Human herpesvirus 8 (HHV-8) encodes four interferon regulatory factor homologues (vIRFs) that interact, in inhibitory fashion, with cellular IRFs and/or a variety of other cellular proteins involved in innate immunity and cellular stress signaling. For vIRF-1 these proteins include CBP/p300 (required for IRF-induced transcription and interferon signaling), p53, p53-activating ATM kinase, BH3-only proteins Bim and Bid, TGF?-activated Smad transcription factors, and retinoic acid and interferon induced protein 19 (GRIM19). Recent affinity precipitation and mass spectrometry (MS) analyses undertaken in our laboratory have identified novel cellular targets of vIRF- 1. Among these are translational initiation factor eIF4A1, poly(rC)-binding proteins 1 and 2, and USP7 (which has since been published by others). Interestingly, eIF4A1 and USP7 were detected at far higher abundance in MS-analyzed vIRF-1 co-precipitates [from HHV-8+ BCBL-1 primary effusion lymphoma (PEL) cells] than were previously reported interactors of vIRF-1 (e.g., CBP/p300). Interactions of vIRF-1 with eIF4A1 and PCBP1 (in addition to other proteins) have been verified by immunoprecipitation experiments. Of note, eIF4A1 expression has been reported to be elevated in several cancers and to enhance translation of mRNAs encoding proteins such as cyclins, CDKs and pro-survival and angiogenic factors involved in cancer development and progression. Regulation by eIF4A1 is through unwinding of poly-G repeat-induced G-quadruplex and other secondary structures in long 5' untranslated regions of client mRNAs. PCBP1/2 contain hnRNP-K homology (KH) domains and are related to hnRNP-K ATP helicase; the functions of the PCBPs are varied but include translational regulation via enhancement of internal ribosome entry site (IRES)-dependent initiation, in addition to mRNA stabilization and transcriptional regulation. We hypothesize that interactions of vIRF-1 with eIF4A1, PCBP1 and PCBP2 regulate translation to promote HHV-8 infected cell survival and function during latency and/or productive replication via enhanced expression of eIF4A1- and PCBP1/2-regulated mRNAs such as Bcl-2, Bcl-x and c-IAP (eIF4A1) and Bag-1, XIAP and c-myc (PCBP1/2). We have identified pro-growth/survival activity of vIRF-1 in latently infected PEL cells and pro-replication activities of vIRF-1 in lytically reactivated PEL and other cell types. We propose here to focus follow-up experimental analysis on vIRF-1-targeted eIF4A1 and PCBPs 1 and 2 to characterize an entirely new mechanism of vIRF-1 function, via translational regulation, which is of significance scientifically and potentially also for future development of anti-viral and therapeutic strategies targeting these interactions in infected cells.
Human herpesvirus 8 (HHV-8) encodes four interferon regulatory factor homologues (vIRFs), one of which, vIRF-1, has demonstrated roles in promoting productive replication and latently infected cell viability, in addition to oncogenic properties. The vIRF-1-targeted cellular proteins and mechanisms underlying these activities are unknown but are likely to be important contributors to HHV-8-associated malignancies Kaposi's sarcoma, primary effusion lymphoma and multicentric Castleman's disease in addition to playing major roles in normal virus biology. The project outlined in this application is focused on the characterization of newly-identified and novel interactions of vIRF-1 with translation initiation factors regulating the expression of pro-survival, oncogenic and other critical cellular proteins; the research findings will not only reveal a new paradigm of vIRF-1 activity, but could potentially be exploited for the development of anti-viral and therapeutic agents targeting these interactions.
