SARS-CoV-2 Nsp8 suppresses MDA5 antiviral immune responses by impairing TRIM4-mediated K63-linked polyubiquitination.

Melanoma differentiation-associated gene-5 (MDA5) acts as a cytoplasmic RNA sensor to detect viral dsRNA and mediates antiviral innate immune responses to infection by RNA viruses. Upon recognition of viral dsRNA, MDA5 is activated with K63-linked polyubiquitination and then triggers the recruitment of MAVS and activation of TBK1 and IKKα/ß, subsequently leading to IRF3 and NF-κB phosphorylation. However, the specific E3 ubiquitin ligase for MDA5 K63-polyubiquitination has not been well characterized. Great numbers of symptomatic and severe infections of SARS-CoV-2 are spreading worldwide, and the poor efficacy of treatment with type I interferon and antiviral immune agents indicates that SARS-CoV-2 escapes from antiviral immune responses via several unknown mechanisms. Here, we report that SARS-CoV-2 nonstructural protein 8 (nsp8) acts as a suppressor of antiviral innate immune and inflammatory responses to promote infection of SARS-CoV-2. It downregulates the expression of type I interferon, IFN-stimulated genes and proinflammatory cytokines by binding to MDA5 and TRIM4 and impairing TRIM4-mediated MDA5 K63-linked polyubiquitination. Our findings reveal that nsp8 mediates innate immune evasion during SARS-CoV-2 infection and may serve as a potential target for future therapeutics for SARS-CoV-2 infectious diseases.

Downregulation of EphA2 stability by RNF5 limits its tumor-suppressive function in HER2-negative breast cancers.

Ephrin receptor A2 (EphA2) plays dual functions in tumorigenesis through ligand-independent tumor promotion or ligand-dependent tumor suppression. However, the regulation of EphA2 tumor-suppressive function remains unclear. Here, we showed that RNF5 interacts with EphA2 and induces its ubiquitination and degradation, decreases the stability and cell surface distribution of EphA2 and alters the balance of its phosphorylation at S897 and Y772. In turn, RNF5 inhibition decreases ERK phosphorylation and increases p53 expression through an increase in the EphA2 level in HER2-negative breast cancer cells. Consequently, RNF5 inhibition increases the adhesion and decreases the migration of HER2-negative breast cancer cells, and RNF5 silencing suppresses the growth of xenograft tumors derived from ER-positive, HER2-negative breast cancer cells with increased EphA2 expression and altered phosphorylation. RNF5 expression is inversely correlated with EphA2 expression in breast cancers, and a high EphA2 level accompanied by a low RNF5 level is related to better survival in patients with ER-positive, HER2-negative breast cancers. These studies revealed that RNF5 negatively regulates EphA2 properties and suppresses its tumor-suppressive function in HER2-negative breast cancers.

Inhibition of AIM2 inflammasome activation by SOX/ORF37 promotes lytic replication of Kaposi's sarcoma-associated herpesvirus.

Inflammasomes are one kind of important innate immune defense against viral and bacterial infections. Several inflammasome-forming sensors detect molecular patterns of invading pathogens and then trigger inflammasome activation and/or pyroptosis in infected cells, and viruses employ unique strategies to hijack or subvert inflammasome activation. Infection with herpesviruses induces the activation of diverse inflammasomes, including AIM2 and IFI16 inflammasomes; however, how Kaposi's sarcoma-associated herpesvirus (KSHV) counteracts inflammasome activation largely remains unclear. Here, we reveal that the KSHV ORF37-encoded SOX protein suppresses AIM2 inflammasome activation independent of its viral DNA exonuclease activity and host mRNA turnover. SOX interacts with the AIM2 HIN domain through the C-terminal Motif VII region and disrupts AIM2:dsDNA polymerization and ASC recruitment and oligomerization. The Y443A or F444A mutation of SOX abolishes the inhibition of AIM2 inflammasome without disrupting SOX nuclease activity, and a short SOX peptide is capable of inhibiting AIM2 inflammasome activation; consequently, infection with SOX-null, Y443A, or F444A Bac16 recombinant viruses results in robust inflammasome activation, suppressed lytic replication, and increased pyroptosis in human lymphatic endothelial cells in an AIM2-dependent manner. These results reveal that KSHV SOX suppresses AIM2 inflammasome activation to promote KSHV lytic replication and inhibit pyroptosis, representing a unique mechanism for evasion of inflammasome activation during KSHV lytic cycle.

Suppression of KSHV lytic replication and primary effusion lymphoma by selective RNF5 inhibition.

Primary effusion lymphoma (PEL), a rare aggressive B-cell lymphoma in immunosuppressed patients, is etiologically associated with oncogenic γ-herpesvirus infection. Chemotherapy is commonly used to treat PEL but usually results in poor prognosis and survival; thus, novel therapies and drug development are urgently needed for PEL treatment. Here, we demonstrated that inhibition of Ring finger protein 5 (RNF5), an ER-localized E3 ligase, suppresses multiple cellular pathways and lytic replication of Kaposi sarcoma-associated herpesvirus (KSHV) in PEL cells. RNF5 interacts with and induces Ephrin receptors A3 (EphA3) and EphA4 ubiquitination and degradation. RNF5 inhibition increases the levels of EphA3 and EphA4, thereby reducing ERK and Akt activation and KSHV lytic replication. RNF5 inhibition decreased PEL xenograft tumor growth and downregulated viral gene expression, cell cycle gene expression, and hedgehog signaling in xenograft tumors. Our study suggests that RNF5 plays the critical roles in KSHV lytic infection and tumorigenesis of primary effusion lymphoma.

Upregulation of ATF4-LAMP3 Axis by ORF45 Facilitates Lytic Replication of Kaposi's Sarcoma-Associated Herpesvirus.

Kaposi's sarcoma-associated herpesvirus (KSHV) is a γ-oncogenic herpesvirus, and both lytic and latent infections play important roles in its pathogenesis and tumorigenic properties. Multiple cellular pathways and diverse mediators are hijacked by viral proteins and are used to support KSHV lytic replication. In previous studies, we revealed that KSHV ORF45 promoted KSHV transcription and translation by inducing sustained p90 ribosomal S6 kinase (RSK) activation and the phosphorylation of its substrates c-Fos and eIF4B. However, the cellular mediators required for lytic replication remain largely unknown. Here, we reveal that ORF45 activates eIF2α phosphorylation and ATF4 translation and then upregulates the expression of lysosome-associated membrane protein 3 (LAMP3) in an ATF4-dependent manner during KSHV lytic replication. Consequently, LAMP3 promotes Akt and ERK activation and then facilitates lytic gene expression and virion production. Furthermore, ATF4 enhances lytic replication through LAMP3, and LAMP3 acts in an ATF4-independent manner. Our findings suggest that the ATF4-LAMP3 axis is upregulated by ORF45 through ER stress activation during the KSHV lytic life cycle and, in turn, facilitates optimal lytic replication. IMPORTANCE The lytic replication of Kaposi's sarcoma-associated herpesvirus (KSHV) reprograms cellular transcription and translation to generate viral proteins and virion particles. Here, we show that the mediator of ER stress ATF4 and the expression of the downstream gene LAMP3 are upregulated by ORF45 during lytic replication. Consequently, increased LAMP3 expression activates Akt and ERK and promotes lytic replication. Although several UPR transcription factors are able to promote KSHV lytic replication, the proviral effect of ATF4 on lytic replication is attenuated by LAMP3 silencing, whereas the effect of LAMP3 does not directly require ATF4 expression, indicating that LAMP3 primarily exerts effects on KSHV lytic replication downstream of ATF4 and ER stress. Taken together, our findings suggest that the ORF45-upregulated ATF4-LAMP3 axis plays an essential role in KSHV lytic replication.

A non-catalytic herpesviral protein reconfigures ERK-RSK signaling by targeting kinase docking systems in the host.

The Kaposi's sarcoma associated herpesvirus protein ORF45 binds the extracellular signal-regulated kinase (ERK) and the p90 Ribosomal S6 kinase (RSK). ORF45 was shown to be a kinase activator in cells but a kinase inhibitor in vitro, and its effects on the ERK-RSK complex are unknown. Here, we demonstrate that ORF45 binds ERK and RSK using optimized linear binding motifs. The crystal structure of the ORF45-ERK2 complex shows how kinase docking motifs recognize the activated form of ERK. The crystal structure of the ORF45-RSK2 complex reveals an AGC kinase docking system, for which we provide evidence that it is functional in the host. We find that ORF45 manipulates ERK-RSK signaling by favoring the formation of a complex, in which activated kinases are better protected from phosphatases and docking motif-independent RSK substrate phosphorylation is selectively up-regulated. As such, our data suggest that ORF45 interferes with the natural design of kinase docking systems in the host.

RTN3 inhibits RIG-I-mediated antiviral responses by impairing TRIM25-mediated K63-linked polyubiquitination.

Upon viral RNA recognition, the RIG-I signalosome continuously generates IFNs and cytokines, leading to neutrophil recruitment and inflammation. Thus, attenuation of excessive immune and inflammatory responses is crucial to restore immune homeostasis and prevent unwarranted damage, yet few resolving mediators have been identified. In the present study, we demonstrated that RTN3 is strongly upregulated during RNA viral infection and acts as an inflammation-resolving regulator. Increased RTN3 aggregates on the endoplasmic reticulum and interacts with both TRIM25 and RIG-I, subsequently impairing K63-linked polyubiquitination and resulting in both IRF3 and NF-κB inhibition. Rtn3 overexpression in mice causes an obvious inflammation resolving phenomenon when challenged with VSV, Rtn3-overexpressing mice display significantly decreased neutrophil numbers and inflammatory cell infiltration, which is accompanied by reduced tissue edema in the liver and thinner alveolar interstitium. Taken together, our findings identify RTN3 as a conserved negative regulator of immune and inflammatory responses and provide insights into the negative feedback that maintains immune and inflammatory homeostasis.

BRLF1-dependent viral and cellular transcriptomes and transcriptional regulation during EBV primary infection in B lymphoma cells.

The immediate-early protein BRLF1 plays important roles in lytic infection of Epstein-Barr virus (EBV), in which it activates lytic viral transcription and replication. However, knowledge of the influence of BRLF1 on cellular gene expression and transcriptional reprogramming during the early lytic cycle remains limited. In the present study, deep RNA-sequencing analysis identified all differentially expressed genes (DEGs) and alternative splicing in B lymphoma cells subjected to wild-type and BRLF1-deficient EBV primary infection. The BRLF1-dependent cellular DEGs were annotated, and major differentially enriched pathways were related to DNA replication and transcription, immune and inflammatory responses, cytokine-receptor interactions and chemokine signaling and metabolic processes. Furthermore, analysis of BRLF1-binding proteins by mass spectrometry shows that BRLF1 binds to and cooperates with several transcription factors and components of the spliceosome and then influences both RNA polymerase II-dependent transcription and pre-mRNA splicing. The RTA-binding RRE motifs or specific motifs of unique cooperative transcription factors in viral and cellular DEG promoter regions indicate that BRLF1 employs different strategies for regulating viral and cellular transcription. Thus, our study characterized BRLF1-dependent cellular and viral transcriptional profile during primary infection and then revealed the comprehensive virus-cell interaction and alterations of transcription during EBV primary infection and lytic replication.

BRLF1 suppresses RNA Pol III-mediated RIG-I inflammasome activation in the early EBV lytic lifecycle.

Latent infection with herpesviruses constitutively activates inflammasomes, while lytic replication suppresses their activation through distinct mechanisms. However, how Epstein-Barr virus (EBV) lytic replication inhibits the activation of inflammasomes remains unknown. Here, we reveal that the EBV immediate-early protein BRLF1 inhibits inflammasome activation, and BRLF1 deficiency significantly increases the activation of inflammasomes and pyroptosis during early lytic lifecycle. BRLF1 interacts with RNA polymerase III subunits to suppress immunostimulatory small RNA transcription, RIG-I inflammasome activation, and antiviral responses. Consequently, BRLF1-deficient EBV primary infection induces robust T-cell and NK cell activation and killing through IL-1ß and IL-18. A BRLF1-derived peptide that inhibits inflammasome activation is sufficient to suppress T-cell and NK cell responses during BRLF1-deficient EBV primary infection in lymphocytes. These results reveal a novel mechanism involved in the evasion of inflammasome activation and antiviral responses during EBV early lytic infection and provide a promising approach for the manipulation of inflammasomes against infection of oncogenic herpesviruses.

Development of an ORF45-Derived Peptide To Inhibit the Sustained RSK Activation and Lytic Replication of Kaposi's Sarcoma-Associated Herpesvirus.

The lytic replication of Kaposi's sarcoma-associated herpesvirus (KSHV) requires sustained extracellular signal-regulated kinase (ERK)-p90 ribosomal S6 kinase (RSK) activation, which is induced by an immediate early (IE) gene-encoded tegument protein called ORF45, to promote the late transcription and translation of viral lytic genes. An ORF45-null or single-point F66A mutation in ORF45 abolishes ORF45-RSK interaction and sustained ERK-RSK activation during lytic reactivation and subsequently results in a significant decrease in late lytic gene expression and virion production, indicating that ORF45-mediated RSK activation plays a critical role in KSHV lytic replication. Here, we demonstrate that a short ORF45-derived peptide in the RSK-binding region is sufficient for disrupting ORF45-RSK interaction, consequently suppressing lytic gene expression and virion production. We designed a nontoxic cell-permeable peptide derived from ORF45, TAT-10F10, which is composed of the ORF45 56 to 76 amino acid (aa) region and the HIV Tat protein transduction domain, and this peptide markedly inhibits KSHV lytic replication in iSLK.219 and BCBL1 cells. Importantly, this peptide enhances the inhibitory effect of rapamycin on KSHV-infected cells and decreases spontaneous and hypoxia-induced lytic replication in KSHV-positive lymphoma cells. These findings suggest that a small peptide that disrupts ORF45-RSK interaction might be a promising agent for controlling KSHV lytic infection and pathogenesis.IMPORTANCE ORF45-induced RSK activation plays an essential role in KSHV lytic replication, and ORF45-null or ORF45 F66A mutagenesis that abolishes sustained RSK activation and RSK inhibitors significantly decreases lytic replication, indicating that the ORF45-RSK association is a unique target for KSHV-related diseases. However, the side effects, low affinity, and poor efficacy of RSK modulators limit their clinical application. In this study, we developed a nontoxic cell-permeable ORF45-derived peptide from the RSK-binding region to disrupt ORF45-RSK associations and block ORF45-induced RSK activation without interfering with S6K1 activation. This peptide effectively suppresses spontaneous, hypoxia-induced, or chemically induced KSHV lytic replication and enhances the inhibitory effect of rapamycin on lytic replication and sensitivity to rapamycin in lytic KSHV-infected cells. Our results reveal that the ORF45-RSK signaling axis and KSHV lytic replication can be effectively targeted by a short peptide and provide a specific approach for treating KSHV lytic and persistent infection.

Niclosamide inhibits lytic replication of Epstein-Barr virus by disrupting mTOR activation.

Infection with the oncogenic γ-herpesviruses Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV) cause several severe malignancies in humans. Inhibition of the lytic replication of EBV and KSHV eliminates the reservoir of persistent infection and transmission, consequently preventing the occurrence of diseases from the sources of infection. Antiviral drugs are limited in controlling these viral infectious diseases. Here, we demonstrate that niclosamide, an old anthelmintic drug, inhibits mTOR activation during EBV lytic replication. Consequently, niclosamide effectively suppresses EBV lytic gene expression, viral DNA lytic replication and virion production in EBV-infected lymphoma cells and epithelial cells. Niclosamide exhibits cytotoxicity toward lymphoma cells and induces irreversible cell cycle arrest in lytically EBV-infected cells. The ectopic overexpression of mTOR reverses the inhibition of niclosamide in EBV lytic replication. Similarly, niclosamide inhibits KSHV lytic replication. Thus, we conclude that niclosamide is a promising candidate for chemotherapy against the acute occurrence and transmission of infectious diseases of oncogenic γ-herpesviruses.

Chloroquine inhibits lytic replication of Kaposi's sarcoma-associated herpesvirus by disrupting mTOR and p38-MAPK activation.

Lytic infection is essential for the persistent infection and pathogenesis of Kaposi's sarcoma-associated herpesvirus (KSHV), and inhibiting KSHV lytic replication may effectively prevent the occurrence of KSHV-related diseases. Chloroquine (CQ), a well-known antimalarial drug and autophagy inhibitor, exerts broad-spectrum antiviral effects and shows anti-cancer therapeutic potential. However, the ability of CQ and its derivatives to control infection of oncogenic γ-herpesvirus remains undefined. Here we reveal that CQ suppresses KSHV lytic gene expression and virion production, and shows cytotoxicity toward KSHV lytically infected B cells at clinically acceptable doses. CQ suppresses mTOR and p38-MAPK pathway activation during KSHV lytic replication but not latent infection. Furthermore, CQ blocks Epstein-Barr virus (EBV) lytic replication via a distinct mechanism that is invoked to block virion production but does not affect viral gene expression. These results suggest that CQ is an effective antiviral drug against KSHV lytic infection. Our findings indicate that CQ treatment should be considered for controlling KSHV-related diseases, particularly for primary use in co-infection of KSHV with malaria.

BZLF1 Attenuates Transmission of Inflammatory Paracrine Senescence in Epstein-Barr Virus-Infected Cells by Downregulating Tumor Necrosis Factor Alpha.

UNLABELLED: Recent studies have shown that inflammatory responses trigger and transmit senescence to neighboring cells and activate the senescence-associated secretory phenotype (SASP). Latent Epstein-Barr virus (EBV) infection induces increased secretion of several inflammatory factors, whereas lytic infections evade the antiviral inflammatory response. However, the changes in and roles of the inflammatory microenvironment during the switch between EBV life cycles remain unknown. In the present study, we demonstrate that latent EBV infection in EBV-positive cells triggers the SASP in neighboring epithelial cells. In contrast, lytic EBV infection abolishes this phenotype. BZLF1 attenuates the transmission of paracrine senescence during lytic EBV infection by downregulating tumor necrosis factor alpha (TNF-α) secretion. A mutant BZLF1 protein, BZLF1Δ207-210, that cannot inhibit TNF-α secretion while maintaining viral transcription, fails to block paracrine senescence, whereas a neutralizing antibody against TNF-α is sufficient to restore its inhibition. Furthermore, latent EBV infection induces oxidative stress in neighboring cells, while BZLF1-mediated downregulation of TNF-α reduces reactive oxygen species (ROS) levels in neighboring cells, and ROS scavengers alleviate paracrine senescence. These results suggest that lytic EBV infection attenuates the transmission of inflammatory paracrine senescence through BZLF1 downregulation of TNF-α secretion and alters the inflammatory microenvironment to allow virus propagation and persistence. IMPORTANCE: The senescence-associated secretory phenotype (SASP), an important tumorigenic process, is triggered and transmitted by inflammatory factors. The different life cycles of Epstein-Barr virus (EBV) infection in EBV-positive cells employ distinct strategies to modulate the inflammatory response and senescence. The elevation of inflammatory factors during latent EBV infection promotes the SASP in uninfected cells. In contrast, during the viral lytic cycle, BZLF1 suppresses the production of TNF-α, resulting in the attenuation of paracrine inflammatory senescence. This finding indicates that EBV evades inflammatory senescence during lytic infection and switches from facilitating tumor-promoting SASP to generating a virus-propagating microenvironment, thereby facilitating viral spread in EBV-associated diseases.

Epstein-Barr Virus BZLF1-Mediated Downregulation of Proinflammatory Factors Is Essential for Optimal Lytic Viral Replication.

UNLABELLED: Elevated secretion of inflammatory factors is associated with latent Epstein-Barr virus (EBV) infection and the pathology of EBV-associated diseases; however, knowledge of the inflammatory response and its biological significance during the lytic EBV cycle remains elusive. Here, we demonstrate that the immediate early transcriptional activator BZLF1 suppresses the proinflammatory factor tumor necrosis factor alpha (TNF-α) by binding to the promoter of TNF-α and preventing NF-κB activation. A BZLF1Δ207-210 mutant with a deletion of 4 amino acids (aa) in the protein-protein binding domain was not able to inhibit the proinflammatory factors TNF-α and gamma interferon (IFN-γ) and reduced viral DNA replication with complete transcriptional activity during EBV lytic gene expression. TNF-α depletion restored the viral replication mediated by BZLF1Δ207-210. Furthermore, a combination of TNF-α- and IFN-γ-neutralizing antibodies recovered BZLF1Δ207-210-mediated viral replication, indicating that BZLF1 attenuates the antiviral response to aid optimal lytic replication primarily through the inhibition of TNF-α and IFN-γ secretion during the lytic cycle. These results suggest that EBV BZLF1 attenuates the proinflammatory responses to facilitate viral replication. IMPORTANCE: The proinflammatory response is an antiviral and anticancer strategy following the complex inflammatory phenotype. Latent Epstein-Barr virus (EBV) infection strongly correlates with an elevated secretion of inflammatory factors in a variety of severe diseases, while the inflammatory responses during the lytic EBV cycle have not been established. Here, we demonstrate that BZLF1 acts as a transcriptional suppressor of the inflammatory factors TNF-α and IFN-γ and confirm that BZLF1-facilitated escape from the TNF-α and IFN-γ response during the EBV lytic life cycle is required for optimal viral replication. This finding implies that the EBV lytic cycle employs a distinct strategy to evade the antiviral inflammatory response.

ORF45-Mediated Prolonged c-Fos Accumulation Accelerates Viral Transcription during the Late Stage of Lytic Replication of Kaposi's Sarcoma-Associated Herpesvirus.

UNLABELLED: Kaposi's sarcoma-associated herpesvirus (KSHV) encodes multiple viral proteins that activate extracellular signal-regulated kinase (ERK)-mitogen-activated protein kinase (MAPK) cascades. One of these viral proteins, ORF45, mediates sustained ERK-p90 ribosomal S6 kinase (RSK) activation during KSHV lytic replication and facilitates viral translation through the phosphorylation of a eukaryotic translation initiation factor, eIF4B. The importance of ERK-RSK activation for KSHV viral transcription has been shown; however, which transcription factor senses the sustained MAPK signaling and leads to viral transcription remains poorly understood. Here we show that the presence of ORF45 leads to the prolonged accumulation of c-Fos during the late stage of KSHV lytic replication through ERK-RSK-dependent phosphorylation and stabilization and that the depletion of c-Fos disrupts viral lytic transcription. Genome-wide screening revealed that c-Fos directly binds to multiple viral gene promoters and enhances viral transcription. Mutation of the ERK-RSK phosphorylation sites of c-Fos restrains KSHV lytic gene expression and virion production. These results indicate that the prolonged accumulation of c-Fos promotes the progression of viral transcription from early to late stages and accelerates viral lytic replication upon sustained ORF45-ERK-RSK activation during the KSHV lytic life cycle. IMPORTANCE: During KSHV lytic replication, transient activation and sustained activation of ERK-RSK induce viral immediate early (IE) transcription and late transcription, respectively. Studies have revealed that ERK-RSK activates several transcription factors involved in IE gene expression, including Ets, AP-1, CREB, and C/EBP, which lead to the transient ERK-RSK activation-dependent IE transcription. Whereas c-Fos acts as a sensor of sustained ERK-RSK activation, ORF45-ERK-RSK signaling mediates c-Fos phosphorylation and accumulation during late KSHV lytic replication, consequently promoting viral transcription through the direct binding of c-Fos to multiple KSHV promoters. This finding indicates that c-Fos mediates distinct viral transcriptional progression following sustained ERK-RSK signaling during the KSHV lytic life cycle.

Activation of p90 ribosomal S6 kinases by ORF45 of Kaposi's sarcoma-associated herpesvirus is critical for optimal production of infectious viruses.

UNLABELLED: We have previously shown that ORF45, an immediate-early and tegument protein of Kaposi's sarcoma-associated herpesvirus (KSHV), causes sustained activation of p90 ribosomal S6 kinases (RSKs) and extracellular regulated kinase (ERK) (E. Kuang, Q. Tang, G. G. Maul, and F. Zhu, J Virol 82:1838-1850, 2008, http://dx.doi.org/10.1128/JVI.02119-07). We now have identified the critical region of ORF45 that is involved in RSK interaction and activation. Alanine scanning mutagenesis of this region revealed that a single F66A point mutation abolished binding of ORF45 to RSK or ERK and, consequently, its ability to activate the kinases. We introduced the F66A mutation into BAC16 (a bacterial artificial chromosome clone containing the entire infectious KSHV genome), producing BAC16-45F66A. In parallel, we also repaired the mutation and obtained a revertant, BAC16-45A66F. The reconstitution of these mutants in iSLK cells demonstrated that the ORF45-F66A mutant failed to cause sustained ERK and RSK activation during lytic reactivation, resulting in dramatic differences in the phosphoproteomic profile between the wild-type virus-infected cells and the mutant virus-infected cells. ORF45 mutation or deletion also was accompanied by a noticeable decreased in viral gene expression during lytic reactivation. Consequently, the ORF45-F66A mutant produced significantly fewer infectious progeny virions than the wild type or the revertant. These results suggest a critical role for ORF45-mediated RSK activation in KSHV lytic replication. IMPORTANCE: KSHV is the causative agent of three human malignancies. KSHV pathogenesis is intimately linked to its ability to modulate the host cell microenvironment and to facilitate efficient production of progeny viral particles. We previously described the mechanism by which the KSHV lytic protein ORF45 activates the cellular kinases ERK and RSK. We now have mapped the critical region of ORF45 responsible for binding and activation of ERK/RSK to a single residue, F66. We mutated this amino acid of ORF45 (F66A) and introduced the mutation into a newly developed bacterial artificial chromosome containing the KSHV genome (BAC16). This system has provided us with a useful tool to characterize the functions of ORF45-activated RSK upon KSHV lytic reactivation. We show that viral gene expression and virion production are significantly reduced by F66A mutation, indicating a critical role for ORF45-activated RSK during KSHV lytic replication.

Regulation of ATG4B stability by RNF5 limits basal levels of autophagy and influences susceptibility to bacterial infection.

Autophagy is the mechanism by which cytoplasmic components and organelles are degraded by the lysosomal machinery in response to diverse stimuli including nutrient deprivation, intracellular pathogens, and multiple forms of cellular stress. Here, we show that the membrane-associated E3 ligase RNF5 regulates basal levels of autophagy by controlling the stability of a select pool of the cysteine protease ATG4B. RNF5 controls the membranal fraction of ATG4B and limits LC3 (ATG8) processing, which is required for phagophore and autophagosome formation. The association of ATG4B with-and regulation of its ubiquitination and stability by-RNF5 is seen primarily under normal growth conditions. Processing of LC3 forms, appearance of LC3-positive puncta, and p62 expression are higher in RNF5(-/-) MEF. RNF5 mutant, which retains its E3 ligase activity but does not associate with ATG4B, no longer affects LC3 puncta. Further, increased puncta seen in RNF5(-/-) using WT but not LC3 mutant, which bypasses ATG4B processing, substantiates the role of RNF5 in early phases of LC3 processing and autophagy. Similarly, RNF-5 inactivation in Caenorhabditis elegans increases the level of LGG-1/LC3::GFP puncta. RNF5(-/-) mice are more resistant to group A Streptococcus infection, associated with increased autophagosomes and more efficient bacterial clearance by RNF5(-/-) macrophages. Collectively, the RNF5-mediated control of membranalATG4B reveals a novel layer in the regulation of LC3 processing and autophagy.

Phosphorylation of eukaryotic translation initiation factor 4B (EIF4B) by open reading frame 45/p90 ribosomal S6 kinase (ORF45/RSK) signaling axis facilitates protein translation during Kaposi sarcoma-associated herpesvirus (KSHV) lytic replication.

Open reading frame 45 (ORF45) of Kaposi sarcoma-associated herpesvirus (KSHV) causes sustained activation of p90 ribosomal S6 kinase (RSK), which is crucial for KSHV lytic replication, but the exact functional roles remain to be determined. To characterize the biological consequence of persistent RSK activation by ORF45, we screened known cellular substrates of RSK. We found that ORF45 induced phosphorylation of eukaryotic translation initiation factor 4B (eIF4B), increased its assembly into translation initiation complex, and subsequently facilitated protein translation. The ORF45/RSK-mediated eIF4B phosphorylation was distinguishable from that caused by the canonical AKT/mammalian target of rapamycin/ribosomal S6 kinase and MEK/ERK/RSK pathways because it was resistant to both rapamycin (an mammalian target of rapamycin inhibitor) and U1026 (an MEK inhibitor). The rapamycin and U1026 doubly insensitive eIF4B phosphorylation was induced during KSHV reactivation but was abolished if either ORF45 or RSK1/2 were ablated by siRNA, a pattern that is correlated with reduced lytic gene expression as we observed previously. Ectopic expression of eIF4B but not its phosphorylation-deficient mutant form increased KSHV lytic gene expression and production of progeny viruses. Together, these results indicated that ORF45/RSK axis-induced eIF4B phosphorylation is involved in translational regulation and is required for optimal KSHV lytic replication.