The Mitochondrial Protein MAVS Stabilizes p53 to Suppress Tumorigenesis.

Recent reports have shown the critical role of the mitochondrial antiviral signaling (MAVS) protein in virus-induced apoptosis, but the involvement of MAVS in tumorigenesis is still poorly understood. Herein, we report that MAVS is a key regulator of p53 activation and is critical for protecting against tumorigenesis. We find that MAVS promotes p53-dependent cell death in response to DNA damage. MAVS interacts with p53 and mediates p53 mitochondrial recruitment under genotoxic stress. Mechanistically, MAVS inhibits p53 ubiquitination by blocking the formation of the p53-murine double-minute 2 (MDM2) complex, leading to the stabilization of p53. Notably, compared with their wild-type littermates, MAVS knockout mice display decreased resistance to azoxymethane (AOM) or AOM/dextran sulfate sodium salt (DSS)-induced colon cancer. MAVS expression is significantly downregulated in human colon cancer tissues. These results unveil roles for MAVS in DNA damage response and tumor suppression.

RNF34 functions in immunity and selective mitophagy by targeting MAVS for autophagic degradation.

Viral infection triggers the formation of mitochondrial antiviral signaling protein (MAVS) aggregates, which potently promote immune signaling. Autophagy plays an important role in controlling MAVS-mediated antiviral signaling; however, the exact molecular mechanism underlying the targeted autophagic degradation of MAVS remains unclear. Here, we investigated the mechanism by which RNF34 regulates immunity and mitophagy by targeting MAVS. RNF34 binds to MAVS in the mitochondrial compartment after viral infection and negatively regulates RIG-I-like receptor (RLR)-mediated antiviral immunity. Moreover, RNF34 catalyzes the K27-/K29-linked ubiquitination of MAVS at Lys 297, 311, 348, and 362 Arg, which serves as a recognition signal for NDP52-dependent autophagic degradation. Specifically, RNF34 initiates the K63- to K27-linked ubiquitination transition on MAVS primarily at Lys 311, which facilitates the autophagic degradation of MAVS upon RIG-I stimulation. Notably, RNF34 is required for the clearance of damaged mitochondria upon viral infection. Thus, we elucidated the mechanism by which RNF34-mediated autophagic degradation of MAVS regulates the innate immune response, mitochondrial homeostasis, and infection.

Tumor Microenvironment Regulation by the Endoplasmic Reticulum Stress Transmission Mediator Golgi Protein 73 in Mice.

The unfolded protein response (UPR) signal in tumor cells activates UPR signaling in neighboring macrophages, which leads to tumor-promoting inflammation by up-regulating UPR target genes and proinflammatory cytokines. However, the molecular basis of this endoplasmic reticulum (ER) stress transmission remains largely unclear. Here, we identified the secreted form of Golgi protein 73 (GP73), a Golgi-associated protein functional critical for hepatocellular carcinoma (HCC) growth and metastasis, is indispensable for ER stress transmission. Notably, ER stressors increased the cellular secretion of GP73. Through GRP78, the secreted GP73 stimulated ER stress activation in neighboring macrophages, which then released cytokines and chemokines involved in the tumor-associated macrophage (TAM) phenotype. Analysis of HCC patients revealed a positive correlation of GP73 with glucose-regulated protein 78 (GRP78) expression and TAM density. High GP73 and CD206 expression was associated with poor prognosis. Blockade of GP73 decreased the density of TAMs, inhibited tumor growth, and prolonged survival in two mouse HCC models. Conclusion: Our findings provide insight into the molecular mechanisms of extracellular GP73 in the amplification and transmission of ER stress signals.

A sorting signal suppresses IFITM1 restriction of viral entry.

The interferon-induced transmembrane proteins (IFITMs) broadly inhibit virus infections, particularly at the viral entry level. However, despite this shared ability to inhibit fusion, IFITMs differ in the potency and breadth of viruses restricted, an anomaly that is not fully understood. Here, we show that differences in the range of viruses restricted by IFITM1 are regulated by a C-terminal non-canonical dibasic sorting signal KRXX that suppresses restriction of some viruses by governing its intracellular distribution. Replacing the two basic residues with alanine (KR/AA) increased restriction of jaagsiekte sheep retrovirus and 10A1 amphotropic murine leukemia virus. Deconvolution microscopy revealed an altered subcellular distribution for KR/AA, with fewer molecules in LAMP1-positive lysosomes balanced by increased levels in CD63-positive multivesicular bodies, where jaagsiekte sheep retrovirus pseudovirions are colocalized. IFITM1 binds to cellular adaptor protein complex 3 (AP-3), an association that is lost when the dibasic motif is altered. Although knockdown of AP-3 itself decreases some virus entry, expression of parental IFITM1, but not its KR/AA mutant, potentiates inhibition of viral infections in AP-3 knockdown cells. By using the substituted cysteine accessibility method, we provide evidence that IFITM1 adopts more than one membrane topology co-existing in cellular membranes. Because the C-terminal dibasic sorting signal is unique to human IFITM1, our results provide novel insight into understanding the species- and virus-specific antiviral effect of IFITMs.

IFN-stimulated gene LY6E in monocytes regulates the CD14/TLR4 pathway but inadequately restrains the hyperactivation of monocytes during chronic HIV-1 infection.

Owing to ongoing recognition of pathogen-associated molecular patterns, immune activation and upregulation of IFN-stimulated genes (ISGs) are sustained in the chronically infected host. Albeit most ISGs are important effectors for containing viral replication, some might exert compensatory immune suppression to limit pathological dysfunctions, although the mechanisms are not fully understood. In this study, we report that the ISG lymphocyte Ag 6 complex, locus E (LY6E) is a negative immune regulator of monocytes. LY6E in monocytes negatively modulated CD14 expression and subsequently dampened the responsiveness to LPS stimulation in vitro. In the setting of chronic HIV infection, the upregulation of LY6E was correlated with reduced CD14 level on monocytes; however, the immunosuppressive effect of LY6E was not adequate to remedy the hyperresponsiveness of activated monocytes. Taken together, the regulatory LY6E pathway in monocytes represents one of negative feedback mechanisms that counterbalance monocyte activation, which might be caused by LPS translocation through the compromised gastrointestinal tract during persistent HIV-1 infection and may serve as a potential target for immune intervention.

HIV-1 Vpr stimulates NF-κB and AP-1 signaling by activating TAK1.

BACKGROUND: The Vpr protein of human immunodeficiency virus type 1 (HIV-1) plays an important role in viral replication. It has been reported that Vpr stimulates the nuclear factor-κB (NF-κB) and activator protein 1 (AP-1) signaling pathways, and thereby regulates viral and host cell gene expression. However, the molecular mechanism behind this function of Vpr is not fully understood. RESULTS: Here, we have identified transforming growth factor-ß-activated kinase 1 (TAK1) as the important upstream signaling molecule that Vpr associates with in order to activate NF-κB and AP-1 signaling. HIV-1 virion-associated Vpr is able to stimulate phosphorylation of TAK1. This activity of Vpr depends on its association with TAK1, since the S79A Vpr mutant lost interaction with TAK1 and was unable to activate TAK1. This association allows Vpr to promote the interaction of TAB3 with TAK1 and increase the polyubiquitination of TAK1, which renders TAK1 phosphorylation. In further support of the key role of TAK1 in this function of Vpr, knockdown of endogenous TAK1 significantly attenuated the ability of Vpr to activate NF-κB and AP-1 as well as the ability to stimulate HIV-1 LTR promoter. CONCLUSIONS: HIV-1 Vpr enhances the phosphorylation and polyubiquitination of TAK1, and as a result, activates NF-κB and AP-1 signaling pathways and stimulates HIV-1 LTR promoter.

N-Myc interactor inhibits prototype foamy virus by sequestering viral Tas protein in the cytoplasm.

UNLABELLED: Foamy viruses (FVs) are complex retroviruses that establish lifelong persistent infection without evident pathology. However, the roles of cellular factors in FV latency are poorly understood. This study revealed that N-Myc interactor (Nmi) could inhibit the replication of prototype foamy virus (PFV). Overexpression of Nmi reduced PFV replication, whereas its depletion by small interfering RNA increased PFV replication. The Nmi-mediated impairment of PFV replication resulted from the diminished transactivation by PFV Tas of the viral long terminal repeat (LTR) and an internal promoter (IP). Nmi was determined to interact with Tas and abrogate its function by sequestration in the cytoplasm. In addition, human and bovine Nmi proteins were found to inhibit the replication of bovine foamy virus (BFV) and PFV. Together, these results indicate that Nmi inhibits both human and bovine FVs by interfering with the transactivation function of Tas and may have a role in the host defense against FV infection. IMPORTANCE: From this study, we report that the N-Myc interactor (Nmi), an interferon-induced protein, can interact with the regulatory protein Tas of the prototype foamy virus and sequester it in the cytoplasm. The results of this study suggest that Nmi plays an important role in maintaining foamy virus latency and may reveal a new pathway in the interferon-mediated antiviral barrier against viruses. These findings are important for understanding virus-host relationships not only with FVs but potentially for other retroviruses as well.

Characterization of a full-length infectious clone of bovine foamy virus 3026.

The biological features of most foamy viruses (FVs) are poorly understood, including bovine foamy virus (BFV). BFV strain 3026 (BFV3026) was isolated from the peripheral blood mononuclear cells of an infected cow in Zhangjiakou, China. A full-length genomic clone of BFV3026 was obtained from BFV3026-infected cells, and it exhibited more than 99% amino acid (AA) homology to another BFV strain isolated in the USA. Upon transfection into fetal canine thymus cells, the full-length BFV3026 clone produced viral structural and auxiliary proteins, typical cytopathic effects, and virus particles. These results demonstrate that the full-length BFV3026 clone is fully infectious and can be used in further BFV3026 research.

Identification of an endocytic signal essential for the antiviral action of IFITM3.

Members of the interferon-induced transmembrane (IFITM) protein family inhibit the entry of a wide range of viruses. Viruses often exploit the endocytosis pathways to invade host cells and escape from the endocytic vesicles often in response to low pH. Localization to these endocytic vesicles is essential for IFITM3 to interfere with the cytosolic entry of pH-dependent viruses. However, the nature of the sorting signal that targets IFITM3 to these vesicles is poorly defined. In this study, we report that IFITM3 possesses a YxxΦ sorting motif, i.e. 20-YEML-23, that enables IFITM3 to undergo endocytosis through binding to the µ2 subunit of the AP-2 complex. IFITM3 accumulates at the plasma membrane as a result of either mutating 20-YEML-23, depleting the µ2 subunit or overexpressing µ2 mutants. Importantly, blocking endocytosis of IFITM3 abrogates its ability to inhibit pH-dependent viruses. We have therefore identified a critical sorting signal, namely 20-YEML-23, that controls both the endocytic trafficking and the antiviral action of IFITM3. This finding also reveals that as an endocytic protein, IFITM3 first arrives at the plasma membrane before it is endocytosed and further traffics to the late endosomes where it acts to impede virus entry.

[Effect of RelB on HIV-1 Vpr-mediated transcription activation and cell G2/M arrest].

Vpr, an auxiliary protein of HIV-1(Human immunodeficiency virus type 1), exerts important functions to promote viral replication and AIDS progression. In this study, we performed a yeast two-hybrid screening assay using human cDNA library to further investigate the molecular mechanism of various functions of Vpr RelB, a key protein in NF-kappaB signaling pathway, was identified as a Vpr interaction protein by co-immunoprecipitation. Further investigations indicated that RelB not only promoted the Vpr-mediated activation of NF-kappaB reporter gene, but also enhanced the transactivation of HIV LTR. Moreover, the results showed that RelB promoted Vpr-induced cell cycle G2/M arrest. Collectively, these results indicated that RelB might interact with Vpr and regulate its transcriptional activation and cell cycle arrest.

HIV-1 Vpr activates both canonical and noncanonical NF-κB pathway by enhancing the phosphorylation of IKKα/ß.

The human immunodeficiency virus type I (HIV-1) Vpr plays an essential role in viral replication. A number of studies have reported that Vpr modulates the nuclear factor-κB (NF-κB) pathway. Yet, the reported effects of Vpr on NF-κB signaling are controversial. In this study, we investigate the interplay between Vpr and NF-κB pathway. We discover that HIV-1 infection elevates the phosphorylation of IκBα and p100, and that this increase is greatly reduced when a Vpr-negative HIV-1 is used for infection. Our data further show that Vpr regulates the activity of IKKα/ß through interactions. In addition, Vpr modulates the phosphorylation of p65 and p100, suggesting that Vpr activates both canonical and noncanonical NF-κB pathway. Knock down of endogenous IKKα/ß result in a decrease in Vpr-mediated NF-κB and HIV-1 LTR activation. Given that Vpr is present in HIV-1 particles, our data suggest that Vpr activates the NF-κB pathway immediately after HIV-1 entry.

Interferon-γ upregulates expression of IFP35 gene in HeLa cells via interferon regulatory factor-1.

BACKGROUND: Interferon-induced 35-kDa protein (IFP35) plays important roles in antiviral defense and the progression of some skin cancer diseases. It can be induced by interferon-γ (IFN-γ) in multiple human cells. However, the mechanisms by which IFN-γ contributes to IFP35 induction remain to be elucidated. METHODS/PRINCIPAL FINDINGS: We identified the transcription start sites of IFP35 by 5' rapid amplification of cDNA ends (RACE) and cloned the promoter of IFP35. Sequence analysis and luciferase assays revealed two GC boxes and an IFN-stimulated response element (ISRE) in the 5' upstream region of the transcription start sites, which were important for the basal transcription of IFP35 gene. Furthermore, we found that interferon regulatory factor 1 (IRF-1) and IRF-2 could bind to IFP35 promoter and upregulate endogenous IFP35 protein level. Depletion of endogenous IRF-1 by interfering RNA reduced the constitutive and IFN-γ-dependent expression of IFP35, whereas depletion of IRF-2 had little effect on IFN-γ-inducible IFP35 expression. Moreover, IRF-1 was recruited to the ISRE site in IFP35 promoter in IFN-γ treated HeLa cells, as demonstrated by electrophoretic mobility shift and chromatin immunoprecipitation assays. CONCLUSIONS/SIGNIFICANCE: These findings provide the first evidence that IRF-1 and IRF-2 are involved in constitutive IFP35 expression in HeLa cells, while IRF-1 also activates IFP35 expression in an IFN-γ-inducible manner. Our data therefore identified a new IRF-1 and IRF-2 target gene, which may expand our current understanding of the versatile functions of IRF-1 and IRF-2.

The N-terminal region of IFITM3 modulates its antiviral activity by regulating IFITM3 cellular localization.

Interferon-inducible transmembrane (IFITM) protein family members IFITM1, -2, and -3 restrict the infection of multiple enveloped viruses. Significant enrichment of a minor IFITM3 allele was recently reported for patients who were hospitalized for seasonal and 2009 H1N1 pandemic flu. This IFITM3 allele lacks the region corresponding to the first amino-terminal 21 amino acids and is unable to inhibit influenza A virus. In this study, we found that deleting this 21-amino-acid region relocates IFITM3 from the endosomal compartments to the cell periphery. This finding likely underlies the lost inhibition of influenza A virus that completes its entry exclusively within endosomes at low pH. Yet, wild-type IFITM3 and the mutant with the 21-amino-acid deletion inhibit HIV-1 replication equally well. Given the pH-independent nature of HIV-1 entry, our results suggest that IFITM3 can inhibit viruses that enter cells via different routes and that its N-terminal region is specifically required for controlling pH-dependent viruses.

Comparative analysis of the fusion efficiency elicited by the envelope glycoprotein V1-V5 regions derived from human immunodeficiency virus type 1 transmitted perinatally.

Understanding the properties of viruses preferentially establishing infection during perinatal transmission of human immunodeficiency virus type 1 (HIV-1) is critical for the development of effective measures to prevent transmission. A previous study demonstrated that the newly transmitted viruses (in infants) of chronically infected mother-infant pairs (MIPs) were fitter in terms of growth, which was imparted by their envelope (Env) glycoprotein V1-V5 regions, than those in the corresponding chronically infected mothers. In order to investigate whether the higher fitness of transmitted viruses was conferred by their higher entry efficiency directed by the V1-V5 regions during perinatal transmission, the fusogenicity of Env containing V1-V5 regions derived from transmitted and non-tranmsmitted viruses of five chronically infected MIPs and two acutely infected MIPs was analysed using two different cell-cell fusion assays. The results showed that, in one chronically infected MIP, a higher fusion efficiency was induced by the infant Env V1-V5 compared with that of the corresponding mother. Moreover, the V4-V5 regions played an important role in discriminating the transmitted and non-transmitted viruses in this pair. However, neither a consistent pattern nor significant differences in fusogenicity mediated by the V1-V5 regions between maternal and infant variants was observed in the other MIPs. This study suggests that there is no consistent and significant correlation between viral fitness selection and entry efficiency directed by the V1-V5 regions during perinatal transmission. Other factors such as the route and timing of transmission may also be involved.

Design, synthesis and antiviral activity of novel quinazolinones.

HIV-1 integrase (IN) is a validated therapeutic target for antiviral drug design. However, the emergence of viral strains resistant to clinically studied IN inhibitors demands the discovery of novel inhibitors that are structurally as well as mechanistically different. Herein, a series of quinazolinones were designed and synthesized as novel HIV-1 inhibitors. The new synthetic route provides a practical method for the preparation of 5-hydroxy quinazolinones. Primary bioassay results indicated that most of the quinazolinones possess anti-HIV activity, especially for compound 11b with 77.5% inhibition rate at 10 µM emerged as a new active lead. Most of the synthesized compounds were also found to exhibit good anti-TMV activity, of which compo und 9a showed similar in vivo anti-TMV activity to commercial plant virucide Ribavirin. This work provides a new and efficient approach to evolve novel multi-functional antiviral agents by rational integration and optimization of previously reported antiviral agents.

Design, synthesis and antiviral activity of novel pyridazines.

A series of pyridazines were prepared and evaluated for their anti-HIV activity. The new synthetic route involving a novel rearrangement reaction provided a practical method for the preparation of 5-hydroxypyridazines. The primary bioassay results indicated that most of the pyridazines possess anti-HIV activity. It ought to been mentioned that the rearranged compounds 35 and 39 exhibited relatively higher HIV inhibitory effect. Most of the synthesized compounds were also found to possess good anti-TMV activity, of which compound 9 showed similar in vivo anti-TMV activity to commercial plant virucide Ribavirin. This work provides a new and efficient approach to evolve novel multi-functional antiviral agents by rational integration and optimization of previously reported antiviral agents.

Hydroxyl may not be indispensable for raltegravir: Design, synthesis and SAR Studies of raltegravir derivatives as HIV-1 inhibitors.

A series of raltegravir derivatives 20-42 were prepared and systematically evaluated for their anti-HIV activity. The bioassay results showed that most of the compounds possess good to excellent anti-HIV activity. Especially, compounds 25 and 35 with subpicomole IC(50) values seemed to be the most potent anti-HIV agents among all of the reported synthesized compounds. These compounds may therefore be considered as new potent anti-HIV agents. The 5-hydroxyl modification of raltegravir derivatives significantly increased the anti-HIV activity, which indicates that the hydroxyl may not be indispensable for raltegravir. The introducing of acyl at 5-position of raltegravir derivatives is favorable for antiviral activity. In addition, a high-throughput cell-based assay method with pseudotyped virus stocks was developed and used to identify HIV inhibitors.

Bovine herpesvirus 1 protein bICP0 represses the transcription of bISG15 in fetal bovine lung cells.

The ubiquitin-like modifier bISG15 is an antiviral protein found in fetal bovine lung (FBL) cells. Bovine Herpesvirus 1(BHV-1), which is a viral pathogen of cattle, can infect FBL cells and induce cytopathic effects. Real-time PCR assays showed that BHV-1's infection could repress the basal or inducible transcription of bISG15 in FBL cells. It demonstrates that this repression effect depends on BHV-1 viral infection and new protein synthesis. Our previous work showed that bIRF-3 was the key factor in the stimulation of bISG15 in FBL cells, so the effect of BHV-1 viral protein on bIRF-3 activating the promoter of bISG15 was confirmed. The luciferase assay showed the BHV-1 viral protein bICP0 inhibited the activation of bISG15 promoter stimulated by bIRF-3. Taken together, our work suggested that BHV-1 had some molecular mechanism to resist the cellular bISG15's antiviral functions.

Lysine acetylation sites in bovine foamy virus transactivator BTas are important for its DNA binding activity.

Cellular acetylation signaling is important for viral gene regulation, particularly during the transactivation of retroviruses. The regulatory protein of bovine foamy virus (BFV), BTas, is a transactivator that augments viral gene transcription from both the long terminal repeat (LTR) promoter and the internal promoter (IP). In this study, we report that the histone acetyltransferase (HAT), p300, specifically acetylates BTas both in vivo and in vitro. Further studies demonstrated that BTas acetylation markedly enhances its transactivation activity. Mutagenesis analysis identified three lysines at positions 66, 109 and 110 in BTas that are acetylated by p300. The K110R mutant lost its binding to BFV promoter as well as its ability to activate BFV promoter. The acetylation of K66 and K109 may contribute to increased BTas binding ability. These results suggest that the p300-acetylated lysines of BTas are important for transactivation of BFV promoters and therefore have an important role in BFV replication.

Tetherin inhibits prototypic foamy virus release.

BACKGROUND: Tetherin (also known as BST-2, CD317, and HM1.24) is an interferon- induced protein that blocks the release of a variety of enveloped viruses, such as retroviruses, filoviruses and herpesviruses. However, the relationship between tetherin and foamy viruses has not been clearly demonstrated. RESULTS: In this study, we found that tetherin of human, simian, bovine or canine origin inhibits the production of infectious prototypic foamy virus (PFV). The inhibition of PFV by human tetherin is counteracted by human immunodeficiency virus type 1 (HIV-1) Vpu. Furthermore, we generated human tetherin transmembrane domain deletion mutant (delTM), glycosyl phosphatidylinositol (GPI) anchor deletion mutant (delGPI), and dimerization and glycosylation deficient mutants. Compared with wild type tetherin, the delTM and delGPI mutants only moderately inhibited PFV production. In contrast, the dimerization and glycosylation deficient mutants inhibit PFV production as efficiently as the wild type tetherin. CONCLUSIONS: These results demonstrate that tetherin inhibits the release and infectivity of PFV, and this inhibition is antagonized by HIV-1 Vpu. Both the transmembrane domain and the GPI anchor of tetherin are important for the inhibition of PFV, whereas the dimerization and the glycosylation of tetherin are dispensable.