Unconventional Sequence Requirement for Viral Late Gene Core Promoters of Murine Gammaherpesvirus 68

Infection with the human gammaherpesviruses, Epstein-Barr virus (EBV) and Kaposi''s sarcoma-associated herpesvirus (KSHV), is associated with several cancers. During lytic replication of herpesviruses, viral genes are expressed in an ordered cascade. However, the mechanism by which late gene expression is regulated has not been well characterized in gammaherpesviruses. In this study, we have investigated the cis element that mediates late gene expression during de novo lytic infection with murine gammaherpesvirus 68 (MHV-68). A reporter system was established and used to assess the activity of viral late gene promoters upon infection with MHV-68. It was found that the viral origin of lytic replication, orilyt, must be on the reporter plasmid to support activation of the late gene promoter. Furthermore, the DNA sequence required for the activation of late gene promoters was mapped to a core element containing a distinct TATT box and its neighboring sequences. The critical nucleotides of the TATT box region were determined by systematic mutagenesis in the reporter system, and the significance of these nucleotides was confirmed in the context of the viral genome. In addition, EBV and KSHV late gene core promoters could be activated by MHV-68 lytic replication, indicating that the mechanisms controlling late gene expression are conserved among gammaherpesviruses. Therefore, our results on MHV-68 establish a solid foundation for mechanistic studies of late gene regulation.     

The M10 Locus of Murine Gammaherpesvirus 68 Contributes to both the Lytic and the Latent Phases of Infection

Murine gammaherpesvirus 68 (MHV-68) is closely related to Epstein-Barr virus and Kaposi''s sarcoma-associated herpesvirus (KSHV) and provides a small-animal model to study the pathogenesis of gammaherpesvirus (γHV) infections. According to the colinear organization of the γHV genomes, the M10 locus is situated at a position equivalent to the K12 locus of KSHV, which codes for proteins of the kaposin family. The M10 locus of MHV-68 has been predicted to code for three overlapping open reading frames (M10a, M10b, and M10c [M10a-c]) with unknown function. In addition, the M10 locus contains a lytic origin of replication (oriLyt). To elucidate the function of the M10 locus during lytic and latent infections, we investigated, both in vitro and in vivo, the following four recombinant viruses which were generated using MHV-68 cloned as a bacterial artificial chromosome: (i) a mutant virus with a deletion which affects both the coding region for M10a-c and the oriLyt; (ii) a revertant virus in which both the M10a-c coding region and the oriLyt were reverted to those of the wild type; (iii) a virus with an ectopic insertion of the oriLyt, which restores the function of the oriLyt but not the M10a-c coding region; and (iv) a mutant virus with a deletion in the oriLyt only. While the mutants were slightly attenuated with regard to lytic replication in cell culture, they showed severe growth defects in vivo. Both lytic replication and latency amplification were strongly reduced. In contrast, both the revertant virus and the virus with the ectopic oriLyt insertion grew very similarly to the parental wild-type virus both in vitro and in vivo. Thus, we provide genetic evidence that mutation of the oriLyt, and not of putative protein coding sequences within the M10a-c region, is responsible for the observed phenotype. We conclude that the oriLyt in the M10 locus plays an important role during infection of mice with MHV-68.Diseases caused by gammaherpesviruses continue to be a challenge for human health. The prototypic gamma-1 herpesvirus Epstein-Barr virus (EBV) is associated with lymphomas and nasopharyngeal carcinoma (22). Human herpesvirus 8 (also called Kaposi''s sarcoma-associated herpesvirus [KSHV]), a gamma-2 herpesvirus, is associated with lymphoproliferative disorders and Kaposi''s sarcoma (24). In vivo studies of gammaherpesvirus pathogenesis have been limited to clinical investigation of the infection because of the restricted host range of these viruses. The murine gammaherpesvirus 68 (MHV-68) is also a member of the gammaherpesvirus subfamily and is closely related to KSHV and EBV. Since there exist no good animal models for KSHV and EBV, MHV-68 serves as a small-animal model to investigate gammaherpesvirus pathogenesis (6, 9, 10, 13, 21, 25, 26, 30). MHV-68 is a natural pathogen of wild rodents (7) and is capable of infecting laboratory mice. The nucleotide sequence of MHV-68 is similar to that of EBV and even more closely related to that of KSHV (29). MHV-68 contains genes which are homologous to cellular genes or to genes of other gammaherpesviruses. In addition, it contains virus-specific genes. Many of the latency- and transformation-associated proteins of the gammaherpesviruses, for example, EBNA and LMP of EBV, appear to be encoded by virus-specific genes, yet it has been suggested that pathogenesis-associated genes of gammaherpesviruses may be contained in similarly positioned genome regions (29). The virus-specific genes of MHV-68 were originally designated M1 to M14 (29). The M10 locus has been predicted to code for three overlapping open reading frames (M10a, M10b, and M10c [M10a-c]) (29). While several MHV-68-specific genes have been shown to code for proteins with important functions, the function of M10 is still unknown. A more recent report even considered M10a-c rather unlikely to code for proteins (21). Importantly, the M10 locus also contains a lytic origin of replication (oriLyt) (3, 8). According to the colinear organization of the gammaherpesvirus genomes, the M10 locus is situated at a position equivalent to that of the K12 locus of KSHV. K12 encodes proteins of the kaposin family. Kaposin proteins are involved in cellular transformation and in stabilization of cytokine mRNAs (16-18,20). Of note, the K12 locus also contains an oriLyt (5).Here, we investigated the function of the M10 locus during lytic and latent infections by studying mutant viruses with deletions in the M10 loci, either affecting both the coding region for M10a-c and the oriLyt or the oriLyt only. While the mutants were slightly attenuated with regard to lytic replication in cell culture, they showed severe growth defects in vivo. Both lytic replication and latency amplification were strongly reduced in mice infected with the mutant viruses. In contrast, a revertant virus in which both the M10a-c coding region and the oriLyt were reverted to those of the wild type and a virus with an ectopic insertion of the oriLyt which restores the function of the oriLyt but not the M10a-c coding region grew very similarly to the parental wild-type virus both in vitro and in vivo. Thus, we provide genetic evidence that mutation of the oriLyt, and not of putative protein coding sequences within the M10a-c region, is responsible for the observed phenotype.     

Perturbation of Lytic and Latent Gammaherpesvirus Infection in the Absence of the Inhibitory Receptor CEACAM1

Control of gammaherpesvirus infections requires a complex, well orchestrated immune response regulated by positive and negative co-signaling molecules. While the impact of co-stimulatory molecules has been addressed in various studies, the role of co-inhibitory receptors has not been tested. The ITIM-bearing CEACAM1 is an inhibitory receptor expressed by a variety of immune cells, including B, T and NK cells. Using Ceacam1^−/− mice, we analyzed the in vivo function of CEACAM1 during acute and latent murine gammaherpesvirus 68 (MHV-68) infection. During acute lytic replication, we observed lower virus titers in the lungs of Ceacam1^−/− mice than in WT mice. In contrast, during latency amplification, Ceacam1^−/− mice displayed increased splenomegaly and a higher latent viral load in the spleen. Analysis of the immune response revealed increased virus-specific antibody levels in Ceacam1^−/− mice, while the magnitude of the T cell-mediated antiviral immune response was reduced. These findings suggest that inhibitory receptors can modulate the efficacy of immune responses against gammaherpesvirus infections.