Young C. Shin

TRIM25 RING-finger E3 ubiquitin ligase is essential for RIG-I-mediated antiviral activity

Retinoic-acid-inducible gene-I (RIG-I; also called DDX58) is a cytosolic viral RNA receptor that interacts with MAVS (also called VISA, IPS-1 or Cardif) to induce type I interferon-mediated host protective innate immunity against viral infection. Furthermore, members of the tripartite motif (TRIM) protein family, which contain a cluster of a RING-finger domain, a B box/coiled-coil domain and a SPRY domain, are involved in various cellular processes, including cell proliferation and antiviral activity. Here we report that the amino-terminal caspase recruitment domains (CARDs) of RIG-I undergo robust ubiquitination induced by TRIM25 in mammalian cells. The carboxy-terminal SPRY domain of TRIM25 interacts with the N-terminal CARDs of RIG-I; this interaction effectively delivers the Lys 63-linked ubiquitin moiety to the N-terminal CARDs of RIG-I, resulting in a marked increase in RIG-I downstream signalling activity. The Lys 172 residue of RIG-I is critical for efficient TRIM25-mediated ubiquitination and for MAVS binding, as well as the ability of RIG-I to induce antiviral signal transduction. Furthermore, gene targeting demonstrates that TRIM25 is essential not only for RIG-I ubiquitination but also for RIG-I-mediated interferon-β production and antiviral activity in response to RNA virus infection. Thus, we demonstrate that TRIM25 E3 ubiquitin ligase induces the Lys 63-linked ubiquitination of RIG-I, which is crucial for the cytosolic RIG-I signalling pathway to elicit host antiviral innate immunity.

Roles of RIG-I N-terminal tandem CARD and splice variant in TRIM25-mediated antiviral signal transduction

The caspase recruitment domain (CARD) of intracellular adaptors and sensors plays a critical role in the assembly of signaling complexes involved in innate host defense against pathogens and in the regulation of inflammatory responses. The cytosolic receptor retinoic acid-inducible gene-I (RIG-I) recognizes viral RNA in a 5′-triphosphate-dependent manner and initiates an antiviral signaling cascade. Upon viral infection, the N-terminal CARDs of RIG-I undergo the K63-linked ubiquitination induced by tripartite motif protein 25 (TRIM25), critical for the interaction of RIG-I with its downstream signaling partner MAVS/VISA/IPS-1/Cardif. Here, we demonstrate the distinct roles of RIG-I first and second CARD in TRIM25-mediated RIG-I ubiquitination: TRIM25 binds the RIG-I first CARD and subsequently ubiquitinates its second CARD. The T55I mutation in RIG-I first CARD abolishes TRIM25 interaction, whereas the K172R mutation in the second CARD eliminates polyubiquitin attachment. The necessity of the intact tandem CARD for RIG-I function is further evidenced by a RIG-I splice variant (SV) whose expression is robustly up-regulated upon viral infection. The RIG-I SV carries a short deletion (amino acids 36–80) within the first CARD and thereby loses TRIM25 binding, CARD ubiquitination, and downstream signaling ability. Furthermore, because of its robust inhibition of virus-induced RIG-I multimerization and RIG-I-MAVS signaling complex formation, this SV effectively suppresses the RIG-I-mediated IFN-β production. This study not only elucidates the vital role of the intact tandem CARD for TRIM25-mediated RIG-I activation but also identifies the RIG-I SV as an off-switch regulator of its own signaling pathway.

Inhibition of the ATM/p53 Signal Transduction Pathway by Kaposi's Sarcoma-Associated Herpesvirus Interferon Regulatory Factor 1

ABSTRACT Infected cells recognize viral replication as a DNA damage stress and elicit the ataxia telangiectasia-mutated (ATM)/p53-mediated DNA damage response signal transduction pathway as part of the host surveillance mechanisms, which ultimately induces the irreversible cell cycle arrest and apoptosis. Viruses have evolved a variety of mechanisms to counteract this host intracellular innate immunity. Kaposis sarcoma-associated herpesvirus (KSHV) viral interferon regulatory factor 1 (vIRF1) interacts with the cellular p53 tumor suppressor through its central DNA binding domain, and this interaction inhibits transcriptional activation of p53. Here, we further demonstrate that KSHV vIRF1 downregulates the total p53 protein level by facilitating its proteasome-mediated degradation. Detailed biochemical study showed that vIRF1 interacted with cellular ATM kinase through its carboxyl-terminal transactivation domain and that this interaction blocked the activation of ATM kinase activity induced by DNA damage stress. As a consequence, vIRF1 expression greatly reduced the level of serine 15 phosphorylation of p53, resulting in an increase of p53 ubiquitination and thereby a decrease of its protein stability. These results indicate that KSHV vIRF1 comprehensively compromises an ATM/p53-mediated DNA damage response checkpoint by targeting both upstream ATM kinase and downstream p53 tumor suppressor, which might circumvent host growth surveillance and facilitate viral replication in infected cells.

Inhibition of Interferon Regulatory Factor 7 (IRF7)-Mediated Interferon Signal Transduction by the Kaposi's Sarcoma-Associated Herpesvirus Viral IRF Homolog vIRF3

ABSTRACT Upon viral infection, the major defense mounted by the host immune system is activation of the interferon (IFN)-mediated antiviral pathway that is mediated by IFN regulatory factors (IRFs). In order to complete their life cycle, viruses must modulate the host IFN-mediated immune response. Kaposis sarcoma-associated herpesvirus (KSHV), a human tumor-inducing herpesvirus, has developed a unique mechanism for antagonizing cellular IFN-mediated antiviral activity by incorporating viral homologs of the cellular IRFs, called vIRFs. Here, we report a novel immune evasion mechanism of KSHV vIRF3 to block cellular IRF7-mediated innate immunity in response to viral infection. KSHV vIRF3 specifically interacts with either the DNA binding domain or the central IRF association domain of IRF7, and this interaction leads to the inhibition of IRF7 DNA binding activity and, therefore, suppression of alpha interferon (IFN-α) production and IFN-mediated immunity. Remarkably, the central 40 amino acids of vIRF3, containing the double α helix motifs, are sufficient not only for binding to IRF7, but also for inhibiting IRF7 DNA binding activity. Consequently, the expression of the double α helix motif-containing peptide effectively suppresses IRF7-mediated IFN-α production. This demonstrates a remarkably efficient means of viral avoidance of host antiviral activity.

Kaposi's Sarcoma–Associated Herpesvirus Viral IFN Regulatory Factor 3 Stabilizes Hypoxia-Inducible Factor-1α to Induce Vascular Endothelial Growth Factor Expression

Kaposis sarcoma-associated herpesvirus (KSHV) is the etiologic agent associated with Kaposis sarcoma, primary effusion lymphoma, and multicentric Castlemans disease. Hypoxia-inducible factor-1 (HIF-1) is the master regulator of both developmental and pathologic angiogenesis, composed of an oxygen-sensitive alpha-subunit and a constitutively expressed beta-subunit. HIF-1 activity in tumors depends on the availability of the HIF-1 alpha subunit, the levels of which are increased under hypoxic conditions. Recent studies have shown that HIF-1 plays an important role in KSHV reactivation from latency and pathogenesis. Here, we report a novel mechanism by which KSHV activates HIF-1 activity. Specific interaction between KSHV viral IFN regulatory factor 3 (vIRF3) and the HIF-1 alpha subunit led to the HIF-1 alpha stabilization and transcriptional activation, which induced vascular endothelial growth factor expression and ultimately facilitated endothelial tube formation. Remarkably, the central domain of vIRF3, containing double alpha-helix motifs, was sufficient not only for binding to HIF-1 alpha but also for blocking its degradation in normoxic conditions. This indicates that KSHV has developed a unique mechanism to enhance HIF-1 alpha protein stability and transcriptional activity by incorporating a viral homologue of cellular IRF gene into its genome, which may contribute to viral pathogenesis.

Kaposi's Sarcoma-Associated Herpesvirus Viral Interferon Regulatory Factor 4 Targets MDM2 To Deregulate the p53 Tumor Suppressor Pathway

ABSTRACT Cells infected by viruses utilize interferon (IFN)-mediated and p53-mediated irreversible cell cycle arrest and apoptosis as part of the overall host surveillance mechanism to ultimately block viral replication and dissemination. Viruses, in turn, have evolved elaborate mechanisms to subvert IFN- and p53-mediated host innate immune responses. Kaposis sarcoma-associated herpesvirus (KSHV) encodes several viral IFN regulatory factors (vIRF1 to vIRF4) within a cluster of loci, their functions being primarily to inhibit host IFN-mediated innate immunity and deregulate p53-mediated cell growth control. Despite its significant homology and similar genomic location to other vIRFs, vIRF4 is distinctive, as it does not target and antagonize host IFN-mediated signal transduction. Here, we show that KSHV vIRF4 interacts with the murine double minute 2 (MDM2) E3 ubiquitin ligase, leading to the reduction of p53, a tumor suppressor, via proteasome-mediated degradation. The central region of vIRF4 is required for its interaction with MDM2, which led to the suppression of MDM2 autoubiquitination and, thereby, a dramatic increase in MDM2 stability. Consequently, vIRF4 expression markedly enhanced p53 ubiquitination and degradation, effectively suppressing p53-mediated apoptosis. These results indicate that KSHV vIRF4 targets and stabilizes the MDM2 E3 ubiquitin ligase to facilitate the proteasome-mediated degradation of p53, perhaps to circumvent host growth surveillance and facilitate viral replication in infected cells. Taken together, the indications are that the downregulation of p53-mediated cell growth control is a common characteristic of the four KSHV vIRFs and that p53 is indeed a key factor in the hosts immune surveillance program against viral infections.

Inhibition of Interferon-Mediated Antiviral Activity by Murine Gammaherpesvirus 68 Latency-Associated M2 Protein

ABSTRACT Upon viral infection, the major defense mounted by the host immune system is the activation of the interferon (IFN)-mediated antiviral pathway. In order to complete their life cycle, viruses that are obligatory intracellular parasites must modulate the host IFN-mediated immune response. Murine gammaherpesvirus 68 (γHV68) infects a wide range of cell types and establishes latent infections in mice. Here we demonstrate that the γHV68 latency-associated M2 protein has a cell-type-dependent localization pattern: M2 is present in the cytoplasm and plasma membrane in lymphocytes, whereas it is present primarily in the nucleus in epithelial and fibroblast cells. A mutational analysis indicated that the internal positively charged amino acids of M2 are required for its nuclear localization in fibroblasts. Purification of the M2 complex showed that M2 specifically interacts with the cellular p32 acidic protein through its central positively charged region and that this interaction recruits the cellular p32 protein to the nucleus in fibroblasts. Regardless of its localization, M2 expression effectively induced the downregulation of STAT1 and/or STAT2 in both A20 B lymphocytes and NIH 3T3 fibroblasts, resulting in the inhibition of IFN-α/β- and IFN-γ-mediated transcriptional activation. Finally, the M2 interaction with the p32 protein appeared to contribute to its ability to inhibit IFN-mediated transcriptional activation. These results indicate that γHV68 harbors a latency-associated M2 gene that antagonizes IFN-mediated host innate immunity and thus could play an important role in the establishment and maintenance of viral latency in infected animals.

A Novel Inhibitory Mechanism of Mitochondrion-Dependent Apoptosis by a Herpesviral Protein

Upon viral infection, cells undergo apoptosis as a defense against viral replication. Viruses, in turn, have evolved elaborate mechanisms to subvert apoptotic processes. Here, we report that a novel viral mitochondrial anti-apoptotic protein (vMAP) of murine γ-herpesvirus 68 (γHV-68) interacts with Bcl-2 and voltage-dependent anion channel 1 (VDAC1) in a genetically separable manner. The N-terminal region of vMAP interacted with Bcl-2, and this interaction markedly increased not only Bcl-2 recruitment to mitochondria but also its avidity for BH3-only pro-apoptotic proteins, thereby suppressing Bax mitochondrial translocation and activation. In addition, the central and C-terminal hydrophobic regions of vMAP interacted with VDAC1. Consequently, these interactions resulted in the effective inhibition of cytochrome c release, leading to the comprehensive inhibition of mitochondrion-mediated apoptosis. Finally, vMAP gene was required for efficient γHV-68 lytic replication in normal cells, but not in mitochondrial apoptosis-deficient cells. These results demonstrate that γHV-68 vMAP independently targets two important regulators of mitochondrial apoptosis-mediated intracellular innate immunity, allowing efficient viral lytic replication.

Formation of Polyomavirus-Like Particles with Different VP1 Molecules That Bind the Urokinase Plasminogen Activator Receptor

ABSTRACT Icosahedral virus-like particles formed by the self-assembly of polyomavirus capsid proteins (Py-VLPs) can serve as useful nanostructures for delivering nucleic acids, proteins, and pharmaceuticals into animal cells and tissues. Four predominant surface-exposed loops in the VP1 structure offer potential sites to display sequences that might contribute new targeting specificities. Introduction into each of these loops of sequences derived from the amino-terminal fragment of urokinase plasminogen activator (uPA) or a related phage display peptide reduced the solubility of VP1 molecules when expressed in insect cells, and insertions into the EF loop reduced VP1 solubility least. Coexpression in insect cells of the uPA-VP1 molecules and VP1 containing a FLAG epitope in the HI loop permitted the formation of heterotypic Py-VLPs containing uPA-VP1 and FLAG-VP1. These heterotypic VLPs bound to uPAR on the surfaces of animal cells. Heterotypic Py-VLPs containing ligands for multiple cell surface receptors should be useful for targeting specific cells and tissues.

Vaccine Protection against Simian Immunodeficiency Virus in Monkeys Using Recombinant Gamma-2 Herpesvirus

ABSTRACT Recombinant strains of replication-competent rhesus monkey rhadinovirus (RRV) were constructed in which strong promoter/enhancer elements were used to drive expression of simian immunodeficiency virus (SIV) Env or Gag or a Rev-Tat-Nef fusion protein. Cultured rhesus monkey fibroblasts infected with each recombinant strain were shown to express the expected protein. Three RRV-negative and two RRV-positive rhesus monkeys were inoculated intravenously with a mixture of these three recombinant RRVs. Expression of SIV Gag was readily detected in lymph node biopsy specimens taken at 3 weeks postimmunization. Impressive anti-SIV cellular immune responses were elicited on the basis of major histocompatibility complex (MHC) tetramer staining and gamma interferon enzyme-linked immunospot (ELISPOT) assays. Responses were much greater in magnitude in the monkeys that were initially RRV negative but were still readily detected in the two monkeys that were naturally infected with RRV at the time of immunization. By 3 weeks postimmunization, responses measured by MHC tetramer staining in the two Mamu-A*01 + RRV-negative monkeys reached 9.3% and 13.1% of all CD8+ T cells in peripheral blood to the Gag CM9 epitope and 2.3% and 7.3% of all CD8+ T cells in peripheral blood to the Tat SL8 epitope. Virus-specific CD8+ T cell responses persisted at high levels up to the time of challenge at 18 weeks postimmunization, and responding cells maintained an effector memory phenotype. Despite the ability of the RRVenv recombinant to express high levels of Env in cultured cells, and despite the appearance of strong anti-RRV antibody responses in immunized monkeys, anti-Env antibody responses were below our ability to detect them. Immunized monkeys, together with three unimmunized controls, were challenged intravenously with 10 monkey infectious doses of SIVmac239. All five immunized monkeys and all three controls became infected with SIV, but peak viral loads were 1.2 to 3.0 log10 units lower and chronic-phase viral loads were 1.0 to 3.0 log10 units lower in immunized animals than the geometric mean of unimmunized controls. These differences were statistically significant. Anti-Env antibody responses following challenge indicated an anamnestic response in the vaccinated monkeys. These findings further demonstrate the potential of recombinant herpesviruses as preventive vaccines for AIDS. We hypothesize that this live, replication-competent, persistent herpesvirus vector could match, or come close to matching, live attenuated strains of SIV in the degree of protection if the difficulty with elicitation of anti-Env antibody responses can be overcome.