encoded by the woodchuck hepadnavirus have provided correlative evidence indicating that the interaction with DDB1 is important for establishment of infection by the virus. In addition, the interaction with DDB1 has been implicated in the nuclear localization of HBx. Because the DDB2 subunit of DDB is required for the nuclear accumulation of DDB1, we investigated the role of DDB2 in the nuclear accumulation of HBx. Here we show that expression of DDB2 increases the nuclear levels of HBx. Several C-terminal deletion mutants of DDB2 that fail to bind DDB1 are able to associate with HBx, suggesting that DDB2 may associate with HBx independently of binding to DDB1. We also show that DDB2 enhances the nuclear accumulation of HBx independently of binding to DDB1, since a mutant that does not bind DDB1 is able to enhance the nuclear accumulation of HBx. HBV infection is associated with liver pathogenesis. We show that the nuclear levels of DDB1 and DDB2 are tightly regulated in hepatocytes. Studies with regenerating mouse liver indicate that during late G1 phase the nuclear levels of both subunits of DDB are transiently increased, followed by a sharp decrease in S phase. Taken together, these results suggest that DDB1 and DDB2 would participate in the nuclear functions of HBx effectively only during the late-G1 phase of the cell cycle.     

The salt dependence of the interferon regulatory factor 1 DNA binding domain binding to DNA reveals ions are localized around protein and DNA

The equilibrium dissociation constant of the DNA binding domain of interferon regulatory factor 1 (IRF1 DBD) for its DNA binding site depends strongly on salt concentration and salt type. These dependencies are consistent with IRF1 DBD binding to DNA, resulting in the release of cations from the DNA and both release of anions from the protein and uptake of a cation by the protein. We demonstrated this by utilizing the fact that the release of fluoride from protein upon complex formation does not contribute to the salt concentration dependence of binding and by studying mutants in which charged residues in IRF1 DBD that form salt bridges with DNA phosphates are changed to alanine. The salt concentration dependencies of the dissociation constants of wild-type IRF1 DBD and the mutants R64A, D73A, K75A, and D73A/K75A were measured in buffer containing NaF, NaCl, or NaBr. The salt concentration and type dependencies of the mutants relative to wild-type IRF1 DBD provide evidence of charge neutralization by solution ions for R64 and by a salt bridge between D73 and K75 in buffer containing chloride or bromide salts. These data also allowed us to determine the number, type, and localization of condensed ions around both IRF1 DBD and its DNA binding site.