Arterivirus and nairovirus ovarian tumor domain-containing Deubiquitinases target activated RIG-I to control innate immune signaling

The innate immune response constitutes the first line of defense against viral infection and is extensively regulated through ubiquitination. The removal of ubiquitin from innate immunity signaling factors by deubiquitinating enzymes (DUBs) therefore provides a potential opportunity for viruses to evade this host defense system. It was previously found that specific proteases encoded by the unrelated arteri- and nairoviruses resemble the ovarian tumor domain-containing (OTU) family of DUBs. In arteriviruses, this domain has been characterized before as a papain-like protease (PLP2) that is also involved in replicase polyprotein processing. In nairoviruses, the DUB resides in the polymerase protein but is not essential for RNA replication. Using both in vitro and cell-based assays, we now show that PLP2 DUB activity is conserved in all members of the arterivirus family and that both arteri- and nairovirus DUBs inhibit RIG-I-mediated innate immune signaling when overexpressed. The potential relevance of RIG-I-like receptor (RLR) signaling for the innate immune response against arterivirus infection is supported by our finding that in mouse embryonic fibroblasts, the production of beta interferon primarily depends on the recognition of arterivirus RNA by the pattern-recognition receptor MDA5. Interestingly, we also found that both arteri- and nairovirus DUBs inhibit RIG-I ubiquitination upon overexpression, suggesting that both MDA5 and RIG-I have a role in countering infection by arteriviruses. Taken together, our results support the hypothesis that arteri- and nairoviruses employ their deubiquitinating potential to inactivate cellular proteins involved in RLR-mediated innate immune signaling, as exemplified by the deubiquitination of RIG-I.     

RIG-I-mediated antiviral signaling is inhibited in HIV-1 infection by a protease-mediated sequestration of RIG-I

The rapid induction of type I interferon (IFN) is essential for establishing innate antiviral responses. During infection, cytoplasmic viral RNA is sensed by two DExD/H box RNA helicases, RIG-I and MDA5, ultimately driving IFN production. Here, we demonstrate that purified genomic RNA from HIV-1 induces a RIG-I-dependent type I IFN response. Both the dimeric and monomeric forms of HIV-1 were sensed by RIG-I, but not MDA5, with monomeric RNA, usually found in defective HIV-1 particles, acting as a better inducer of IFN than dimeric RNA. However, despite the presence of HIV-1 RNA in the de novo infection of monocyte-derived macrophages, HIV-1 replication did not lead to a substantial induction of IFN signaling. We demonstrate the existence of an evasion mechanism based on the inhibition of the RIG-I sensor through the action of the HIV-1 protease (PR). Indeed, the ectopic expression of PR resulted in the inhibition of IFN regulatory factor 3 (IRF-3) phosphorylation and decreased expression of IFN and interferon-stimulated genes. A downregulation of cytoplasmic RIG-I levels occurred in cells undergoing a single-cycle infection with wild-type provirus BH10 but not in cells transfected with a protease-deficient provirus, BH10-PR(-). Cellular fractionation and confocal microscopy studies revealed that RIG-I translocated from the cytosol to an insoluble fraction during the de novo HIV-1 infection of monocyte-derived macrophages, in the presence of PR. The loss of cytoplasmic RIG-I was prevented by the lysosomal inhibitor E64, suggesting that PR targets RIG-I to the lysosomes. This study reveals a novel PR-dependent mechanism employed by HIV-1 to counteract the early IFN response to viral RNA in infected cells.     

Heterogeneity and Breadth of Host Antibody Response to KSHV Infection Demonstrated by Systematic Analysis of the KSHV Proteome

The Kaposi sarcoma associated herpesvirus (KSHV) genome encodes more than 85 open reading frames (ORFs). Serological evaluation of KSHV infection now generally relies on reactivity to just one latent and/or one lytic protein (commonly ORF73 and K8.1). Most of the other polypeptides encoded by the virus have unknown antigenic profiles. We have systematically expressed and purified products from 72 KSHV ORFs in recombinant systems and analyzed seroreactivity in US patients with KSHV-associated malignancies, and US blood donors (low KSHV seroprevalence population). We identified several KSHV proteins (ORF38, ORF61, ORF59 and K5) that elicited significant responses in individuals with KSHV-associated diseases. In these patients, patterns of reactivity were heterogeneous; however, HIV infection appeared to be associated with breadth and intensity of serological responses. Improved antigenic characterization of additional ORFs may increase the sensitivity of serologic assays, lead to more rapid progresses in understanding immune responses to KSHV, and allow for better comprehension of the natural history of KSHV infection. To this end, we have developed a bead-based multiplex assay detecting antibodies to six KSHV antigens.     

Establishment and maintenance of the innate antiviral response to West Nile Virus involves both RIG-I and MDA5 signaling through IPS-1

RIG-I and MDA5, two related pathogen recognition receptors (PRRs), are known to be required for sensing various RNA viruses. Here we investigated the roles that RIG-I and MDA5 play in eliciting the antiviral response to West Nile virus (WNV). Functional genomics analysis of WNV-infected fibroblasts from wild-type mice and RIG-I null mice revealed that the normal antiviral response to this virus occurs in two distinct waves. The initial response to WNV resulted in the expression of interferon (IFN) regulatory factor 3 target genes and IFN-stimulated genes, including several subtypes of alpha IFN. Subsequently, a second phase of IFN-dependent antiviral gene expression occurred very late in infection. In cells lacking RIG-I, both the initial and the secondary responses to WNV were delayed, indicating that RIG-I plays a critical role in initiating innate immunity against WNV. However, another PRR(s) was able to trigger a response to WNV in the absence of RIG-I. Disruption of both MDA5 and RIG-I pathways abrogated activation of the antiviral response to WNV, suggesting that MDA5 is involved in the host's defense against WNV infection. In addition, ablation of the function of IPS-1, an essential RIG-I and MDA5 adaptor molecule, completely disabled the innate antiviral response to WNV. Our data indicate that RIG-I and MDA5 are responsible for triggering downstream gene expression in response to WNV infection by signaling through IPS-1. We propose a model in which RIG-I and MDA5 operate cooperatively to establish an antiviral state and mediate an IFN amplification loop that supports immune effector gene expression during WNV infection.     

The ubiquitin ligase Riplet is essential for RIG-I-dependent innate immune responses to RNA virus infection

RNA virus infection is recognized by the RIG-I-like receptors RIG-I and MDA5, which induce antiviral responses including the production of type I interferons (IFNs) and proinflammatory cytokines. RIG-I is regulated by Lys63-linked polyubiquitination, and three E3 ubiquitin ligases, RNF125, TRIM25, and Riplet, are reported to target RIG-I for ubiquitination. To examine the importance of Riplet in?vivo, we generated Riplet-deficient mice. Fibroblasts, macrophages, and conventional dendritic cells from Riplet-deficient animals were defective for the production of IFN and other cytokines in response to infection with several RNA viruses. However, Riplet was dispensable for the production of IFN in response to B-DNA and DNA virus infection. Riplet deficiency abolished RIG-I activation during RNA virus infection, and the mutant mice were more susceptible to vesicular stomatitis virus infection than wild-type mice. These data indicate that Riplet is essential for regulating RIG-I-mediated innate immune response against RNA virus infection in?vivo.     

Virion proteins of Kaposi's sarcoma-associated herpesvirus

The proteins that compose a herpesvirus virion are thought to contain the functional information required for de novo infection, as well as virion assembly and egress. To investigate functional roles of Kaposi's sarcoma-associated herpesvirus (KSHV) virion proteins in viral productive replication and de novo infection, we attempted to identify virion proteins from purified KSHV by a proteomic approach. Extracellular KSHV virions were purified from phorbol-12-tetradecanoate-13-acetate-induced BCBL-1 cells through double-gradient ultracentrifugation, and their component proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Thirty prominent protein bands were excised and subjected to high-performance liquid chromatography ion trap mass spectrometric analysis. This study led to the identification of 24 virion-associated proteins. These include five capsid proteins, eight envelope glycoproteins, six tegument proteins, and five proteins whose locations in the virions have not yet been defined. Putative tegument proteins encoded by open reading frame 21 (ORF21), ORF33, and ORF45 were characterized and found to be resistant to protease digestion when purified virions were treated with trypsin, confirming that they are located within the virion particles. The ORF64-encoded large tegument protein was found to be associated with capsid but sensitive to protease treatment, suggesting its unique structure and array in KSHV virions. In addition, cellular beta-actin and class II myosin heavy chain type A were found inside KSHV virions and associated with tegument-capsid structure. Identification of KSHV virion proteins makes it possible to study the functional roles of these virion proteins in KSHV replication and pathogenicity.     

Adenovirus virus-associated RNAs induce type I interferon expression through a RIG-I-mediated pathway

The current study demonstrates that adenovirus virus-associated RNA (VA) is recognized by retinoic acid-inducible gene I (RIG-I), a cytosolic pattern recognition receptor, and activates RIG-I downstream signaling, leading to the induction of type I interferons (IFNs), similarly to Epstein-Barr virus-encoded small RNA. Further analysis revealed that adenovirus infection leads to biphasic type I IFN induction at 12 to 24 h and 48 to 60 h postinfection. The later induction coincided with VA expression and was reduced by virus UV inactivation or RIG-I silencing. These results suggest that VA-mediated RIG-I activation is involved in activating innate immune responses during adenovirus infection.     

Computational analysis predicts the Kaposi's sarcoma-associated herpesvirus tegument protein ORF63 to be alpha helical

The innate immune response provides our first line of defence against infection. Over the course of evolution, pathogens have evolved numerous strategies to either avoid activating or to limit the effectiveness of the innate immune system. The Kaposi's sarcoma-associated herpesvirus (KSHV) contains tegument proteins in the virion that contribute to immune evasion and aid the establishment of viral infection. For example, the KSHV tegument protein ORF63 modulates inflammasome activation to inhibit the innate immune response against the virus. Understanding the likely structure of proteins involved in immune evasion enables potential mechanisms of action to be proposed. To understand more fully how ORF63 modulates the innate immune system we have utilized widely available bioinformatics tools to analyze the primary protein sequence of ORF63 and to predict its secondary and tertiary structure. We found that ORF63 is predicted to be almost entirely alpha-helical and may possess similarity to HEAT repeat containing proteins. Consequently, ORF63 is unlikely to be a viral homolog of the NLR protein family. ORF63 may inhibit the innate immune response by flexibly interacting with its target protein and inhibiting the recruitment of protein co-factors and/or conformational changes required for immune signaling.     

RIG-I, MDA5 and TLR3 synergistically play an important role in restriction of dengue virus infection

Dengue virus (DV) infection is one of the most common mosquito-borne viral diseases in the world. The innate immune system is important for the early detection of virus and for mounting a cascade of defense measures which include the production of type 1 interferon (IFN). Hence, a thorough understanding of the innate immune response during DV infection would be essential for our understanding of the DV pathogenesis. A recent application of the microarray to dengue virus type 1 (DV1) infected lung carcinoma cells revealed the increased expression of both extracellular and cytoplasmic pattern recognition receptors; retinoic acid inducible gene-I (RIG-I), melanoma differentiation associated gene-5 (MDA-5) and Toll-like receptor-3 (TLR3). These intracellular RNA sensors were previously reported to sense DV infection in different cells. In this study, we show that they are collectively involved in initiating an effective IFN production against DV. Cells silenced for these genes were highly susceptible to DV infection. RIG-I and MDA5 knockdown HUH-7 cells and TLR3 knockout macrophages were highly susceptible to DV infection. When cells were silenced for only RIG-I and MDA5 (but not TLR3), substantial production of IFN-β was observed upon virus infection and vice versa. High susceptibility to virus infection led to ER-stress induced apoptosis in HUH-7 cells. Collectively, our studies demonstrate that the intracellular RNA virus sensors (RIG-I, MDA5 and TLR3) are activated upon DV infection and are essential for host defense against the virus.     

Virion-wide protein interactions of Kaposi's sarcoma-associated herpesvirus

Herpesvirus virions are highly organized structures built through specific protein-protein interactions. Thus, revelation of the protein interactions among virion proteins will shed light on the processes and the mechanisms of virion formation. Recently, we identified 24 virion proteins of Kaposi's sarcoma-associated herpesvirus (KSHV), using a proteomic approach (F. X. Zhu et al., J. Virol. 79:800-811, 2005). In the current study, a comprehensive analysis of protein-protein interaction between KSHV virion proteins was carried out using yeast two-hybrid (Y2H) and coimmunoprecipitation (co-IP) approaches. Every pairwise combination between KSHV tegument and capsid proteins, between tegument and envelope proteins, and among tegument proteins was tested for possible binary interaction. Thirty-seven protein-protein interactions were identified by both Y2H and co-IP analyses. The results revealed interactions between tegument and capsid proteins such as that of open reading frame 64 (ORF64) with ORF25 (major capsid protein [MCP]), ORF62 (triplex-1 [TRI-1]), and ORF26 (TRI-2). Many interactions were detected among the tegument proteins. ORF64 was found to interact with several tegument proteins including ORF11, ORF21, ORF33, ORF45, ORF63, ORF75, and ORF64 itself, suggesting that ORF64 may serve as a hub protein and play a role in recruiting tegument proteins during tegumentation and virion assembly. Our investigation also revealed redundant interactions between tegument proteins and envelope glycoproteins. These interactions are believed to contribute to final envelopment in virion assembly. Overall, this study allows us to establish a virion-wide protein interaction map, which provides insight into the architecture of the KSHV virion and sets up a foundation for exploring the functions of these proteins in viral particle assembly.     

The ORF45 protein of Kaposi's sarcoma-associated herpesvirus is associated with purified virions

Kaposi's sarcoma-associated herpesvirus (KSHV) ORF45 is encoded by an immediate-early gene in the KSHV genome. This protein was recently shown to interact with interferon regulatory factor 7 and inhibit virus-mediated alpha/beta interferon induction (Zhu et al., Proc. Natl. Acad. Sci. USA 99:5573-5578, 2002). ORF45 was characterized as a phosphorylated protein, and it is localized in the cytoplasm of infected cells. In this report, we provide evidence that ORF45 is associated with KSHV virions. (i) ORF45 was detected in gradient-purified virions by Western blotting along with known structural proteins of KSHV including gB, K8.1, and major capsid protein. In contrast, ORF50/Rta, K8alpha, and ORF59/PF8 were not detected in the same virion preparation. (ii) ORF45 comigrates with KSHV virions in sucrose gradient ultracentrifugation. (iii) Virion-associated ORF45 was resistant to trypsin digestion but became sensitive after the virions were treated with detergent which destroys the viral envelope. (iv) ORF45 remained associated with tegument-nucleocapsid complex when virion-specific glycoproteins were removed after detergent treatment. (v) An ORF45 protein band was visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of extensively purified KSHV virions and identified by mass spectrometry. (vi) By immunoelectron microscopy, virus-like structures were specifically stained by anti-ORF45 antibody. Based on the evidence, we conclude that ORF45 is associated with purified KSHV virions and appears to be a tegument protein. The presence of ORF45 in KSHV virions raised the possibility that this protein may be delivered to host cells at the start of infection and therefore have the opportunity to act at the very early stage of the infection, suggesting an important role of ORF45 in KSHV primary infection.     

RIG-I mediates the co-induction of tumor necrosis factor and type I interferon elicited by myxoma virus in primary human macrophages

The sensing of pathogen infection and subsequent triggering of innate immunity are key to controlling zoonotic infections. Myxoma virus (MV) is a cytoplasmic DNA poxvirus that in nature infects only rabbits. Our previous studies have shown that MV infection of primary mouse cells is restricted by virus-induced type I interferon (IFN). However, little is known about the innate sensor(s) involved in activating signaling pathways leading to cellular defense responses in primary human immune cells. Here, we show that the complete restriction of MV infection in the primary human fibroblasts requires both tumor necrosis factor (TNF) and type I IFN. We also demonstrate that MV infection of primary human macrophages (pHMs) activates the cytoplasmic RNA sensor called retinoic acid inducible gene I (RIG-I), which coordinately induces the production of both TNF and type I IFN. Of note, RIG-I sensing of MV infection in pHMs initiates a sustained TNF induction through the sequential involvement of the downstream IFN-regulatory factors 3 and 7 (IRF3 and IRF7). Thus, RIG-I-mediated co-induction of TNF and type I IFN by virus-infected pHMs represents a novel innate defense mechanism to restrict viral infection in human cells. These results also reveal a new regulatory mechanism for TNF induction following viral infection.