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

Abstract

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...

Modulation of oncogenic signaling networks by Kaposi’s sarcoma-associated herpesvirus

“…KSHV ORF36 encodes a serine/threonine viral protein kinase (vPK) conserved among the herpesviruses and has been shown to activate JNK, a MAPK family member (Hamza et al, 2004; Avey et al, 2016; Bhatt et al, 2016). Though vPK is classified as a late lytic gene, it can be expressed in hypoxic conditions.…”

“…Though vPK is classified as a late lytic gene, it can be expressed in hypoxic conditions. Knock down of vPK negatively affects lytic replication and attenuates expression of several late genes (Avey et al, 2016). A ~3 to 10 fold reduction in viral progeny production and infection was observed in deficient or kinase-dead vPK viruses.…”

“…ORF45 is a lytic immediate early protein located in the cytoplasm and nucleus of infected cells (Avey et al, 2015; Avey et al, 2016). While ORF45 does not contain any intrinsic phosphorylation activity, ORF45 does mediate the activity of several kinases.…”

“…Suggestive of ORF45’s role in mediating protein synthesis, expression of ORF45 promotes the association of cellular and viral mRNAs with higher 5’ UTR complexity to polysomes. ORF45 also interacts and augments the activity of vPK in a poorly understood ORF45-RSK independent manner (Avey et al, 2016). …”

Generation of a KSHV K13 deletion mutant for vFLIP function study

“…With BAC16‐based mutagenesis, Fanxiu Zhu et al found that disruption of ORF45 caused a decrease in yield of progeny viruses and reduced infectivity . Furthermore, to investigate the roles of ORF36 that interacts with ORF45 in the context of KSHV lytic replication, they also engineered both ORF36‐null and kinase‐dead mutants by BAC16 system and figured out that ORF36‐null/mutant virions were moderately defective in viral particle production and were further deficient in primary infection . BAC16‐based mutagenesis has also been employed to several studies of KSHV‐encoded 25 mature miRNAs derived from 12 pre‐miRNAs in viral latency and the development of KSHV‐related malignancies By producing BAC16 miR‐K3 mutant viruses, we previously demonstrated that deletion of miR‐K3 from the KSHV genome attenuated KSHV‐induced cell migration and invasion, and also disrupted KSHV latency and impaired angiogenesis .…”