CXCL1 confers a survival advantage in Kaposi's sarcoma-associated herpesvirus-infected human endothelial cells through STAT3 phosphorylation.

Many cytokines produced by Kaposi's sarcoma-associated herpesvirus (KSHV)-infected cells have been shown to participate in the pathogenesis of KSHV. Determination of the exact role of cytokines in Kaposi's sarcoma (KS) pathogenesis is limited, however, by the difficulty to manipulate the target genes in human endothelial cells. In this study, we sought to elucidate the role of cytokines in KSHV-infected human immortalized endothelial cell line (HuARLT cells) by knockout (KO) of the corresponding target genes using the CRISPR/Cas9 system. The cytokine production profile of KSHV-infected HuARLT cells was analyzed using a protein array, and several cytokines were found to be highly upregulated following KSHV infection. This study focused on CXCL1, which was investigated by knocked out in HuARLT cells. KSHV-infected CXCL1 KO cells underwent increased cell death compared to KSHV-infected wild-type (WT) cells and mock-infected CXCL1 KO cells. Lytic replication was not observed in KSHV-infected WT nor CXCL1 KO cells. Phosphorylation of STAT3 was significantly suppressed in KSHV-infected CXCL1 KO cells. Additionally, inhibitors of STAT3 and CXCL1 induced cell death in KSHV-infected endothelial cells. Our results show that CXCL1 production is required for the survival of KSHV-infected endothelial cells, and the CXCL1 to STAT3 phosphorylation signaling pathway may be a therapeutic target for KS.

HMGB1 Knockout Decreases Kaposi's Sarcoma-Associated Herpesvirus Virion Production in iSLK BAC16 Cells by Attenuating Viral Gene Expression.

Multiple host proteins affect the gene expression of Kaposi's sarcoma-associated herpesvirus (KSHV) during latent and lytic replication. High-mobility group box 1 (HMGB1) serves as a highly conserved chromosomal protein inside the cell and a prototypical damage-associated molecular pattern molecule outside the cell. HMGB1 has been shown to play a pathogenic role in viral infectious diseases and to regulate the lytic replication of KSHV. However, its functional effects on the KSHV life cycle in KSHV-infected cells have not been fully elucidated. Here, we explored the role of intracellular and extracellular HMGB1 in KSHV virion production by employing CRISPR/Cas9-mediated HMGB1 knockout in the KSHV-producing iSLK BAC16 cell line. Intracellular HMGB1 formed complexes with various proteins, and the abundance of HMGB1-interacting proteins changed during latent and lytic replication. Moreover, extracellular HMGB1 was found to enhance lytic replication by phosphorylating JNK. Of note, the expression of viral genes was attenuated during lytic replication in HMGB1 knockout iSLK BAC16 cells, with significantly decreased production of infectious virions compared to that of wild-type cells. Collectively, our results demonstrate that HMGB1 is an important cellular cofactor that affects the generation of infectious KSHV progeny during lytic replication. IMPORTANCE The high-mobility group box 1 (HMGB1) protein has many intra- and extracellular biological functions with an intricate role in various diseases. In certain viral infections, HMGB1 affects the viral life cycle and pathogenesis. In this study, we explored the effects of HMGB1 knockout on the production of Kaposi's sarcoma-associated herpesvirus (KSHV). HMGB1 knockout decreased virion production in KSHV-producing cells by decreasing the expression of viral genes. The processes by which HMGB1 affects KSHV production may occur inside or outside infected cells. For instance, several cellular and viral proteins interacted with intracellular HMGB1 in a nucleosomal complex, whereas extracellular HMGB1 induced JNK phosphorylation, thereby enhancing lytic replication. Our results suggest that both intracellular and extracellular HMGB1 are necessary for efficient KSHV replication. Thus, HMGB1 may represent an effective therapeutic target for the regulation of KSHV production.

Rab27b regulates extracellular vesicle production in cells infected with Kaposi's sarcoma-associated herpesvirus to promote cell survival and persistent infection.

Extracellular vesicles (EVs) play a crucial role in cell-to-cell communication. EVs and viruses share several properties related to their structure and the biogenesis machinery in cells. EVs from virus-infected cells play a key role in virus spread and suppression using various loading molecules, such as viral proteins, host proteins, and microRNAs. However, it remains unclear how and why viruses regulate EV production inside host cells. The purpose of this study is to investigate the molecular mechanisms underlying EV production and their roles in Kaposi's sarcoma-associated herpesvirus (KSHV)-infected cells. Here, we found that KSHV induced EV production in human endothelial cells via Rab-27b upregulation. The suppression of Rab27b expression in KSHV-infected cells enhanced cell death by increasing autophagic flux and autolysosome formation. Our results indicate that Rab27b regulates EV biogenesis to promote cell survival and persistent viral infection during KSHV infection, thereby providing novel insights into the crucial role of Rab-27b in the KSHV life cycle.

Dimethyl Sulfoxide Enhances Kaposi's Sarcoma-Associated Herpesvirus Production During Lytic Replication.

Kaposi's sarcoma-associated herpesvirus (KSHV) is an etiologic agent of Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman disease. In studies of KSHV, efficient virus production and isolation are essential. Reactivation of KSHV can be initiated by treating latently infected cells with chemicals, such as 12-O-tetradecanoyl-phorbol-13-acetate and sodium butyrate. These chemicals have been used as tools to induce lytic replication and viral production in KSHV-producing cell lines. Dimethyl sulfoxide (DMSO) is an organosulfur compound that is frequently used as an aprotic solvent similar to water. In experiments exploring signaling pathways in KSHV-infected cells, DMSO treatment alone as a vehicle affected the lytic gene expression of KSHV. However, to the best of our knowledge, the effects of DMSO on KSHV-producing cells have not yet been reported. Therefore, in this study, we investigated whether DMSO could be used as a reagent to enhance viral production during lytic replication in KSHV-producing cells and assessed the underlying mechanisms. The effects of DMSO on KSHV production were analyzed in iSLK BAC16 cells, which have been widely used for recombinant KSHV production. We found that the production of KSHV virions was significantly increased by treatment with DMSO during the induction of lytic replication. Mechanistically, lytic genes of KSHV were enhanced by DMSO treatment, which was correlated with virion production. Additionally, DMSO induced the phosphorylation of JNK during lytic replication, and inhibition of JNK abolished the effects of DMSO on lytic replication and virion production. Our findings showed that additional treatment with DMSO during the induction of lytic replication significantly improved the yield of KSHV production.

Effects of different separation methods on the physical and functional properties of extracellular vesicles.

Extracellular vesicles (EVs) are small vesicles secreted from cells. They have crucial biological functions in intercellular communications and may even be biomarkers for cancer. The various methods used to isolate EVs from body fluid and cell culture supernatant have been compared in prior studies, which determined that the component yield and physical properties of isolated EVs depend largely on the isolation method used. Several novel and combined methods have been recently developed, which have not yet been compared to the established methods. Therefore, the purpose of this study is to compare the physical and functional differences in EVs isolated using a differential centrifugation method, the precipitation-based Invitrogen kit, the ExoLutE kit, and the Exodisc, of which the latter two were recently developed. We investigated the properties of EVs isolated from non-infected and Kaposi's sarcoma-associated herpesvirus-infected human umbilical vein endothelial cells using each method and determined the yields of DNA, RNA, and proteins using quantitative polymerase chain reaction and bicinchoninic acid assays. Additionally, we determined whether the biological activity of EVs correlated with the quantity or physical properties of the EVs isolated using different methods. We found that Exodisc was the most suitable method for obtaining large quantities of EVs, which might be useful for biomarker investigations, and that the EVs separated using Exodisc exhibited the highest complement activation activity. However, we also found that the functional properties of EVs were best maintained when differential centrifugation was used. Effective isolation is necessary to study EVs as tools for diagnosing cancer and our findings may have relevant implications in the field of oncology by providing researchers with data to assist their selection of a suitable isolation method.

Extracellular Vesicles From KSHV-Infected Cells Stimulate Antiviral Immune Response Through Mitochondrial DNA.

Kaposi's Sarcoma-associated herpesvirus (KSHV) is the etiologic agent of Kaposi's sarcoma, which is the most common cancer in acquired immune deficiency syndrome patients. KSHV contains a variety of immunoregulatory proteins. There have been many studies on the modulation of antiviral response by these immunoregulatory proteins of KSHV. However, the antiviral effects of extracellular vesicles (EVs) during de novo KSHV infection have not been investigated to our best knowledge. In this study, we showed that KSHV-infected cells induce interferon-stimulated genes (ISGs) response but not type I interferon in uninfected bystander cells using EVs. mRNA microarray analysis showed that ISGs and IRF-activating genes were prominently activated in EVs from KSHV-infected cells (KSHV EVs)-treated human endothelial cells, which were validated by RT-qPCR and western blot analysis. We also found that this response was not associated with cell death or apoptosis by virus infection. Mechanistically, the cGAS-STING pathway was linked with these KSHV EVs-mediated ISGs expressions, and mitochondrial DNA on the surface of KSHV EVs was one of the causative factors. Besides, KSHV EVs-treated cells showed lower infectivity for KSHV and viral replication activity than mock EVs-treated cells. Our results indicate that EVs from KSHV-infected cells could be an initiating factor for the innate immune response against viral infection, which may be critical to understanding the microenvironment of virus-infected cells.