The Viral Bcl-2 Homologs of Kaposi's Sarcoma-Associated Herpesvirus and Rhesus Rhadinovirus Share an Essential Role for Viral Replication

Abstract

KS-Bcl-2 is a Kaposi's sarcoma-associated herpesvirus (KSHV)-encoded viral Bcl-2 (vBcl-2) homolog which has apoptosis-and autophagy-inhibiting activity when expressed in transfected cells. However, little is known about its function during viral infection. As KS-Bcl-2 is expressed during the lytic replication cycle, we used constitutively lytic and inducibly lytic KSHV mutants to investigate the role of KSBcl-2 during the lytic cycle. We show that KSHV cannot complete the lytic replication cycle and produce infectious progeny in the absence of KS-Bcl-2, indicating that the protein is essential for KSHV replication. Replacement of the KS-Bcl-2 coding sequence, ORF16, by sequences encoding a potent cellular apoptosis and autophagy inhibitor, Bcl-XL, or the cytomegalovirus mitochondrial inhibitor of apoptosis, vMIA, did not rescue KSHV replication, suggesting that KS-Bcl-2 has a function that goes beyond apoptosis and autophagy inhibition. Strikingly, the vBcl-2 proteins of the related ␥ 2 -herpesviruses murine herpesvirus 68 and herpesvirus saimiri did not rescue the replication of a KS-Bcl-2 deletion mutant, but rhesus rhadinovirus (RRV) vBcl-2 did. Deletion of ORF16 from the RRV genome abrogated viral replication, but its replacement by KSHV ORF16 rescued RRV replication, indicating that the essential vBcl-2 function is conserved between these two primate rhadinoviruses. We further show that the KSHV and RRV Bcl-2 homologs localize to the mitochondria and nuclei of infected cells. Deletion of 17 amino acids from the N terminus of KS-Bcl-2 abrogates nuclear localization and KSHV replication, suggesting that KS-Bcl-2 might execute its essential function in the nuclei of infected cells.IMPORTANCE Several viruses express proteins homologous to cellular Bcl-2. Viral Bcl-2 proteins have functions similar to those of cellular Bcl-2: they can inhibit apoptosis, a form of programmed cell death, and autophagy, a self-degradative process for the disposal of dysfunctional or unwanted components. This study shows that the vBcl-2 proteins of KSHV and RRV differ from other vBcl-2 proteins in that they are essential for viral replication. The essential function is separate from the apoptosis-and autophagy-inhibiting activity but correlates with an unusual localization within the cell nucleus, suggesting that these proteins exert a novel function in the nucleus.KEYWORDS KS-Bcl-2, Kaposi's sarcoma-associated herpesvirus, rhesus rhadinovirus, apoptosis, autophagy, vBcl-2, viral replication K aposi's sarcoma-associated herpesvirus (KSHV), also referred to as human herpesvirus 8 (HHV-8), is a ␥ 2 -herpesvirus and the causative agent of Kaposi's sarcoma (KS), primary effusion lymphoma, and multicentric Castleman's disease (1-4).The KSHV life cycle is divided into two distinct phases, latency and lytic replication, which allow the virus to establish a long-term persistent infection. During latency, viral genomes are maintained within the host cell nucleus as circular episomes that are

Kaposi’s Sarcoma-Associated Herpesvirus Lytic Replication Is Independent of Anaphase-Promoting Complex Activity

“…The first lytic KSHV mutants were generated by M. Budt et al (35) utilizing the KSHV-BAC36 and A. Gallo et al (36) utilizing the KSHV-BAC16 backbone by inserting a constitutively active promoter upstream of ORF50. We constructed a similar KSHV mutant utilizing the KSHV-BAC16, hereafter referred to as KSHV-Lyt in order to preserve the nomenclature suggested in the earlier studies (35,36) (Fig 1A). However, instead of reconstituting the virus using RPE-1 cells as done previously (35,36), we reconstituted the KSHV-Lyt in LECs.…”

“…We constructed a similar KSHV mutant utilizing the KSHV-BAC16, hereafter referred to as KSHV-Lyt in order to preserve the nomenclature suggested in the earlier studies (35,36) (Fig 1A). However, instead of reconstituting the virus using RPE-1 cells as done previously (35,36), we reconstituted the KSHV-Lyt in LECs. Two weeks after the KSHV-Lyt DNA transfection, we noticed the appearance of the first plaques, which continued to expand until all cells were infected ( Fig 1B).…”

Targeting apoptosis pathways in infections

“…77 Besides targeting mitochondria and inhibiting apoptosis, these viral BCL-2 orthologs may also fulfill additional roles, as replacement with mammalian pro-survival BCL-2 members is insufficient to rescue viral replication. 84 Other viruses also inhibit BAX and BAK activation even though the encode proteins that show very little similarities to mammalian BCL-2 family members, as is the case in poxviruses. [85][86][87] This suggests that inhibition of BAX/BAK-mediated apoptosis is important to establish viral infections and to promote efficient viral replication.…”

To keep the host alive – the role of viral Bcl-2 proteins

“…Surprisingly, KSBcl-2 differs from other vBcl-2 proteins in that it is essential for viral replication, as its knockout dramatically lowers KSHV lytic gene expression, viral DNA replication, and progeny virus production (Gelgor et al, 2015;Liang et al, 2015b). Most importantly, it has been found that this novel essential function of KSBcl-2 in lytic replication is separate from the apoptosis-and autophagy-inhibiting activity but correlates with an unusual localization within the cell nucleus (in addition to mitochondria) of infected cells, suggesting that this protein might execute its essential function in the nucleus (Gallo et al, 2017).…”