No abstract

Elevated secretion of inflammatory factors is associated with latent Epstein-Barr virus (EBV) infection and the pathology of EBV-asso- Infection by the Epstein-Barr virus (EBV) causes infectious mononucleosis and several malignant cancers, including Burkitt's lymphoma, Hodgkin's lymphoma, nasopharyngeal carcinoma (NPC), and gastric carcinoma, as well as posttransplant lymphomas (1-5). EBV infection is persistent worldwide, but the frequency of EBV-associated NPC is highest in southern China, while Burkitt's lymphoma is most commonly found in equatorial Africa (2, 3). Although the exact mechanism by which EBV causes tumorigenesis remains to be fully defined, two important cofactors are strongly involved in EBV pathogenesis: genetic susceptibility and local diet. Unique polymorphisms of NPC-associated EBV have been identified in Chinese individuals, indicating the existence of EBV variants with higher pathogenic potential for NPC than that seen in the typical Western strains that cause infectious mononucleosis (6-8).Latent infection with limited gene expression is the default EBV cycle, whereas the lytic cycle is essential for transmission (1, 9). Lytic replication during primary infection or reactivation from the latent cycle is initiated by the expression of the immediate early (IE) viral transactivators BZLF1 and BRLF1. BZLF1, an EBV-encoded transcription factor of the basic-leucine zipper (b-ZIP) family, activates both viral and cellular genes by binding to BZLF1-responsive elements (ZREs), including several transcription factors and inflammatory factors (10).Inflammatory mediators have complex roles in cancer and infectious diseases, either limiting or promoting these disorders (11-15). Several proinflammatory factors have been fully characterized in experimental and clinical studies, including tumor necrosis factor alpha (TNF-␣), interferon gamma (IFN-␥), interleukin-1␣ (IL-1␣), and IL-1␤. TNF-␣ serves as an antiviral immune factor operating via two different mechanisms: induction of apoptosis in infected cells and activation of the antiviral response in uninfected cells (16)(17)(18)(19). For successful infection and replication, viruses employ multiple strategies to escape or hijack the host defenses, including innate immunity and the inflammatory response (15,17,20). The EBV lytic cycle evades the host inflammatory responses through the activity of BZLF1, which inhibits both IFN-␥ signaling and tumor necrosis factor receptor 1 (TNFR1) signaling (21-23). BZLF1 suppresses the NF-B signaling pathway by directly binding the p65 subunit (24, 25), acting as an

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-1b 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.

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.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections have resulted in a number of severe cases of COVID-19 and deaths worldwide. However, knowledge of SARS-CoV-2 infection, diseases and therapy remains limited, underlining the urgency of fundamental studies and drug development. Studies have shown that induction of autophagy and hijacking of autophagic machinery are essential for infection and replication of SARS-CoV-2; however, the mechanism of this manipulation and function of autophagy during SARS-CoV-2 infection remain unclear. In the present study, we identified ORF3 as an inducer of autophagy and revealed that ORF3 localizes to the ER and induces FAM134B-related ERphagy through the HMGB1-Beclin1 pathway. As a consequence, ORF3 induces ER stress and inflammatory responses through ERphagy and sensitizes cells to ER stress-induced cell death, suggesting that SARS-CoV-2 ORF3 hijacks ERphagy and then harms ER homeostasis to induce inflammatory responses through excessive ER stress. These findings reveal a sequential induction of ERphagy, ER stress and acute inflammatory responses during SARS-CoV-2 infection and provide therapeutic potential for ERphagy and ER stress-related drugs for COVID-19 treatment and prevention.ImportanceSARS-CoV-2 infection and replication require autophagosome-like double-membrane vacuoles. Inhibition of autophagy suppresses viral replication, indicating the essential role of autophagy in SARS-CoV-2 infection. However, how SARS-CoV-2 hijacks autophagy and the function of autophagy in the disease progression remain unknown. Here, we reveal that SARS-CoV-2 ORF3 induces ERphagy and consequently induces ER stress to trigger acute inflammatory responses and enhance sensitivity to ER stress-induced apoptosis. Our studies uncover ERphagy-induced inflammatory responses during SARS-CoV-2 infection and provide a promising therapeutic approach for treating SARS-CoV-2 infection and inflammatory responses in COVID-19 by manipulating autophagy and ER stress.