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...
Background: Diseases caused by gammaherpesviruses continue to be a challenge for human health and antiviral treatment. Most of the commonly used antiviral drugs are directed against viral gene products. However, the emergence of drug-resistant mutations ma limit the effectiveness of these drugs. Since viruses require a host cell to propagate, the search for host cell targets is an interestin alternative. Methods: In this study, we infected three different cell types (fibroblasts, endothelial precursor cells and macrophages with a murine gammaherpesvirus and analysed the host cell response for changes either common to all or unique to a particular cell type using oligonucleotide microarrays. Results: The analysis revealed a number of genes whose transcription was significantly up- or down-regulated in either one or two of the cell types tested. After infection, only two genes, Lman1 (also known as ERGIC53) an synaptobrevin-like 1 (sybl1) were significantly up-regulated in all three cell types, suggestive for a general role for the virus life cycl independent of the cell type. Both proteins have been implicated in cellular exocytosis and transport of glycoproteins through the secre tory pathway. To test the significance of the observed up-regulation, the functionality of these proteins was modulated, and the effect on virus replication was monitored. Inhibition of either Lman1 or sybl1 resulted in a significant reduction in virus production. Conclusions: This suggests that proteins of the secretory pathway which appear to be rate limiting for virus production may represent new targets for intervention.
BackgroundGammaherpesviruses cause important infections of humans, in particular in immunocompromised patients. The genomes of gammaherpesviruses contain variable numbers of internal repeats whose precise role for in vivo pathogenesis is not well understood.Methodology/Principal FindingsWe used infection of laboratory mice with murine gammaherpesvirus 68 (MHV-68) to explore the biological role of the 40 bp internal repeat of MHV-68. We constructed several mutant viruses partially or completely lacking this repeat. Both in vitro and in vivo, the loss of the repeat did not substantially affect lytic replication of the mutant viruses. However, the extent of splenomegaly, which is associated with the establishment of latency, and the number of ex vivo reactivating and genome positive splenocytes were reduced. Since the 40 bp repeat is part of the hypothetical open reading frame (ORF) M6, it might function as part of M6 or as an independent structure. To differentiate between these two possibilities, we constructed an N-terminal M6STOP mutant, leaving the repeat structure intact but rendering ORF M6 unfunctional. Disruption of ORF M6 did neither affect lytic nor latent infection. In contrast to the situation in lytically infected NIH3T3 cells, the expression of the latency-associated genes K3 and ORF72 was reduced in the latently infected murine B cell line Ag8 in the absence of the 40 bp repeat.Conclusions/SignificanceThese data suggest that the 40 bp repeat contributes to latency amplification and might be involved in the regulation of viral gene expression.
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