The Virion-Associated Open Reading Frame 49 of Murine Gammaherpesvirus 68 Promotes Viral Replication both In Vitro and In Vivo as a Derepressor of RTA

Abstract

Replication and transcription activator (RTA), an immediate-early gene, is a key molecular switch to evoke lytic replication of gammaherpesviruses. Open reading frame 49 (ORF49) is conserved among gammaherpesviruses and shown to cooperate with RTA in regulating virus lytic replication. Here we show a molecular mechanism and in vivo functions of murine gammaherpesvirus 68 (MHV-68 or ␥HV-68) ORF49. MHV-68 ORF49 was transcribed and translated as a late gene. The ORF49 protein was associated with a virion, interacting with the ORF64 large tegument protein and the ORF25 capsid protein. Moreover, ORF49 directly bound to RTA and its negative cellular regulator, poly(ADP-ribose) polymerase-1 (PARP-1), and disrupted the interactions of RTA and PARP-1. Productive replication of an ORF49-deficient mutant virus (49S) was attenuated in vivo as well as in vitro. Likewise, latent infection was also impaired in the spleen of 49S-infected mice. Taken together, our results suggest that the virion-associated ORF49 protein may promote virus replication both in vitro and in vivo by providing an optimal environment in the early phase of virus infection as a derepressor of RTA.

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

“…PF-8 was sufficient to induce PARP-1 downregulation. In addition to RTA and ORF49, ORF59 was shown to interact with PARP-1 in a genome-wide yeast two-hybrid screen of MHV-68 (8,11,24), although this interaction has not been validated in mammalian cells. KSHV ORF59 encodes PF-8, which is essential for virus genome replication (13)(14)(15)25) as an early late protein (26,27).…”

“…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 further promoting lytic replication by enhancing RTA-mediated transactivation (11). Here we report another viral strategy that modulates the activity of PARP-1 to facilitate the lytic replication of gammaherpesvirus.…”

“…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)(6)(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)(9)(10)(11). PARP-1 is also known to be a component of the viral replication complex, increasing viral genome replication (9,12).…”

Structure of the Open Reading Frame 49 Protein Encoded by Kaposi's Sarcoma-Associated Herpesvirus

“…In KSHV, this is governed by the transcriptional control facilitated by RTA, and studies have demonstrated that ORF49 KSHV and its homologs are able to cooperate with RTA to enhance its transcriptional activation (13,(16)(17)(18)(20)(21)(22). However, the underlying mechanism of this process is still unclear, with limited functional studies and lack of structural information.…”

“…Another KSHV-encoded protein, the open reading frame 49 protein (ORF49 KSHV ), has been shown to upregulate the transcriptional activity of RTA (18). The protein homologs of ORF49 KSHV in Epstein-Barr virus (EBV) and murine herpesvirus 68 (MHV-68) display similar effects in the respective viruses (19)(20)(21)(22). ORF49 KSHV has been demonstrated to enhance lytic replication in KSHV, while the lack of the ORF49 MHV-68 protein has been shown to attenuate viral growth of MHV-68 (23).…”