KSHV RTA Induces Degradation of the Host Transcription Repressor ID2 To Promote the Viral Lytic Cycle.

The immediate early viral protein replication and transcription activator (RTA) of Kaposi's sarcoma-associated herpesvirus (KSHV) is essential for activating the lytic cycle of KSHV. RTA induces the KSHV lytic cycle by several mechanisms, acting as a viral transcription factor that directly induces viral and host genes and acting as a viral E3 ubiquitin ligase by degrading host proteins that block viral lytic replication. Recently, we have characterized the global gene expression changes in primary effusion lymphoma (PEL) upon lytic reactivation of KSHV, which also led to the identification of rapidly downregulated genes such as ID2, an inhibitor of basic helix-loop-helix transcription factors. Here, we demonstrate that ID2 overexpression in PEL ablates KSHV lytic reactivation, indicating that ID2 inhibits the KSHV lytic cycle. Furthermore, we show that while ID2 is highly expressed during latency, its protein level is rapidly reduced by 4 h postinduction during lytic reactivation. Our results indicate that RTA binds to ID2 and induces its degradation during the KSHV lytic cycle by N-terminal ubiquitination through the ubiquitin-proteasome pathway. Importantly, we found that not only KSHV RTA but also its Epstein-Barr virus (EBV) and murine gammaherpesvirus 68 (MHV68) homologs interact with ID2, and they can induce the degradation of all four members of the ID protein family, suggesting an evolutionarily conserved interplay between gammaherpesvirus RTAs and ID proteins. Taken together, we propose that ID2 acts as a repressor of the KSHV lytic cycle, which is counteracted by its RTA-mediated degradation. We also predict that ID proteins may act as restriction factors of the lytic phase of the other gammaherpesviruses as well. IMPORTANCE In addition to its transcription regulatory role, RTA is also known to have an E3 ubiquitin ligase activity, which RTA utilizes for inducing protein degradation. However, it is still largely unknown what host factors are downregulated during KSHV lytic reactivation by RTA-mediated protein degradation and what the biological significance of the degradation of these host factors is. In this study, we discovered that RTA employs N-terminal ubiquitination to induce degradation of ID2, a potent transcription repressor of host genes, via the ubiquitin-proteasome pathway to promote KSHV lytic reactivation in PEL cells. Furthermore, we found that not only KSHV RTA but also RTA of EBV and MHV68 gammaherpesviruses can induce the degradation of all four human ID proteins, indicating that the interplay between gammaherpesvirus RTAs and ID proteins is evolutionarily conserved.

KDM2B Overexpression Facilitates Lytic De Novo KSHV Infection by Inducing AP-1 Activity Through Interaction with the SCF E3 Ubiquitin Ligase Complex.

It is still largely unknown what host factors are involved in controlling the expression of the lytic viral gene RTA during primary infection, which determines if Kaposi's sarcoma-associated herpesvirus (KSHV) establishes latent or lytic infection. We have recently identified the histone demethylase KDM2B as a repressor of RTA expression during both de novo KSHV infection and latency based on an epigenetic factor siRNA screen. Here, we report that surprisingly, KDM2B overexpression can promote lytic de novo infection by using a mechanism that differs from what is needed for its repressor function. Our study revealed that while the DNA-binding and demethylase activities of KDM2B linked to its transcription repressive function are dispensable, its C-terminal F-box and LRR domains are required for the lytic infection-inducing function of KDM2B. We found that overexpressed KDM2B increases the half-life of the AP-1 subunit c-Jun protein and induces the AP-1 signaling pathway. This effect is dependent upon the binding of KDM2B to the SKP1-CUL1-F-box (SCF) E3 ubiquitin ligase complex via its F-box domain. Importantly, the inhibition of AP-1 reduces KDM2B-mediated lytic de novo KSHV infection. Overall, our findings indicate that KDM2B may induce the degradation of some host factors by using the SCF complex resulting in the enrichment of c-Jun. This leads to increased AP-1 transcriptional activity, which facilitates lytic gene expression following de novo infection interfering with the establishment of viral latency.SignificanceThe expression of epigenetic factors is often dysregulated in cancers or upon specific stress signals, which often results in a display of non-canonical functions of the epigenetic factors that are independent from their chromatin-modifying roles. We have previously demonstrated that KDM2B normally inhibits KSHV lytic cycle using its histone demethylase activity. Surprisingly, we found that KDM2B overexpression can promote lytic de novo infection, which does not require its histone demethylase or DNA-binding functions. Instead, KDM2B uses the SKP1-CUL1-F-box (SCF) E3 ubiquitin ligase complex to induce AP-1 transcriptional activity, which promotes lytic gene expression. This is the first report that demonstrates a functional link between SFCKDM2B and AP-1 in the regulation of KSHV lytic cycle.

Comparative analysis of the viral interferon regulatory factors of KSHV for their requisite for virus production and inhibition of the type I interferon pathway.

Unique among human viruses, Kaposi's sarcoma-associated herpesvirus (KSHV) encodes several homologs of cellular interferon regulatory factors (vIRFs). Since KSHV expresses multiple factors that can inhibit interferon (IFN) signaling to promote virus production, it is still unclear to what extent vIRFs contribute to these specific processes during KSHV infection. To study the function of vIRFs during viral infection, we engineered 3xFLAG-tagged-vIRF and vIRF-knockout recombinant KSHV clones, which were utilized to test vIRF expression, as well as their requirement for viral replication, virus production, and inhibition of the type I IFN pathway in different models of lytic KSHV infection. Our data show that all vIRFs can be expressed as lytic viral proteins, yet were dispensable for KSHV production and inhibition of type I IFN. Nevertheless, as vIRFs were able to suppress IFN-stimulated antiviral genes, vIRFs may still promote the KSHV lytic cycle in the presence of an ongoing antiviral response.

Characterization of de novo lytic infection of dermal lymphatic microvascular endothelial cells by Kaposi's sarcoma-associated herpesvirus.

The biology of primary lytic Kaposi's sarcoma-associated herpesvirus (KSHV) infection is still not well understood, which is largely attributed to the lack of cell lines permissive to robust lytic KSHV infection in vitro. Our study demonstrates that primary human dermal lymphatic microvascular endothelial cells (HDLMEC) support lytic KSHV replication following de novo infection, resulting in robust KSHV production, indicating that HDLMECs are suitable for studying the regulation of primary lytic KSHV infection. Importantly, by utilizing lytically infected HDLMECs, we show for the first time that the KSHV latent genes LANA and viral cyclin are required for lytic replication during de novo lytic infection, a function of these latent genes that has not yet been recognized. Since Kaposi's sarcoma is considered to be originated from infected lymphatic endothelial cells, HDLMECs represent a valuable in vitro cell culture model for investigating lytic KSHV infection, which has been understudied in KSHV pathogenesis.

Genome-Wide Identification of Direct RTA Targets Reveals Key Host Factors for Kaposi's Sarcoma-Associated Herpesvirus Lytic Reactivation.

Kaposi's sarcoma-associated herpesvirus (KSHV) is a human oncogenic virus, which maintains the persistent infection of the host by intermittently reactivating from latently infected cells to produce viral progenies. While it is established that the replication and transcription activator (RTA) viral transcription factor is required for the induction of lytic viral genes for KSHV lytic reactivation, it is still unknown to what extent RTA alters the host transcriptome to promote KSHV lytic cycle and viral pathogenesis. To address this question, we performed a comprehensive time course transcriptome analysis during KSHV reactivation in B-cell lymphoma cells and determined RTA-binding sites on both the viral and host genomes, which resulted in the identification of the core RTA-induced host genes (core RIGs). We found that the majority of RTA-binding sites at core RIGs contained the canonical RBP-Jκ-binding DNA motif. Subsequently, we demonstrated the vital role of the Notch signaling transcription factor RBP-Jκ for RTA-driven rapid host gene induction, which is consistent with RBP-Jκ being essential for KSHV lytic reactivation. Importantly, many of the core RIGs encode plasma membrane proteins and key regulators of signaling pathways and cell death; however, their contribution to the lytic cycle is largely unknown. We show that the cell cycle and chromatin regulator geminin and the plasma membrane protein gamma-glutamyltransferase 6, two of the core RIGs, are required for efficient KSHV reactivation and virus production. Our results indicate that host genes that RTA rapidly and directly induces can be pivotal for driving the KSHV lytic cycle.IMPORTANCE The lytic cycle of KSHV is involved not only in the dissemination of the virus but also viral oncogenesis, in which the effect of RTA on the host transcriptome is still unclear. Using genomics approaches, we identified a core set of host genes which are rapidly and directly induced by RTA in the early phase of KSHV lytic reactivation. We found that RTA does not need viral cofactors but requires its host cofactor RBP-Jκ for inducing many of its core RIGs. Importantly, we show a critical role for two of the core RIGs in efficient lytic reactivation and replication, highlighting their significance in the KSHV lytic cycle. We propose that the unbiased identification of RTA-induced host genes can uncover potential therapeutic targets for inhibiting KSHV replication and viral pathogenesis.