Phosphorylation of Kaposi's Sarcoma-Associated Herpesvirus Processivity Factor ORF59 by a Viral Kinase Modulates Its Ability To Associate with RTA and
            <i>ori</i>
            Lyt

Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of three human malignancies. KSHV ORF36 encodes a serine/threonine viral protein kinase, which is conserved throughout all herpesviruses. Although several studies have identified the viral and cellular substrates of conserved herpesvirus protein kinases (CHPKs), the precise functions of KSHV ORF36 during lytic replication remain elusive. Here, we report that ORF36 interacts with another lytic protein, ORF45, in a manner dependent on ORF36 kinase activity. We mapped the regions of ORF36 and ORF45 involved in the binding. Their association appears to be mediated by electrostatic interactions, since deletion of either the highly basic N terminus of ORF36 or an acidic patch of ORF45 abolished the binding. In addition, the dephosphorylation of ORF45 protein dramatically reduced its association with ORF36. Importantly, ORF45 enhances both the stability and kinase activity of ORF36. Consistent with previous studies of CHPK homologs, we detected ORF36 protein in extracellular virions. To investigate the roles of ORF36 in the context of KSHV lytic replication, we used bacterial artificial chromosome mutagenesis to engineer both ORF36-null and kinase-dead mutants. We found that ORF36-null/mutant virions are moderately defective in viral particle production and are further deficient in primary infection. In summary, our results uncover a functionally important interaction between ORF36 and ORF45 and indicate a significant role of ORF36 in the production of infectious progeny virions. IMPORTANCEKaposi's sarcoma-associated herpesvirus (KSHV) is a human tumor virus with a significant public health burden. KSHV ORF36 encodes a serine/threonine viral protein kinase, whose functions throughout the viral life cycle have not been elucidated. Here, we report that ORF36 interacts with another KSHV protein, ORF45. We mapped the regions of ORF36 and ORF45 involved in their association and further characterized the consequences of this interaction. We engineered ORF36 mutant viruses in order to investigate the functional roles of ORF36 in the context of KSHV lytic replication, and we confirmed that ORF36 is a component of KSHV virions. Moreover, we found that ORF36 mutants are defective in virion production and primary infection. In summary, we discovered and characterized a functionally important interaction between KSHV ORF36 and ORF45, and our results suggest a significant role of ORF36 in the production of infectious progeny virions, a process critical for KSHV pathogenesis. K aposi's sarcoma-associated herpesvirus (KSHV) is a human tumor virus and the causative agent of Kaposi's sarcoma (KS), as well as two lymphoproliferative disorders (1-3). All herpesviruses encode at least one serine/threonine protein kinase that is conserved throughout the three subfamilies (alpha-, beta-, and gammaherpesviruses), collectively referred to as conserved herpesvirus protein kinases (CHPKs) (reviewed in references 4 and 5). Orthologs of CHPKs include UL13 of herpes simp...

Section: Discussionsupporting

confidence: 80%

mentioning

confidence: 99%

Discovery of a Coregulatory Interaction between Kaposi's Sarcoma-Associated Herpesvirus ORF45 and the Viral Protein Kinase ORF36

Avey

Tepper

Pifer

et al. 2016

“…However, using a model of primary lytic replication, we have shown a direct correlation between the expression of ORF59, the induction of the lytic program of replication, and the production of infectious virions of a primate homolog of KSHV (33). Furthermore, previous studies have shown that an ORF59 deletion mutant of KSHV is defective in virion production, substantiating the critical role of ORF59 in the lytic cycle of replication (34).…”

supporting

confidence: 65%

Full-Length Isoforms of Kaposi's Sarcoma-Associated Herpesvirus Latency-Associated Nuclear Antigen Accumulate in the Cytoplasm of Cells Undergoing the Lytic Cycle of Replication

Garrigues

Howard

Barcy

et al. 2017

The latency-associated nuclear antigen (LANA) of the Kaposi's sarcomaassociated herpesvirus (KSHV) performs a variety of functions to establish and maintain KSHV latency. During latency, LANA localizes to discrete punctate spots in the nucleus, where it tethers viral episomes to cellular chromatin and interacts with nuclear components to regulate cellular and viral gene expression. Using highly sensitive tyramide signal amplification, we determined that LANA localizes to the cytoplasm in different cell types undergoing the lytic cycle of replication after de novo primary infection and after spontaneous, tetradecanoyl phorbol acetate-, or open reading frame 50 (ORF50)/replication transactivator (RTA)-induced activation. We confirmed the presence of cytoplasmic LANA in a subset of cells in lytically active multicentric Castleman disease lesions. The induction of cellular migration by scratch-wounding confluent cell cultures, culturing under subconfluent conditions, or induction of cell differentiation in primary cultures upregulated the number of cells permissive for primary lytic KSHV infection. The induction of lytic replication was characterized by high-level expression of cytoplasmic LANA and nuclear ORF59, a marker of lytic replication. Subcellular fractionation studies revealed the presence of multiple isoforms of LANA in the cytoplasm of ORF50/RTA-activated Vero cells undergoing primary infection. Mass spectrometry analysis demonstrated that cytoplasmic LANA isoforms were full length, containing the N-terminal nuclear localization signal. These results suggest that trafficking of LANA to different subcellular locations is a regulated phenomenon, which allows LANA to interact with cellular components in different compartments during both the latent and the replicative stages of the KSHV life cycle. IMPORTANCE Kaposi's sarcoma-associated herpesvirus (KSHV) causes AIDS-related malignancies, including lymphomas and Kaposi's sarcoma. KSHV establishes lifelong infections using its latency-associated nuclear antigen (LANA). During latency, LANA localizes to the nucleus, where it connects viral and cellular DNA complexes and regulates gene expression, allowing the virus to maintain long-term infections. Our research shows that intact LANA traffics to the cytoplasm of cells undergoing permissive lytic infections and latently infected cells in which the virus is induced to replicate. This suggests that LANA plays important roles in the cytoplasm and nuclear compartments of the cell during different stages of the KSHV life cycle. Determining cytoplasmic function and mechanism for regulation of the nuclear localiza-

“…KSHV ORF59 encodes viral processivity factor 8 (PF-8), which assists viral DNA polymerase with DNA processivity activity and plays an essential role in virus genome replication (13)(14)(15). PF-8 expression and function are RTA dependent in forming the replication complex (13)(14)(15)(25)(26)(27), while PF-8's activity and expression are regulated by a viral kinase (ORF36) and ORF57, respectively (16)(17)(18). In addition, PF-8 interacts with Ku70 and Ku86, impairing nonhomologous end joining during lytic replication (34).…”

Section: Discussionmentioning

confidence: 99%

“…KSHV PF-8 is known to play an essential role in virus genome replication by translocating viral DNA polymerase into the nucleus and assisting it with its DNA processivity activity (13)(14)(15). Meanwhile, PF-8 requires RTA for its expression and binding to oriLyt to form the replication complex (16)(17)(18). Our finding that PARP-1 degradation by PF-8 further enhanced RTA-mediated transactivation suggests an intricate feed-forward loop of the lytic replication of the virus.…”

mentioning

confidence: 99%

Downregulation of Poly(ADP-Ribose) Polymerase 1 by a Viral Processivity Factor Facilitates Lytic Replication of Gammaherpesvirus

Cheong

Park

Kang

et al. 2015

In Kaposi's sarcoma-associated herpesvirus (KSHV), poly(ADP-ribose) polymerase 1 (PARP-1) acts as an inhibitor of lytic replication. Here, we demonstrate that KSHV downregulated PARP-1 upon reactivation. The viral processivity factor of KSHV (PF-8) interacted with PARP-1 and was sufficient to degrade PARP-1 in a proteasome-dependent manner; this effect was conserved in murine gammaherpesvirus 68. PF-8 knockdown in KSHV-infected cells resulted in reduced lytic replication upon reactivation with increased levels of PARP-1, compared to those in control cells. PF-8 overexpression reduced the levels of the poly(ADPribosyl)ated (PARylated) replication and transcription activator (RTA) and further enhanced RTA-mediated transactivation. These results suggest a novel viral mechanism for overcoming the inhibitory effect of a host factor, PARP-1, thereby promoting the lytic replication of gammaherpesvirus. IMPORTANCEGammaherpesviruses are important human pathogens, as they are associated with various kinds of tumors and establish latency mainly in host B lymphocytes. Replication and transcription activator (RTA) of Kaposi's sarcoma-associated herpesvirus (KSHV) is a central molecular switch for lytic replication, and its expression is tightly regulated by many host and viral factors. In this study, we investigated a viral strategy to overcome the inhibitory effect of poly(ADP-ribose) polymerase 1 (PARP-1) on RTA's activity. PARP-1, an abundant multifunctional nuclear protein, was downregulated during KSHV reactivation. The viral processivity factor of KSHV (PF-8) directly interacted with PARP-1 and was sufficient and necessary to degrade PARP-1 protein in a proteasome-dependent manner. PF-8 reduced the levels of PARylated RTA and further promoted RTA-mediated transactivation. As this was also conserved in another gammaherpesvirus, murine gammaherpesvirus 68, our results suggest a conserved viral modulation of a host inhibitory factor to facilitate its lytic replication. Gammaherpesviruses, such as Epstein-Barr virus (EBV), Kaposi's sarcoma-associated herpesvirus (KSHV), and murine gammaherpesvirus 68 (MHV-68), are associated with various malignancies and lymphoproliferative disorders (1, 2). Several cellular factors are known to regulate the lytic replication of gammaherpesvirus, ultimately contributing to viral pathogenesis (3, 4). Poly(ADP-ribose) polymerase 1 (PARP-1) is an abundant nuclear protein that catalyzes the poly(ADP-ribosyl)ation of target proteins acting in DNA repair, cell cycle control, apoptosis, and gene expression (5-7). In KSHV and MHV-68 infections, PARP-1 interacts with the poly(ADP-ribosyl)ate replication and transcription activator (RTA), the molecular switch of lytic replication, thereby repressing lytic viral replication (8-11). PARP-1 is also known to be a component of the viral replication complex, increasing viral genome replication (9, 12). Previously, we showed that open reading frame 49 (ORF49) of MHV-68 interacts with PARP-1, disrupting interactions between RTA and PARP-1 and furthe...