KDM5A/B contribute to HIV-1 latent infection and survival of HIV-1 infected cells.

Combinational antiretroviral therapy (cART) suppresses human immunodeficiency virus type 1 (HIV-1) viral replication and pathogenesis in acquired immunodeficiency syndrome (AIDS) patients. However, HIV-1 remains in the latent stage of infection by suppressing viral transcription, which hinders an HIV-1 cure. One approach for an HIV-1 cure is the "shock and kill" strategy. The strategy focuses on reactivating latent HIV-1, inducing the viral cytopathic effect and facilitating the immune clearance for the elimination of latent HIV-1 reservoirs. Here, we reported that the H3K4 trimethylation (H3K4me3)-specific demethylase KDM5A/B play a role in suppressing HIV-1 Tat/LTR-mediated viral transcription in HIV-1 latent cells. Furthermore, we evaluated the potential of KDM5-specific inhibitor JQKD82 as an HIV-1 "shock and kill" agent. Our results showed that JQKD82 increases the H3K4me3 level at HIV-1 5' LTR promoter regions, HIV-1 reactivation, and the cytopathic effects in an HIV-1-latent T cell model. In addition, we identified that the combination of JQKD82 and AZD5582, a non-canonical NF-κB activator, generates a synergistic impact on inducing HIV-1 lytic reactivation and cell death in the T cell. The latency-reversing potency of the JQKD82 and AZD5582 pair was also confirmed in peripheral blood mononuclear cells (PBMCs) isolated from HIV-1 aviremic patients and in an HIV-1 latent monocyte. In latently infected microglia (HC69) of the brain, either deletion or inhibition of KDM5A/B results in a reversal of the HIV-1 latency. Overall, we concluded that KDM5A/B function as a host repressor of the HIV-1 lytic reactivation and thus promote the latency and the survival of HIV-1 infected reservoirs.

SARS-CoV-2 Nsp14 protein associates with IMPDH2 and activates NF-κB signaling.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection leads to NF-κB activation and induction of pro-inflammatory cytokines, though the underlying mechanism for this activation is not fully understood. Our results reveal that the SARS-CoV-2 Nsp14 protein contributes to the viral activation of NF-κB signaling. Nsp14 caused the nuclear translocation of NF-κB p65. Nsp14 induced the upregulation of IL-6 and IL-8, which also occurred in SARS-CoV-2 infected cells. IL-8 upregulation was further confirmed in lung tissue samples from COVID-19 patients. A previous proteomic screen identified the putative interaction of Nsp14 with host Inosine-5'-monophosphate dehydrogenase 2 (IMPDH2), which is known to regulate NF-κB signaling. We confirmed the Nsp14-IMPDH2 protein interaction and identified that IMPDH2 knockdown or chemical inhibition using ribavirin (RIB) and mycophenolic acid (MPA) abolishes Nsp14- mediated NF-κB activation and cytokine induction. Furthermore, IMPDH2 inhibitors (RIB, MPA) or NF-κB inhibitors (bortezomib, BAY 11-7082) restricted SARS-CoV-2 infection, indicating that IMPDH2-mediated activation of NF-κB signaling is beneficial to viral replication. Overall, our results identify a novel role of SARS-CoV-2 Nsp14 in inducing NF-κB activation through IMPDH2 to promote viral infection.

FACT subunit SUPT16H associates with BRD4 and contributes to silencing of interferon signaling.

FACT (FAcilitates Chromatin Transcription) is a heterodimeric protein complex composed of SUPT16H and SSRP1, and a histone chaperone participating in chromatin remodeling during gene transcription. FACT complex is profoundly regulated, and contributes to both gene activation and suppression. Here we reported that SUPT16H, a subunit of FACT, is acetylated in both epithelial and natural killer (NK) cells. The histone acetyltransferase TIP60 contributes to the acetylation of SUPT16H middle domain (MD) at lysine 674 (K674). Such acetylation of SUPT16H is recognized by bromodomain protein BRD4, which promotes protein stability of SUPT16H in both epithelial and NK cells. We further demonstrated that SUPT16H-BRD4 associates with histone modification enzymes (HDAC1, EZH2), and further regulates their activation status and/or promoter association as well as affects the relevant histone marks (H3ac, H3K9me3 and H3K27me3). BRD4 is known to profoundly regulate interferon (IFN) signaling, while such function of SUPT16H has never been explored. Surprisingly, our results revealed that SUPT16H genetic knockdown via RNAi or pharmacological inhibition by using its inhibitor, curaxin 137 (CBL0137), results in the induction of IFNs and interferon-stimulated genes (ISGs). Through this mechanism, depletion or inhibition of SUPT16H is shown to efficiently inhibit infection of multiple viruses, including Zika, influenza, and SARS-CoV-2. Furthermore, we demonstrated that depletion or inhibition of SUPT16H also causes the remarkable activation of IFN signaling in NK cells, which promotes the NK-mediated killing of virus-infected cells in a co-culture system using human primary NK cells. Overall, our studies unraveled the previously un-appreciated role of FACT complex in coordinating with BRD4 and regulating IFN signaling in both epithelial and NK cells, and also proposed the novel application of the FACT inhibitor CBL0137 to treat viral infections.

Polyamine biosynthesis and eIF5A hypusination are modulated by the DNA tumor virus KSHV and promote KSHV viral infection.

Polyamines are critical metabolites involved in various cellular processes and often dysregulated in cancers. Kaposi's sarcoma-associated Herpesvirus (KSHV), a defined human oncogenic virus, leads to profound alterations of host metabolic landscape to favor development of KSHV-associated malignancies. In our studies, we identified that polyamine biosynthesis and eIF5A hypusination are dynamically regulated by KSHV infection through modulation of key enzymes (ODC1 and DHPS) of these pathways. During KSHV latency, ODC1 and DHPS are upregulated along with increase of hypusinated eIF5A (hyp-eIF5A), while hyp-eIF5A is further induced along with reduction of ODC1 and intracellular polyamines during KSHV lytic reactivation. In return these metabolic pathways are required for both KSHV lytic reactivation and de novo infection. Further analysis unraveled that synthesis of critical KSHV latent and lytic proteins (LANA, RTA) depends on hypusinated-eIF5A. We also demonstrated that KSHV infection can be efficiently and specifically suppressed by inhibitors targeting these pathways. Collectively, our results illustrated that the dynamic and profound interaction of a DNA tumor virus (KSHV) with host polyamine biosynthesis and eIF5A hypusination pathways promote viral propagation, thus defining new therapeutic targets to treat KSHV-associated malignancies.

Inhibition of polo-like kinase 1 (PLK1) facilitates reactivation of gamma-herpesviruses and their elimination.

Both Kaposi's sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV) establish the persistent, life-long infection primarily at the latent status, and associate with certain types of tumors, such as B cell lymphomas, especially in immuno-compromised individuals including people living with HIV (PLWH). Lytic reactivation of these viruses can be employed to kill tumor cells harboring latently infected viral episomes through the viral cytopathic effects and the subsequent antiviral immune responses. In this study, we identified that polo-like kinase 1 (PLK1) is induced by KSHV de novo infection as well as lytic switch from KSHV latency. We further demonstrated that PLK1 depletion or inhibition facilitates KSHV reactivation and promotes cell death of KSHV-infected lymphoma cells. Mechanistically, PLK1 regulates Myc that is critical to both maintenance of KSHV latency and support of cell survival, and preferentially affects the level of H3K27me3 inactive mark both globally and at certain loci of KSHV viral episomes. Furthremore, we recognized that PLK1 inhibition synergizes with STAT3 inhibition to efficiently induce KSHV reactivation. We also confirmed that PLK1 depletion or inhibition yields the similar effect on EBV lytic reactivation and cell death of EBV-infected lymphoma cells. Lastly, we noticed that PLK1 in B cells is elevated in the context of HIV infection and caused by HIV Nef protein to favor KSHV/EBV latency.

SARS-CoV-2 Nsp14 activates NF-κB signaling and induces IL-8 upregulation.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection leads to NF-κB activation and induction of pro-inflammatory cytokines, though the underlying mechanism for this activation is not fully understood. Our results reveal that the SARS-CoV-2 Nsp14 protein contributes to the viral activation of NF-κB signaling. Nsp14 caused the nuclear translocation of NF-κB p65. Nsp14 induced the upregulation of IL-6 and IL-8, which also occurred in SARS-CoV-2 infected cells. IL-8 upregulation was further confirmed in lung tissue samples from COVID-19 patients. A previous proteomic screen identified the putative interaction of Nsp14 with host Inosine-5'-monophosphate dehydrogenase 2 (IMPDH2) protein, which is known to regulate NF-κB signaling. We confirmed the Nsp14-IMPDH2 protein interaction and found that IMPDH2 knockdown or chemical inhibition using ribavirin (RIB) and mycophenolic acid (MPA) abolishes Nsp14-mediated NF-κB activation and cytokine induction. Furthermore, IMDPH2 inhibitors (RIB, MPA) efficiently blocked SARS-CoV-2 infection, indicating that IMDPH2, and possibly NF-κB signaling, is beneficial to viral replication. Overall, our results identify a novel role of SARS-CoV-2 Nsp14 in causing the activation of NF-κB.

FACT subunit SUPT16H associates with BRD4 and contributes to silencing of antiviral interferon signaling.

FACT ( FA cilitates C hromatin T ranscription) is a heterodimeric protein complex composed of SUPT16H and SSRP1, and a histone chaperone participating in chromatin remodeling during gene transcription. FACT complex is profoundly regulated, and contributes to both gene activation and suppression. Here we reported that SUPT16H, a subunit of FACT, is acetylated at lysine 674 (K674) of middle domain (MD), which involves TIP60 histone acetyltransferase. Such acetylation of SUPT16H is recognized by bromodomain protein BRD4, which promotes protein stability of SUPT16H. We further demonstrated that SUPT16H-BRD4 associates with histone modification enzymes (EZH2, HDAC1) and affects histone marks (H3K9me3, H3K27me3 and H3ac). BRD4 is known to profoundly regulate interferon (IFN) signaling, while such function of SUPT16H has never been explored. Surprisingly, our results revealed that SUPT16H genetic knockdown via RNAi or pharmacological inhibition by using its inhibitor, curaxin 137 (CBL0137), results in the induction of IFNs and interferon-stimulated genes (ISGs). Through this mechanism, CBL0137 is shown to efficiently inhibit infection of multiple viruses, including Zika, influenza, and SARS-CoV-2. Furthermore, we demonstrated that CBL0137 also causes the remarkable activation of IFN signaling in natural killer (NK) cells, which promotes the NK-mediated killing of virus-infected cells in a co-culture system using human primary NK cells. Overall, our studies unraveled the previously un-appreciated role of FACT complex in regulating IFN signaling in both epithelial and NK cells, and also proposed the novel application of CBL0137 to treat viral infections.

Profiling of immune related genes silenced in EBV-positive gastric carcinoma identified novel restriction factors of human gammaherpesviruses.

EBV-associated gastric cancer (EBVaGC) is characterized by high frequency of DNA methylation. In this study, we investigated how epigenetic alteration of host genome contributes to pathogenesis of EBVaGC through the analysis of transcriptomic and epigenomic datasets from NIH TCGA (The Cancer Genome Atlas) consortium. We identified that immune related genes (IRGs) is a group of host genes preferentially silenced in EBV-positive gastric cancers through DNA hypermethylation. Further functional characterizations of selected IRGs reveal their novel antiviral activity against not only EBV but also KSHV. In particular, we showed that metallothionein-1 (MT1) and homeobox A (HOXA) gene clusters are down-regulated via EBV-driven DNA hypermethylation. Several MT1 isoforms suppress EBV lytic replication and release of progeny virions as well as KSHV lytic reactivation, suggesting functional redundancy of these genes. In addition, single HOXA10 isoform exerts antiviral activity against both EBV and KSHV. We also confirmed the antiviral effect of other dysregulated IRGs, such as IRAK2 and MAL, in scenario of EBV and KSHV lytic reactivation. Collectively, our results demonstrated that epigenetic silencing of IRGs is a viral strategy to escape immune surveillance and promote viral propagation, which is overall beneficial to viral oncogenesis of human gamma-herpesviruses (EBV and KSHV), considering that these IRGs possess antiviral activities against these oncoviruses.

HCV RNA traffic and association with NS5A in living cells.

The spatiotemporal dynamics of Hepatitis C Virus (HCV) RNA localisation are poorly understood. To address this we engineered HCV genomes harbouring MS2 bacteriophage RNA stem-loops within the 3'-untranslated region to allow tracking of HCV RNA via specific interaction with a MS2-Coat-mCherry fusion protein. Despite the impact of these insertions on viral fitness, live imaging revealed that replication of tagged-HCV genomes induced specific redistribution of the mCherry-tagged-MS2-Coat protein to motile and static foci. Further analysis showed that HCV RNA was associated with NS5A in both static and motile structures while a subset of motile NS5A structures was devoid of HCV RNA. Further investigation of viral RNA traffic with respect to lipid droplets (LDs) revealed HCV RNA-positive structures in close association with LDs. These studies provide new insights into the dynamics of HCV RNA traffic with NS5A and LDs and provide a platform for future investigations of HCV replication and assembly.

Dynamic imaging of the hepatitis C virus NS5A protein during a productive infection.

UNLABELLED: Hepatitis C virus (HCV) NS5A is essential for viral genome replication within cytoplasmic replication complexes and virus assembly at the lipid droplet (LD) surface, although its definitive functions are poorly understood. We developed approaches to investigate NS5A dynamics during a productive infection. We report here that NS5A motility and efficient HCV RNA replication require the microtubule network and the cytoplasmic motor dynein and demonstrate that both motile and relatively static NS5A-positive foci are enriched with host factors VAP-A and Rab5A. Pulse-chase imaging revealed that newly synthesized NS5A foci are small and distinct from aged foci, while further studies using a unique dual fluorescently tagged infectious HCV chimera showed a relatively stable association of NS5A foci with core-capped LDs. These results reveal new details about the dynamics and maturation of NS5A and the nature of potential sites of convergence of HCV replication and assembly pathways. IMPORTANCE: Hepatitis C virus (HCV) is a major cause of serious liver disease worldwide. An improved understanding of the HCV replication cycle will enable development of novel and improved antiviral strategies. Here we have developed complementary fluorescent labeling and imaging approaches to investigate the localization, traffic and interactions of the HCV NS5A protein in living, virus-producing cells. These studies reveal new details as to the traffic, composition and biogenesis of NS5A foci and the nature of their association with putative sites of virus assembly.

Viperin is induced following dengue virus type-2 (DENV-2) infection and has anti-viral actions requiring the C-terminal end of viperin.

The host protein viperin is an interferon stimulated gene (ISG) that is up-regulated during a number of viral infections. In this study we have shown that dengue virus type-2 (DENV-2) infection significantly induced viperin, co-incident with production of viral RNA and via a mechanism requiring retinoic acid-inducible gene I (RIG-I). Viperin did not inhibit DENV-2 entry but DENV-2 RNA and infectious virus release was inhibited in viperin expressing cells. Conversely, DENV-2 replicated to higher tires earlier in viperin shRNA expressing cells. The anti-DENV effect of viperin was mediated by residues within the C-terminal 17 amino acids of viperin and did not require the N-terminal residues, including the helix domain, leucine zipper and S-adenosylmethionine (SAM) motifs known to be involved in viperin intracellular membrane association. Viperin showed co-localisation with lipid droplet markers, and was co-localised and interacted with DENV-2 capsid (CA), NS3 and viral RNA. The ability of viperin to interact with DENV-2 NS3 was associated with its anti-viral activity, while co-localisation of viperin with lipid droplets was not. Thus, DENV-2 infection induces viperin which has anti-viral properties residing in the C-terminal region of the protein that act to restrict early DENV-2 RNA production/accumulation, potentially via interaction of viperin with DENV-2 NS3 and replication complexes. These anti-DENV-2 actions of viperin show both contrasts and similarities with other described anti-viral mechanisms of viperin action and highlight the diverse nature of this unique anti-viral host protein.