Murine Gammaherpesvirus 68 Contains Two Functional Lytic Origins of Replication

Abstract

A 1.25-kbp DNA fragment from the right side of the genome containing the lytic origin of replication (oriLyt) of murine gammaherpesvirus 68 (MHV-68) has been identified by a plasmid replication assay. Here we show that a mutant MHV-68 with a deletion of an essential part of this oriLyt, generated by using an MHV-68 bacterial artificial chromosome, was only slightly attenuated and still able to replicate but that a mutant containing an additional deletion on the left side of the genome was replication deficient. The newly identified region was sufficient to support plasmid replication, thus providing evidence for a second oriLyt.Murine gammaherpesvirus 68 (MHV-68) is a member of the gammaherpesvirus subfamily and is closely related to Kaposi's sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus. Since there are no good animal models for KSHV and Epstein-Barr virus, MHV-68 serves as a model to investigate gammaherpesvirus pathogenesis in vivo (8,(14)(15)(16)18). Since MHV-68 replicates well in tissue culture, it is a good model to study early stages of gammaherpesvirus infection as well as mechanisms controlling viral replication during the lytic cycle. Using in vitro plasmid replication assays which were carried out in the presence of viral replication proteins, Deng and colleagues mapped one lytic origin of replication (oriLyt) to a 1.25-kbp region in the right side of the MHV-68 genome (7). The 1.25-kbp sequence between nucleotides 100,723 and 101,974 was shown to be responsible for replication, and nucleotides 101,248 to 101,974 were identified as being absolutely essential.To evaluate the impact of the reported oriLyt, we generated a mutant genome with a deletion in its essential part, using an MHV-68 bacterial artificial chromosome (BAC) (1). We generated this BAC mutant (mutant A) (Table 1) lacking nucleotide positions 101,530 to 101,731 by a two-step replacement procedure which does not leave behind any foreign sequence (3, 13). For this purpose, the HindIII D fragment of MHV-68 (9) was cloned into the shuttle plasmid pST76K-SR. Subsequently, nucleotides 101,530 to 101,731 were removed from this fragment by digestion with SfiI and BspEI, followed by blunting of the ends and religation. Surprisingly, we were able to reconstitute a mutant virus which was only slightly attenuated. This finding suggested the presence of a second oriLyt in MHV-68.The structure of the MHV-68 oriLyt is similar to that in KSHV, which contains two oriLyts, with one on either side of the genome, sharing an almost identical 1,153-bp sequence and a 600-bp GC-rich downstream repeat sequence (2). In contrast to the case for KSHV, such a duplicated oriLyt sequence has not been identified in the left side of the MHV-68 genome (7). However, MHV-68 contains a GC-rich 40-bp repeat sequence in the left side of the genome (nucleotides 26,778 to 28,191) (17). In addition, the genomic coordinates 101,765 to 101,624 (belonging to the essential part of the oriLyt) have been found to be Ͼ70% identical to coordinates 26,232 to 26,374 (5). T...

Replication and Transcription Activator (RTA) of Murine Gammaherpesvirus 68 Binds to an RTA-Responsive Element and Activates the Expression of ORF18

“…In addition, it was shown previously that KSHV RTA binds to the left oriLyt and regulates oriLyt-associated transcription (60,61). As for MHV-68, we and another group have previously identified two oriLyt regions (2,20,28). We have further mapped the minimal region of the left oriLyt to nucleotides (nt) 25695 to 26883 (28).…”

A Gammaherpesviral Internal Repeat Contributes to Latency Amplification

“…Defects in latent gene expression and impaired immune evasion might explain the phenotype of the Delta 40 bp repeat mutant, yet there are also other possibilities: First, we have recently demonstrated that the 40 bp repeat forms part of one lytic origin of MHV-68 [59]. Thus, although having no effect in vitro , deletion of the 40 bp repeat might lead to impairment of viral reactivation in vivo .…”

Identification and functional characterization of the left origin of lytic replication of murine gammaherpesvirus 68

“…While this manuscript was in preparation, Adler et al reported identification of a relatively large region which contains a second ori Lyt in MHV-68 (Adler et al, 2007). In our independent study, we first employed a high density transposon-mediated mutagenesis method to fine-map cis -elements essential for the MHV-68 right ori Lyt.…”