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.     

Signaling cascades triggered by bacterial metabolic end products during reactivation of Kaposi's sarcoma-associated herpesvirus

The present studies explore the role of polymicrobial infection in the reactivation of Kaposi's sarcoma-associated herpesvirus (KSHV) and analyze signaling pathways activated upon this induction. We hypothesized that activation of the cellular stress-activated mitogen-activated protein kinase (MAPK) p38 pathway would play a key role in the bacterium-mediated disruption of viral latency similar to that of previously reported results obtained with other inducers of gammaherpesvirus lytic replication. KSHV within infected BCBL-1 cells was induced to replicate following exposure to metabolic end products from gram-negative or -positive bacteria that were then simultaneously exposed to specific inhibitors of signal transduction pathways. We have determined that bacterium-mediated induction of lytic KSHV infection is significantly reduced by the inhibition of the p38 MAPK pathway. In contrast, inhibition of the phosphatidylinositol 3-kinase pathway did not impair induction of lytic replication or p38 phosphorylation. Protein kinase C, though activated, was not the major pathway used for bacterium-induced viral reactivation. Furthermore, hyperacetylation of histones 3 and 4 was detected. Collectively, our results show that metabolic end products from these pathogens induce lytic replication of KSHV in BCBL-1 cells primarily via the activation of a stress-activated MAPK pathway. Importantly, we demonstrate for the first time a mechanism by which polymicrobial bacterial infections result in KSHV reactivation and pathogenesis.     

Kaposi's sarcoma-associated herpesvirus-encoded latency-associated nuclear antigen inhibits lytic replication by targeting Rta: a potential mechanism for virus-mediated control of latency

Like other herpesviruses, Kaposi's sarcoma-associated herpesvirus (KSHV, also designated human herpesvirus 8) can establish a latent infection in the infected host. During latency a small number of genes are expressed. One of those genes encodes latency-associated nuclear antigen (LANA), which is constitutively expressed in cells during latent as well as lytic infection. LANA has previously been shown to be important for the establishment of latent episome maintenance through tethering of the viral genome to the host chromosomes. Under specific conditions, KSHV can undergo lytic replication, with the production of viral progeny. The immediate-early Rta, encoded by open reading frame 50 of KSHV, has been shown to play a critical role in switching from viral latent replication to lytic replication. Overexpression of Rta from a heterologous promoter is sufficient for driving KSHV lytic replication and the production of viral progeny. In the present study, we show that LANA down-modulates Rta's promoter activity in transient reporter assays, thus repressing Rta-mediated transactivation. This results in a decrease in the production of KSHV progeny virions. We also found that LANA interacts physically with Rta both in vivo and in vitro. Taken together, our results demonstrate that LANA can inhibit viral lytic replication by inhibiting expression as well as antagonizing the function of Rta. This suggests that LANA may play a critical role in maintaining latency by controlling the switch between viral latency and lytic replication.     

Activation of p90 ribosomal S6 kinase by ORF45 of Kaposi's sarcoma-associated herpesvirus and its role in viral lytic replication

The extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) pathway is essential for infection by a variety of viruses. The p90 ribosomal S6 kinases (RSKs) are direct substrates of ERK and functional mediators of ERK MAPK signaling, but their roles in viral infection have never been examined. We demonstrate that ORF45 of Kaposi's sarcoma-associated herpesvirus (KSHV) interacts with RSK1 and RSK2 and strongly stimulates their kinase activities. The activation of RSK by ORF45 is correlated with ERK activation but does not require MEK. We further demonstrate that RSK1/RSK2 is activated during KSHV primary infection and reactivation from latency; a subset of RSK1/RSK2 is present in the viral replication compartment in the nucleus. Depletion of RSK1/RSK2 by small interfering RNA or the specific inhibitor BI-D1870 suppresses KSHV lytic gene expression and progeny virion production, suggesting an essential role of RSK1/RSK2 in KSHV lytic replication.