Human herpesvirus 8 (HHV-8

Nasopharyngeal carcinoma, Kaposi's sarcoma, and B-cell lymphomas are human malignancies associated with gammaherpesvirus infections. Members of this virus family are characterized by their ability to establish latent infections in lymphocytes. The latent viral genome expresses very few gene products. The infected cells are therefore poorly recognized by the host immune system, allowing the virus to persist for long periods of time. We sought to identify the cell-specific factors that allow these viruses to redirect their life cycle from productive replication to latency. We find that the cellular transcription factor NF-B can regulate this process. Epithelial cells and fibroblasts support active (lytic) gammaherpesvirus replication and have low NF-B activity. However, overexpression of NF-B in these cells inhibits the replication of the gammaherpesvirus murine herpesvirus 68 (MHV68). In addition, overexpression of NF-B inhibits the activation of lytic promoters from MHV68 and human gammaherpesviruses Kaposi's sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV). In lymphocytes latently infected with KSHV or EBV, the level of NF-B activity is high, and treatment of these cells with an NF-B inhibitor leads to lytic protein synthesis consistent with virus reactivation. These results suggest that high levels of NF-B can inhibit gammaherpesvirus lytic replication and may therefore contribute to the establishment and maintenance of viral latency in lymphocytes. They also suggest that NF-B may be a novel target for the disruption of virus latency and therefore the treatment of gammaherpesvirus-related malignancies.

␥-Herpesviruses, Epstein-Barr virus, and Kaposi's sarcoma-associated herpesvirus are important human pathogens, because they are involved in tumor development. Murine ␥-herpesvirus-68 (MHV-68 or ␥HV-68) has emerged as a small animal model system for the study of ␥-herpesvirus pathogenesis and host-virus interactions. To identify the genes required for viral replication in vitro and in vivo, we generated 1,152 mutants using signature-tagged transposon mutagenesis on an infectious bacterial artificial chromosome of MHV-68. Almost every ORF was mutated by random insertion. For each ORF, a mutant with an insertion proximal to the N terminus of each ORF was examined for the ability to grow in fibroblasts. Our results indicate that 41 genes are essential for in vitro growth, whereas 26 are nonessential and 6 attenuated. Replication-competent mutants were pooled to infect mice, which led to the discovery of ORF 54 being important for MHV-68 to replicate in the lung. This genetic analysis of a tumor-associated herpesvirus at the whole genome level validates signature-tagged transposon mutagenesis screening as an effective genetic system to identify important virulent genes in vivo and define interactions with the host immune system. functional mapping ͉ herpesvirus ͉ bacterial artificial chromosome ͉ deoxyuridine-triphosphatase ͉ transposition