The Kaposi's sarcoma-associated herpesvirus (KSHV) open reading frame 16 (orf16) encodes a viral Bcl-2 (vBcl-2) protein which shares sequence and functional homology with the Bcl-2 family. Like its cellular homologs, vBcl-2 protects various cell types from apoptosis and can also negatively regulate autophagy. vBcl-2 is transcribed during lytic infection; however, its exact function has not been determined to date. By using bacterial artificial chromosome 16 (BAC16) clone carrying the full-length KSHV genome, we have generated recombinant KSHV mutants that fail to express vBcl-2 or express mCherry-tagged vBcl-2. We show that the vBcl-2 protein is expressed at relatively low levels during lytic induction and that a lack of vBcl-2 largely reduces the efficiency of KSHV reactivation in terms of lytic gene expression, viral DNA replication, and production of infectious particles. In contrast, the establishment of latency was not affected by the absence of vBcl-2. Our findings suggest an important role for vBcl-2 during initial phases of lytic reactivation and/or during subsequent viral propagation. Given the known functions of vBcl-2 in regulating apoptosis and autophagy, which involve its direct interaction with cellular proteins and thus require high levels of protein expression, it appears that vBcl-2 may have additional regulatory functions that do not depend on high levels of protein expression. IMPORTANCEThe present study shows for the first time the expression of endogenous vBcl-2 protein in KSHV-infected cell lines and demonstrates the importance of vBcl-2 during the initial phases of lytic reactivation and/or during its subsequent propagation. It is suggested that vBcl-2 has additional regulatory functions beyond apoptosis and autophagy repression that do not depend on high levels of protein expression. K aposi's sarcoma-associated herpesvirus (KSHV), also referred to as human herpesvirus 8 (HHV-8), is a gamma-2 herpesvirus. KSHV is causally linked to particular human cancers, including Kaposi's sarcoma (KS), primary effusion lymphoma (PEL), and plasmablastic multicentric Castleman's disease (1-7). Among human viruses, KSHV is most closely related to the Epstein-Barr virus (EBV), a tumorigenic gamma-1 herpesvirus known to be associated with lymphomas and nasopharyngeal carcinoma. Like all other herpesviruses, the infectious cycle of KSHV includes two alternative infection phases, latent and lytic. Latent infection allows the virus to establish long-term persistent infection and involves the presence of viral episomes and expression of a small set of viral genes. Lytic infection is needed for the maintenance of viral reservoirs and for virus spread and involves a temporally regulated cascade of viral gene expression and DNA replication, leading to the production and release of new virions. KSHV exists mainly in its latent form in KS lesions and in PEL, yet a small subpopulation of cells undergoes lytic replication. Analogously, most KSHV-infected cultured cells are latently infected. Lytic virus react...

Kaposi's sarcoma-associated herpesvirus (KSHV) is implicated in the etiology of several human malignancies. KSHV open reading frame (orf) 35 encodes a conserved gammaherpesvirus protein with an, as yet, unknown function. Employing the bacterial artificial chromosome (BAC) system, we generated a recombinant viral clone that fails to express ORF35 (BAC16-ORF35-stop) but preserves intact adjacent and overlapping reading frames. Using this construct, we studied the role of this previously uncharacterized gene product during lytic reactivation of KSHV. Upon lytic reactivation, the ORF35-stop recombinant virus displayed significantly reduced lytic viral gene expression, viral DNA replication, and progeny virus production as compared to control wild-type virus. Exogenous expression of ORF35-Flag reversed the effects of ORF35 deficiency. These results demonstrate that ORF35 is important for efficient lytic virus reactivation.

Kaposi’s sarcoma-associated herpesvirus (KSHV) is a human tumorigenic virus exhibiting two forms of infection, latent and lytic. Latent infection is abortive and allows the virus to establish lifelong infection, while lytic infection is productive, and is needed for virus dissemination within the host and between hosts. Latent infection may reactivate and switch towards the lytic cycle. This switch is a critical step in the maintenance of long-term infection and for the development of KSHV-related neoplasms. In this study, we examined the effect of nucleolar stress, manifested by failure in ribosome biogenesis or function and often coupled with p53 activation, on lytic reactivation of KSHV. To this end, we induced nucleolar stress by treatment with Actinomycin D, CX-5461 or BMH-21. Treatment with these compounds alone did not induce the lytic cycle. However, enhancement of the lytic cycle by these compounds was evident when combined with expression of the viral protein K-Rta. Further experiments employing combined treatments with Nutlin-3, knock-down of p53 and isogenic p53+/+ and p53-/- cells indicated that the enhancement of lytic reactivation by nucleolar stress does not depend on p53. Thus, our study identifies nucleolar stress as a novel regulator of KSHV infection, which synergizes with K-Rta expression to increase lytic reactivation. This suggests that certain therapeutic interventions, which induce nucleolar stress, may affect the outcome of KSHV infection.