Kaposi’s Sarcoma-Associated Herpesvirus (KSHV) is an oncogenic virus which has adapted unique mechanisms to modulate the cellular microenvironment of its human host. The pathogenesis of KSHV is intimately linked to its manipulation of cellular signaling pathways, including the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) pathway. We have previously shown that KSHV ORF45 contributes to the sustained activation of both ERK and p90 ribosomal S6 kinase (RSK, a major functional mediator of ERK/MAPK signaling) during KSHV lytic replication. ORF45-activated RSK is required for optimal KSHV lytic gene expression and progeny virion production, though the underlying mechanisms downstream of this activation are still unclear. We hypothesized that the activation of RSK by ORF45 causes differential phosphorylation of cellular and viral substrates, affecting biological processes essential for efficient KSHV lytic replication. Accordingly, we observed widespread and significant differences in protein phosphorylation upon induction of lytic replication. Mass-spectrometry-based phosphoproteomic screening identified putative substrates of ORF45-activated RSK in KSHV-infected cells. Bioinformatic analyses revealed that nuclear proteins, including several transcriptional regulators, were overrepresented among these candidates. We validated the ORF45/RSK-dependent phosphorylation of several putative substrates by employing KSHV BAC mutagenesis, kinase inhibitor treatments, and/or CRISPR-mediated knockout of RSK in KSHV-infected cells. Furthermore, we assessed the consequences of knocking out these substrates on ORF45/RSK-dependent regulation of gene expression and KSHV progeny virion production. Finally, we show data to support that ORF45 regulates the translational efficiency of a subset of viral/cellular genes with complex secondary structure in their 5’ UTR. Altogether, these data shed light on the mechanisms by which KSHV ORF45 manipulates components of the host cell machinery via modulation of RSK activity. Thus, this study has important implications for the pathobiology of KSHV and other diseases in which RSK activity is dysregulated.

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...

Induction of matrix maturation and mineralization in calcified tissues is important for patients with primary bone tumors and other bone deficiencies, e.g., osteoporosis. For the former it signifies a better prognosis in osteosarcoma, and for the latter it might improve bone remodeling. In the present study we exposed osteosarcoma cells (Saos2), normal bone cells, and marrow stroma to two different tyrosine kinase (TK) inhibitors: AG-555 and AG-1478. These tyrphostins differ in their effect on signal transduction downstream to the TK receptor (RTK): AG-1478 inhibits src family TKs whereas AG-555 inhibits nuclear TKs. We found that both tyrphostins at 50 microM increased specific alkaline phosphatase (ALP) activity in Saos2 cells. AG-555 abrogated mineralization whereas AG-1478 increased it. Similarly, in human bone-derived cell cultures the same dose of tyrphostins had an opposing effect on mineralization but, in contrast to AG-555, AG-1478 positively selected cells with ALP activity. These tyrphostins also differed in their effect on rat marrow stromal cells. AG-555 decreased cell counts unselectively, whereas the decreased cell counts by AG-1478 resulted in selection of osteoprogenitor cells as indicated by a concordant increase in specific ALP activity. The effect of a lower dose of AG-1478, 5 microM, on the increase in mineralization exceeded its own efficiency in selecting cells with specific ALP activity. Our results indicate that AG-1478 selects and preserves the osteoblastic phenotype, at doses moderately higher than those required to induce mineralization, and substantially higher than the doses required for RTK inhibition. Identification of downstream molecular targets for AG-1478, in marrow stromal cells, might prove useful in designing more selective drugs, capable of separating proliferative from differentiation-inducing activities.

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