Alternatively Initiated Gene 50/RTA Transcripts Expressed during Murine and Human Gammaherpesvirus Reactivation from Latency

Herpesviruses contain large, double-stranded DNA genomes that encode an extensive array of viral proteins required for efficient infection and persistence in host organisms. These viruses establish life-long latent infection in a variety of cell types, a characteristic that is often accompanied by periodic virus reactivation and resumption of lytic replication and egress. Unlike the alpha- and betaherpesviruses, which exhibit neuronal or broad cellular tropism, the gammaherpesviruses are mainly lymphotropic, infecting and establishing latency in B or T lymphocytes. Gammaherpesviruses are associated with the development of lymphomas in both their natural host and in animal models and have therefore been subject to intensive study in an effort to understand, treat, and prevent disease. The gammaherpesvirus type 1 Epstein-Barr virus (EBV) is implicated in the development of several human malignancies, including Burkitt's lymphoma, Hodgkin's lymphoma, and nasopharyngeal carcinoma. Kaposi's sarcoma-associated herpesvirus (KSHV), a gammaherpesvirus type 2, is likewise associated with tumor development in Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease. Although cell culture systems exist to study cells latently infected with these viruses, the narrow tropisms of both EBV and KSHV have necessitated the use of animal models to intimately study the process of primary virus infection and events that contribute to the perpetuation of viral latency in vivo. Murine gammaherpesvirus 68 (MHV68) is a naturally occurring rodent pathogen that mimics several key aspects of both EBV and KSHV infection following experimental infections of inbred mice (5, 34, 41). Substantial sequence homology is shared among the murine and primate gammaherpesviruses in the region of open reading frame 50 (ORF50), which encodes the key lytic switch protein RTA (45). Importantly, in all the characterized gammaherpesviruses the gene 50 transcript contains a short first exon that splices to a long coding exon (Fig. ​(Fig.1).1). For the type 2 gammaherpesviruses the ORF50/RTA reading frame is extended by the exon 1/exon 2 splice, with the translation initiation site located near the 3′ end of exon 1. In the case of the type 1 gammaherpesvirus EBV, exon 1 does not contribute to the RTA coding sequence. Finally, the position of the short first exon is also well-conserved among all the gammaherpesviruses, being located in the region between ORFs 48 and 49 (Fig. ​(Fig.1).1). RTA plays a pivotal role in lytic replication in both human and murine gammaherpesviral infections, and ectopic expression of RTA is sufficient to trigger reactivation into the lytic cycle in latently infected cell lines (33, 40, 49). Because gammaherpesviruses are thought to first infect permissive cells, followed by establishment of a latent infection within lymphocytes, potentially antigenic lytic gene expression must be carefully controlled. This is important both during initial infection, to allow for efficient establishment of latency, as well as during latency when the capability to reactivate remains. This level of regulation is accomplished for both latent and lytic genes by several mechanisms shared between EBV, KSHV, and MHV68, including multiple promoter usage, extensive alternative splicing, and epigenetic modifications (1, 9, 12, 35-37, 47). We demonstrate here that expression of the MHV68 RTA homolog can be driven from a newly defined promoter upstream of the promoter previously identified and that this expression is sufficient to allow for lytic replication and establishment of latency. We also identified an additional exon included in the RTA-encoding transcript generated from this promoter, and we provide evidence that the existence and utility of both the distal promoter and additional exon are conserved among the murine and human gammaherpesviruses. We provide evidence that DNA methylation may serve as a regulatory mechanism for silencing the distal gene 50 promoter during latent infection and may potentially account for the severe reactivation defect observed in mice infected with a gene 50 mutant MHV68 lacking the proximal promoter. Finally, we demonstrate that although the G50pKO virus is defective for reactivation from latently infected splenocytes, this virus is capable of reactivation from peritoneal exudate cells (PECs), indicating that activity of the newly identified distal promoter is regulated in a cell-type-specific manner during latent infection. FIG. 1. Conservation of G50/BRLF1/Rta coding region among gammaherpesviruses. Schematic representation of the G50/BRLF1/Rta coding regions of MHV68, EBV, KSHV, and HVS. The nucleotide positions for G50 exons 1 and 2 are provided. The box drawn around exon 1 demonstrates ...