Herpes simplex virus 1 infection induces ubiquitination of UBE1a.

Herpes simplex virus 1 (HSV-1) is a human DNA virus that causes cold sores, keratitis, meningitis, and encephalitis. Ubiquitination is a post-translational protein modification essential for regulation of cellular events, such as proteasomal degradation, signal transduction, and protein trafficking. The process is also involved in events for establishing viral infection and replication. The first step in ubiquitination involves ubiquitin (Ub) binding with Ub-activating enzyme (E1, also termed UBE1) via a thioester linkage. Our results show that HSV-1 infection alters protein ubiquitination pattern in host cells, as evidenced by MS spectra and co-immunoprecipitation assays. HSV-1 induced ubiquitination of UBE1a isoform via an isopeptide bond with Lys604. Moreover, we show that ubiquitination of K604 in UBE1a enhances UBE1a activity; that is, the activity of ubiquitin-transfer to E2 enzyme. Subsequently, we investigated the functional role of UBE1a and ubiquitination of K604 in UBE1a. We found that UBE1-knockdown increased HSV-1 DNA replication and viral production. Furthermore, overexpression of UBE1a, but not a UBE1a K604A mutant, suppressed viral replication. Furthermore, we found that UBE1a and ubiquitination at K604 in UBE1a retarded expression of HSV-1 major capsid protein, ICP5. Our findings show that UBE1a functions as an antiviral factor that becomes activated upon ubiquitination at Lys604.

UBE1a Suppresses Herpes Simplex Virus-1 Replication.

Herpes simplex virus-1 (HSV-1) is the causative agent of cold sores, keratitis, meningitis, and encephalitis. HSV-1-encoded ICP5, the major capsid protein, is essential for capsid assembly during viral replication. Ubiquitination is a post-translational modification that plays a critical role in the regulation of cellular events such as proteasomal degradation, protein trafficking, and the antiviral response and viral events such as the establishment of infection and viral replication. Ub-activating enzyme (E1, also named UBE1) is involved in the first step in the ubiquitination. However, it is still unknown whether UBE1 contributes to viral infection or the cellular antiviral response. Here, we found that UBE1a suppressed HSV-1 replication and contributed to the antiviral response. The UBE1a inhibitor PYR-41 increased HSV-1 production. Immunofluorescence analysis revealed that UBE1a highly expressing cells presented low ICP5 expression, and vice versa. UBE1a inhibition by PYR-41 and shRNA increased ICP5 expression in HSV-1-infected cells. UBE1a reduced and retarded ICP5 protein expression, without affecting transcription of ICP5 mRNA or degradation of ICP5 protein. Additionally, UBE1a interacted with ICP27, and both partially co-localized at the Hsc70 foci/virus-induced chaperone-enriched (VICE) domains. PYR-41 reduced the co-localization of UBE1a and ICP27. Thus, our findings provide insights into the mechanism of UBE1a in the cellular response to viral infection.

Arctigenin induces the apoptosis of primary effusion lymphoma cells under conditions of glucose deprivation.

Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of primary effusion lymphoma (PEL) and Kaposi's sarcoma. PEL is a type of non-Hodgkin's B-cell lymphoma, affecting immunosuppressed individuals, such as post-transplant or AIDS patients. However, since PEL is resistant to chemotherapeutic regimens, new effective treatment strategies are required. Arctigenin, a natural lignan compound found in the plant Arctium lappa, has been widely investigated as a potential anticancer agent in the clinical setting. In the present study, we examined the cytotoxic effects of arctigenin by cell viability assay and found that arctigenin markedly inhibited the proliferation of PEL cells compared with KSHV-uninfected B-lymphoma cells under conditions of glucose deprivation. Arctigenin decreased cellular ATP levels, disrupted mitochondrial membrane potential and triggered caspase-9-mediated apoptosis in the glucose-deprived PEL cells. In addition, western blot analysis using phospho-specific antibodies were used to evaluate activity changes in the signaling pathways of interest. As a result, arctigenin suppressed the activation of the extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (p38 MAPK) signaling pathways by inhibiting ERK and p38 MAPK phosphorylation in the glucose-deprived PEL cells. We confirmed that an inhibitor of ERK (U0126) or p38 MAPK (SB202190 and SB203580) suppressed the proliferation of the BC3 PEL cells compared with the KSHV-negative DG75 cells. Moreover, RT-PCR and luciferase reporter assay revealed that arctigenin and p38 MAPK inhibition by SB202190 or SB203580 downregulated the transcriptional expression of unfolded protein response (UPR)­related molecules, including GRP78 and ATF6α under conditions of glucose deprivation. Finally, we confirmed that arctigenin did not affect KSHV replication in PEL cells, suggesting that arctigenin treatment for PEL does not contribute to the risk of de novo KSHV production. These data thus indicate that arctigenin may serve as a lead compound for the development of novel and effective drugs for the treatment of PEL.