Development of an ORF45-Derived Peptide To Inhibit the Sustained RSK Activation and Lytic Replication of Kaposi's Sarcoma-Associated Herpesvirus

Abstract

The lytic replication of Kaposi’s sarcoma-associated herpesvirus (KSHV) requires sustained extracellular signal-regulated kinase (ERK)-p90 ribosomal S6 kinase (RSK) activation, which is induced by an immediate early (IE) gene-encoded tegument protein called ORF45, to promote the late transcription and translation of viral lytic genes. An ORF45-null or single-point F66A mutation in ORF45 abolishes ORF45-RSK interaction and sustained ERK-RSK activation during lytic reactivation and subsequently results in a significant decrease in late lytic gene expression and virion production, indicating that ORF45-mediated RSK activation plays a critical role in KSHV lytic replication. Here, we demonstrate that a short ORF45-derived peptide in the RSK-binding region is sufficient for disrupting ORF45-RSK interaction, consequently suppressing lytic gene expression and virion production. We designed a nontoxic cell-permeable peptide derived from ORF45, TAT-10F10, which is composed of the ORF45 56 to 76 amino acid (aa) region and the HIV Tat protein transduction domain, and this peptide markedly inhibits KSHV lytic replication in iSLK.219 and BCBL1 cells. Importantly, this peptide enhances the inhibitory effect of rapamycin on KSHV-infected cells and decreases spontaneous and hypoxia-induced lytic replication in KSHV-positive lymphoma cells. These findings suggest that a small peptide that disrupts ORF45-RSK interaction might be a promising agent for controlling KSHV lytic infection and pathogenesis. IMPORTANCE ORF45-induced RSK activation plays an essential role in KSHV lytic replication, and ORF45-null or ORF45 F66A mutagenesis that abolishes sustained RSK activation and RSK inhibitors significantly decreases lytic replication, indicating that the ORF45-RSK association is a unique target for KSHV-related diseases. However, the side effects, low affinity, and poor efficacy of RSK modulators limit their clinical application. In this study, we developed a nontoxic cell-permeable ORF45-derived peptide from the RSK-binding region to disrupt ORF45-RSK associations and block ORF45-induced RSK activation without interfering with S6K1 activation. This peptide effectively suppresses spontaneous, hypoxia-induced, or chemically induced KSHV lytic replication and enhances the inhibitory effect of rapamycin on lytic replication and sensitivity to rapamycin in lytic KSHV-infected cells. Our results reveal that the ORF45-RSK signaling axis and KSHV lytic replication can be effectively targeted by a short peptide and provide a specific approach for treating KSHV lytic and persistent infection.

Suppression of KSHV lytic replication and primary effusion lymphoma by selective RNF5 inhibition

“…We further investigated whether KSHV gene expression was affected by RNF5 loss in BCBL1 cells. Approximately 5% of BCBL1 cells undergo ORF45-positive spontaneous lytic replication [ 39 ], low spontaneous expression of KSHV lytic genes was observed in BCBL1 cells, and the 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment greatly induced lytic gene expression [ 40 ]. Both spontaneous and TPA-induced lytic gene expression levels of ORF45 and K8 were decreased in RNF5 KO BCBL1 cells compared with control cells ( Fig 1E ), whereas the latent gene expression level of LANA was not affected.…”

Murine gammaherpesvirus 68 ORF45 stimulates B2 retrotransposon and pre-tRNA activation in a manner dependent on mitogen-activated protein kinase (MAPK) signaling

“…In KSHV, ORF36 is a substrate of ORF45-activated kinases and ORF45 enhances the kinase activity of ORF36; although untested, this may similarly occur for the MHV68 homologs. KSHV ORF45 causes sustained activation of p90 ribosomal kinases (RSKs) and extracellular regulated kinases (ERKs) (59)(60)(61)(62)(63)(64), and subsequent studies also identified that KSHV ORF45, ORF36, and RSK form a stable complex (57). Another function of KSHV ORF45 is suppression of interferon regulatory factor 7 (IRF7), a crucial regulator of type I interferon gene expression (78)(79)(80).…”

“…signaling KSHV ORF45 interacts with the cellular p90 ribosomal S6 kinase (RSK1/2) and forms a ternary complex with the extracellular signal-regulated kinase (ERK2) of the mitogen-activated protein kinase (MAPK) pathway, strongly stimulating their kinase activities (59)(60)(61)(62)(63)(64)(65). This correlates with sustained phosphorylation of RSK1/2 and ERK1/2, which are functional mediators of MAPK/ERK signaling, and are important for MHV68 lytic infection (66).…”

Recent Advances in Developing Treatments of Kaposi’s Sarcoma Herpesvirus-Related Diseases

“…BI-D1870, a RSK1/2 inhibitor, suppresses KSHV lytic gene expression and virus production. The RSK1/2 serine/threonine kinase is activated by its interaction with KSHV pORF45 and a small peptide blocking this interaction has been shown to inhibit viral lytic gene expression and viral progeny formation [133]. Furthermore, crizotinib, an inhibitor of ALK and c-Met, suppresses the growth of PEL cells in a mouse xenograft model [134].…”