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ABSTRACT Over the last 2 years, several global virus-host interactome studies have been published with SARS-CoV-2 proteins with the purpose of better understanding how specific viral proteins can subvert or utilize different cellular processes to promote viral infection and pathogenesis. However, most of the virus–host protein interactions have not yet been confirmed experimentally, and their biological significance is largely unknown. The goal of this study was to verify the interaction of NSP5, the main protease of SARS-CoV-2, with the host epigenetic factor histone deacetylase 2 (HDAC2) and test if HDAC2 is required for NSP5-mediated inhibition of the type I interferon signaling pathway. Our results show that NSP5 can significantly reduce the expression of a subset of immune response genes such as IL-6, IL-1β, and IFNβ, which requires NSP5’s protease activity. We also found that NSP5 can inhibit Sendai virus-, RNA sensor-, and DNA sensor-mediated induction of IFNβ promoter, block the IFN response pathway, and reduce the expression of IFN-stimulated genes. We also provide evidence for HDAC2 interacting with IRF3, and NSP5 can abrogate their interaction by binding to both IRF3 and HDAC2. In addition, we found that HDAC2 plays an inhibitory role in the regulation of IFNβ and IFN-induced promoters, but our results indicate that HDAC2 is not involved in NSP5-mediated inhibition of IFNβ gene expression. Taken together, our data show that NSP5 interacts with HDAC2 but NSP5 inhibits the IFNβ gene expression and interferon-signaling pathway in an HDAC2-independent manner. IMPORTANCE SARS-CoV-2 has developed multiple strategies to antagonize the host antiviral response, such as blocking the IFN signaling pathway, which favors the replication and spreading of the virus. A recent SARS-CoV-2 protein interaction mapping revealed that the main viral protease NSP5 interacts with the host epigenetic factor HDAC2, but the interaction was not confirmed experimentally and its biological importance remains unclear. Here, we not only verified the interaction of HDAC2 with NSP5, but we also found that HDAC2 also binds to IRF3, and NSP5 can disrupt the IRF3-HDAC2 complex. Furthermore, our results show that NSP5 can efficiently repress the IFN signaling pathway regardless of whether viral infections, RNA, or DNA sensors activated it. However, our data indicate that HDAC2 is not involved in NSP5-mediated inhibition of IFNβ promoter induction and IFNβ gene expression.

Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic virus that causes lifelong infection in humans by establishing latency after primary infection. Latent infection is a prerequisite for both persistent infection and the development of KSHV-associated cancers. While viral lytic genes are transiently expressed after primary infection, their expression is significantly restricted and concomitant with the binding of host epigenetic repressors Polycomb Repressive Complex 1 and 2 (PRC1 and PRC2) to lytic genes. PRC1 and PRC2 mediate the repressive histone marks H2AK119ub and H3K27me3, respectively, and maintain heterochromatin structure on KSHV lytic genes to inhibit their expression. In contrast to PRC2, little is known about the recruitment and role of PRC1 factors on the KSHV genome following de novo infection. Thus, the goal of this study was to examine the function of PRC1 factors in the establishment of KSHV latency. To address this question, we performed an shRNA screen targeting 7 different components of the canonical and non-canonical PRC1 complexes during primary KSHV infection. We found that RYBP, a main subunit of the non-canonical PRC1 complexes, is a potent repressor of KSHV lytic genes that can bind to the viral genome and inhibit lytic genes as early as 4 hours post infection. Surprisingly, our ChIP analyses showed that RYBP binds to lytic viral gene promoters in a PRC1-independent manner, does not affect PRC1 activity on the KSHV genome, and can reduce the level of histone marks associated with transcription elongation. Our data also suggest that RYBP can repress the viral lytic cycle after primary infection by inhibiting the transcription elongation of the lytic cycle inducer KSHV gene RTA. Based on our results we propose that RYBP uses a PRC1-independent mechanism to block KSHV RTA expression thereby promoting the establishment of KSHV latency following de novo infection.

The impact of different stress conditions on the oncogenic Kaposi's sarcoma-associated herpesvirus (KSHV) primary infection that can occur in vivo remains largely unknown. We hypothesized that KSHV can establish a latency or lytic cycle following de novo infection, depending on the conditions of the cellular environment. Previous studies showed that hypoxia is a natural stress condition that promotes lytic reactivation and contributes to KSHV pathogenesis, but its effect on de novo KSHV infection is unknown. To test the effect of hypoxia on KSHV infection, we infected cells under normoxia and hypoxia, performed a comparative analysis of viral gene expression and viral replication, and tested chromatinization of the KSHV genome during infection. We found that hypoxia induces viral lytic gene expression and viral replication following de novo infection in several biologically relevant cell types, in which the virus normally establishes latency under normoxia. We also found that hypoxia reduces the level of repressive heterochromatin and promotes the formation of a transcriptionally permissive chromatin on the incoming viral DNA during infection. We demonstrate that silencing hypoxia-inducible factor-1a (HIF-1a) during hypoxia abrogates lytic KSHV infection, while the overexpression of HIF-1a under normoxia is sufficient to drive lytic KSHV infection. Also, we determined that the DNA-binding domain and the N-terminal but not the C-terminal transactivation domain of HIF-1a are required for HIF-1a-induced lytic gene expression. Altogether, our data indicate that HIF-1a accumulation, which can be induced by hypoxia, prevents the establishment of latency and promotes lytic KSHV infection following primary infection. [ABSTRACT FROM AUTHOR]

It is still largely unknown what host factors are involved in controlling the expression of the lytic viral gene RTA during primary Kaposi's sarcoma-associated herpesvirus (KSHV) infection, which determines if KSHV establishes latent or lytic infection. We have recently identified the histone demethylase KDM2B as a repressor of RTA expression during both de novo KSHV infection and latency based on an epigenetic factor short interfering RNA screen. Here, we report that, surprisingly, KDM2B overexpression can promote lytic de novo infection by using a mechanism that differs from what is needed for its repressor function. Our study revealed that while the DNA-binding and demethylase activities of KDM2B linked to its transcription repressive function are dispensable, its C-terminal F-box and LRR domains are required for the lytic infection-inducing function of KDM2B. We found that overexpressed KDM2B increases the half-life of the AP-1 subunit c-Jun protein and induces the AP-1 signaling pathway. This effect depends on the binding of KDM2B to the SKP1-CUL1-F-box (SCF) E3 ubiquitin ligase complex via its F-box domain. Importantly, the inhibition of AP-1 reduces KDM2B-mediated lytic de novo KSHV infection. Overall, our findings indicate that KDM2B overexpression induces the degradation of some host factors by using the SCF complex, resulting in the enrichment of c-Jun. This leads to increased AP-1 transcriptional activity, which facilitates lytic gene expression following de novo infection interfering with the establishment of viral latency. [ABSTRACT FROM AUTHOR]

Kaposi sarcoma‐associated herpesvirus (KSHV) is the causative agent of Kaposi sarcoma and several other tumors and hyperproliferative diseases seen predominantly in human immunodeficiency virus‐infected and other immunocompromised persons. There is an increasing body of evidence showing that hypoxia and hypoxia‐inducible factors (HIFs) play important roles in the biology of KSHV and in the pathogenesis of KSHV‐induced diseases. Hypoxia and HIFs can induce lytic activation of KSHV and KSHV can in turn lead to a hypoxic‐like state in infected cells. In this review, we describe the complex interactions between KSHV biology, the cellular responses to hypoxia, and the pathogenesis of KSHV‐induced diseases. We also describe how interference with HIFs can lead to decreased tumor growth and/or death of infected cells and KSHV‐induced tumors. Finally, we show how these observations may lead to novel strategies for the treatment of KSHV‐induced diseases. [ABSTRACT FROM AUTHOR]

Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic virus that causes lifelong infection in humans by establishing latency after primary infection. Latent infection is a prerequisite for both persistent infection and the development of KSHV-associated cancers. While viral lytic genes are transiently expressed after primary infection, their expression is significantly restricted and concomitant with the binding of host epigenetic repressors Polycomb Repressive Complex 1 and 2 (PRC1 and PRC2) to lytic genes. PRC1 and PRC2 mediate the repressive histone marks H2AK119ub and H3K27me3, respectively, and maintain heterochromatin structure on KSHV lytic genes to inhibit their expression. In contrast to PRC2, little is known about the recruitment and role of PRC1 factors on the KSHV genome following de novo infection. Thus, the goal of this study was to examine the function of PRC1 factors in the establishment of KSHV latency. To address this question, we performed an shRNA screen targeting 7 different components of the canonical and non-canonical PRC1 complexes during primary KSHV infection. We found that RYBP, a main subunit of the non-canonical PRC1 complexes, is a potent repressor of KSHV lytic genes that can bind to the viral genome and inhibit lytic genes as early as 4 hours post infection. Surprisingly, our ChIP analyses showed that RYBP binds to lytic viral gene promoters in a PRC1-independent manner, does not affect PRC1 activity on the KSHV genome, and can reduce the level of histone marks associated with transcription elongation. Our data also suggest that RYBP can repress the viral lytic cycle after primary infection by inhibiting the transcription elongation of the lytic cycle inducer KSHV gene RTA. Based on our results we propose that RYBP uses a PRC1-independent mechanism to block KSHV RTA expression thereby promoting the establishment of KSHV latency following de novo infection. Author summary: It has been shown that cellular epigenetic factors play an important role in regulating the chromatin structure of KSHV DNA by controlling the expression of viral genes following primary infection. The repression of lytic genes during latency is modulated by various inhibitory epigenetic mechanisms, including PRC1- and PRC2-mediated chromatin modifications. In this study, we identified RYBP, a major component of many PRC1 complexes, as a novel repressor of KSHV lytic genes during primary KSHV infection. Previous studies have shown that RYBP regulates the binding and enzymatic activity of PRC1 complexes, yet surprisingly, our data indicates that both the binding and repressive function of RYBP on KSHV lytic genes are PRC1-independent. We found that RYBP can inhibit the transcription elongation of the lytic cycle inducer viral gene RTA during de novo infection, thereby promoting the establishment of KSHV latency. Altogether, our study revealed a novel, non-canonical role of RYBP in the regulation of KSHV gene silencing during de novo viral infection. [ABSTRACT FROM AUTHOR]

The pathophysiological functions of matriptase, a type 2 transmembrane serine protease, rely primarily on its enzymatic activity, which is under tight control through multiple mechanisms. Among those regulatory mechanisms, the control of zymogen activation is arguably the most important. Matriptase zymogen activation not only generates the mature active enzyme but also initiates suppressive mechanisms, such as rapid inhibition by HAI-1, and matriptase shedding. These tightly coupled events allow the potent matriptase tryptic activity to fulfill its biological functions at the same time as limiting undesired hazards. Matriptase is converted to the active enzyme via a process of autoactivation, in which the activational cleavage is thought to rely on the interactions of matriptase zymogen molecules and other as yet identified proteins. Matriptase autoactivation can occur spontaneously and is rapidly followed by the formation and then shedding of matriptase-HAI-1 complexes, resulting in the presence of relatively low levels of the complex on cells. Activation can also be induced by several non-protease factors, such as the exposure of cells to a mildly acidic buffer, which rapidly causes high-level matriptase zymogen activation in almost all cell lines tested. In the current study, the structural requirements for this acid-induced zymogen activation are compared with those required for spontaneous activation through a systematic analysis of the impact of 18 different mutations in various structural domains and motifs on matriptase zymogen activation. Our study reveals that both acid-induced matriptase activation and spontaneous activation depend on the maintenance of the structural integrity of the serine protease domain, non-catalytic domains, and posttranslational modifications. The common requirements of both modes of activation suggest that acid-induced matriptase activation may function as a physiological mechanism to induce pericellular proteolysis by accelerating matriptase autoactivation. [ABSTRACT FROM AUTHOR]

Hepatitis delta virus (HDV) is a satellite of hepatitis B virus that increases the severity of acute and chronic liver disease. HDV produces three processed RNAs that accumulate in infected cells: the circular genome, the circular antigenome, which serves as a replication intermediate, and lesser amounts of the mRNA, which encodes the sole viral protein, hepatitis delta antigen (HDAg). The HDV genome and antigenome RNAs form ribonucleoprotein complexes with HDAg. Although HDAg is required for HDV replication, it is not known how the relative amounts of HDAg and HDV RNA affect replication, nor whether HDAg synthesis is regulated by the virus. Using a novel transfection system in which HDV replication is initiated using in vitro-synthesized circular HDV RNAs, HDV replication was found to depend strongly on the relative amounts of HDV RNA and HDAg. HDV controls these relative amounts via differential effects of HDAg on the production of HDV mRNA and antigenome RNA, both of which are synthesized from the genome RNA template. mRNA synthesis is favored at low HDAg levels but becomes saturated at high HDAg concentrations. Antigenome RNA accumulation increases linearly with HDAg and dominates at high HDAg levels. These results provide a conceptual model for how HDV antigenome RNA production and mRNA transcription are controlled from the earliest stage of infection onwards and also demonstrate that, in this control, HDV behaves similarly to other negative strand RNA viruses, even though there is no genetic similarity between them. [ABSTRACT FROM AUTHOR]