Kaposi's sarcoma-associated herpesvirus transactivator RTA promotes degradation of the repressors to regulate viral lytic replication

Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8 (KSHV/HHV-8) RTA is an important protein involved in the induction of KSHV lytic replication from latency through activation of the lytic cascade. A number of cellular and viral proteins, including K-RBP, have been found to repress RTA-mediated transactivation and KSHV lytic replication. However, it is unclear as to how RTA overcomes the suppression during lytic reactivation. In this study, we found that RTA can induce K-RBP degradation through the ubiquitin-proteasome pathway and that two regions in RTA are responsible. Moreover, we found that RTA can promote the degradation of several other RTA repressors. RTA mutants that are defective in inducing K-RBP degradation cannot activate RTA responsive promoter as efficiently as wild-type RTA. Interference of the ubiquitin-proteasome pathway affected RTA-mediated transactivation and KSHV reactivation from latency. Our results suggest that KSHV RTA can stimulate the turnover of repressors to modulate viral reactivation. Since herpes simplex virus type 1 transactivator ICP0 and human cytomegalovirus transactivator pp71 also stimulate the degradation of cellular silencers, it is possible that the promotion of silencer degradation by viral transactivators may be a common mechanism for regulating the lytic replication of herpesviruses.     

Activation of Kaposi's sarcoma-associated herpesvirus (human herpesvirus 8) lytic replication by human cytomegalovirus

The majority of Kaposi's sarcoma-associated herpesvirus (KSHV)-infected cells identified in vivo contain latent KSHV, with lytic replication in only a few percent of cells, as is the case for the cells of Kaposi's sarcoma (KS) lesions. Factors that influence KSHV latent or lytic replication are not well defined. Because persons with KS are often immunosuppressed and susceptible to many infectious agents, including human cytomegalovirus (HCMV), we have investigated the potential for HCMV to influence the replication of KSHV. Important to this work was the construction of a recombinant KSHV, rKSHV.152, expressing the green fluorescent protein (GFP) and neo (conferring resistance to G418). The expression of GFP was a marker of KSHV infection in cells of both epithelial and endothelial origin. The rKSHV.152 virus was used to establish cells, including human fibroblasts (HF), containing only latent KSHV, as demonstrated by latency-associated nuclear antigen expression and Gardella gel analysis. HCMV infection of KSHV latently infected HF activated KSHV lytic replication with the production of infectious KSHV. Dual-color immunofluorescence detected both the KSHV lytic open reading frame 59 protein and the HCMV glycoprotein B in coinfected cells, and UV-inactivated HCMV did not activate the production of infectious KSHV-GFP. In addition, HCMV coinfection increased the production of KSHV from endothelial cells and activated lytic cycle gene expression in keratinocytes. These data demonstrate that HCMV can activate KSHV lytic replication and suggest that HCMV could influence KSHV pathogenesis.     

Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV, HHV-8)

Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV or human herpesvirus 8, HHV-8) are members of gamma-herpes virus family. Both viruses infect to B cells and cause malignancies such as lymphoma. Since EBV and HHV-8 are so-called 'oncovirus', their oncogenecities have been focused in the researches on EBV and KSHV for a long time. EBV was discovered in 1964, whereas KSHV was identified in 1994. However, KSHV was analyzed rapidly in these fifteen years. One of the recent progresses in the research on EBV and KSHV is that virus-encoded small RNAs were identified in their genomes and characterized. EBV is the first human virus in whose genome microRNA was identified. The oncogenecity of EBV and KSHV remains unclear. Here, I discuss the pathogenesis by EBV and KSHV with special reference to recent progress in this field.     

Poly(ADP-ribose) polymerase 1 binds to Kaposi's sarcoma-associated herpesvirus (KSHV) terminal repeat sequence and modulates KSHV replication in latency

During latency, Kaposi's sarcoma-associated herpesvirus (KSHV) is thought to replicate once and to be partitioned in synchrony with the cell cycle of the host. In this replication cycle, the KSHV terminal repeat (TR) sequence functions as a replication origin, assisted by the latency-associated nuclear antigen (LANA). Thus, TR seems to function as a cis element for the replication and partitioning of the KSHV genome. Viral replication and partitioning are also likely to require cellular factors that interact with TR in either a LANA-dependent or -independent manner. Here, we sought to identify factors that associate with TR by using a TR DNA column and found that poly(ADP-ribose) polymerase 1 (PARP1) and known replication factors, including ORC2, CDC6, and Mcm7, bound to TR. PARP1 bound directly to a specific region within TR independent of LANA, and LANA was poly(ADP-ribosyl)ated by PARP1. Drugs such as hydroxyurea and niacinamide, which raise or lower PARP activity, respectively, affected the virus copy number in infected cells. Thus, the poly(ADP-ribosyl)ation status of LANA appears to affect the replication and/or maintenance of the viral genome. Drugs that specifically up-regulate PARP activity may lead to the disappearance of latent KSHV.     

The N-terminal peptide of the Kaposi's sarcoma-associated herpesvirus (KSHV)-cyclin determines substrate specificity

Cyclin-dependent kinases (Cdks) are activated by cyclin binding and phosphorylation by the Cdk-activating kinase (CAK). Activation of Cdk6 by the D-type cyclins requires phosphorylation of Cdk6 by CAK on threonine 177. In contrast, Cdk6 is activated by the Kaposi's sarcoma-associated herpesvirus (KSHV)-cyclin in the absence and presence of CAK phosphorylation. The activity of Cdk6.KSHV-cyclin complexes was investigated here by analyzing mutants of the KSHV-cyclin and Cdk6 in vitro as well as in U2OS cells. Deletion of the N terminus of the KSHV-cyclin affects the substrate specificity indicating that the N terminus is required for phosphorylation of histone H1 but not for other substrates. Mutation of residues in the region 180-200 of the KSHV-cyclin decreases the binding affinity to Cdk6 in U2OS cells but increases the activity of Cdk6.KSHV-cyclin complexes in vitro indicating that low affinity binding of cyclins to the Cdk subunit might favor increased on- or off-rates of Cdk substrates. Expression of high levels of p16(INK4a) in cells leads to the formation of a heterotrimeric complex composed of Cdk6, KSHV-cyclin, and p16(INK4a). Some of the Cdk6 .KSHV-cyclin.p16 complexes were found to be active indicating that there might be different modes of p16 binding to Cdk6.cyclin complexes.     

Evaluation of the lytic origins of replication of Kaposi's sarcoma-associated virus/human herpesvirus 8 in the context of the viral genome

The lytic origins of DNA replication for human herpesvirus 8 (HHV8), oriLyt-L and oriLyt-R, are located between open reading frames K4.2 and K5 and ORF69 and vFLIP, respectively. These lytic origins were elucidated using a transient replication assay. Although this assay is a powerful tool for identifying many herpesvirus lytic origins, it is limited in its ability to evaluate the activity of replication origins in the context of the viral genome. To this end, we investigated the ability of a recombinant HHV8 bacterial artificial chromosome (BAC) to replicate in the absence of oriLyt-R, oriLyt-L, or both oriLyt regions. We generated the HHV8 BAC recombinants (BAC36-DeltaOri-R, BAC36-DeltaOri-L, and BAC36-DeltaOri-RL), which removed one or all of the identified lytic origins. An evaluation of these recombinant BACs revealed that oriLyt-L was sufficient to propagate the viral genome, whereas oriLyt-R alone failed to direct the amplification of viral DNA.     

Requirement of a 12-base-pair TATT-containing sequence and viral lytic DNA replication in activation of the Kaposi's sarcoma-associated herpesvirus K8.1 late promoter

Kaposi's sarcoma-associated herpesvirus (KSHV) K8.1 late promoter consists of a minimal 24-bp sequence, with a TATA-like, 12-bp promoter core, AATATTAAAGGG, and is active on a reporter only in butyrate-induced KSHV-infected cells. The activity of the K8.1 promoter can be enhanced (>15-fold) by the KSHV left-end lytic origin of DNA replication (oriLyt-L) sequence while providing inefficient replication of plasmid DNA and is inhibited by viral DNA replication inhibitors, suggesting that activation of the K8.1 promoter on the reporter is involved in KSHV lytic DNA replication largely by trans.