Murine gammaherpesvirus 68 (MHV-68 [also referred to as ␥HV68]) is phylogenetically related to Kaposi's sarcoma-associated herpesvirus (KSHV [also referred to as HHV-8]) and Epstein-Barr virus (EBV).Gammaherpesviruses are known to establish latency in lymphocytes and are associated with tumorigenesis (5-7, 10, 48). Two important human pathogens in the gammaherpesvirus subfamily of herpesviruses are Kaposi's sarcoma-associated herpesvirus (KSHV [also referred to as HHV-8]) and EpsteinBarr virus (EBV). KSHV and EBV are associated with several malignancies, including B-cell lymphomas, nasopharyngeal carcinoma, and Kaposi's sarcoma (22,23,27,30,32). Studies of KSHV and EBV are limited by the lack of cell lines able to support efficient productive infection and by the restricted host ranges of the viruses (11, 33). Murine gammaherpesvirus 68 (MHV-68 [also referred to as ␥HV68]) is another member of the gammaherpesvirus subfamily. However, in vitro cell culture systems are available to study productive de novo infection by MHV-68, as well as latency and reactivation (34, 40). MHV-68 forms plaques on monolayers of many cell lines, making it possible to genetically manipulate the viral genome. MHV-68 establishes lytic and latent infections in laboratory mice (47), providing a system for examining host-virus interactions (24,25,36,42,43). These characteristics of MHV-68 make it possible to examine the functions of individual viral genes at various points during the viral life cycle, including de novo infection. De novo infection analyses have not been possible for other gammaherpesviruses such as EBV and KSHV.Herpesviruses have two distinct life cycle phases, latency and lytic replication. Reactivation from latency to lytic replication is essential for transmission of the virus from host to host and thus is one important aspect of herpesvirus biology. A viral protein, replication and transcription activator (RTA) is primarily encoded by open reading frame (ORF) 50, which is well conserved among gammaherpesviruses. RTA is necessary and sufficient to reactivate MHV-68 and drive the lytic cycle to completion in latently infected B cells (14,19,54,55). Similarly, KSHV RTA has been shown to be sufficient to reactivate the virus from latently infected B cells derived from KSHVassociated lymphomas (20,46). Although two EBV proteins, RTA and ZEBRA, function in a cooperative manner to reactivate the viral lytic cycle (3, 18, 21), RTA alone can disrupt latency in some latently infected cell lines (31, 56). These studies indicate that RTA of gammaherpesviruses plays a conserved role in virus reactivation.We have constructed custom membrane arrays representing nearly all of the known and predicted MHV-68 ORFs to explore the patterns of viral gene expression. To illustrate the value of genome-wide transcription analysis, we used the MHV-68 DNA arrays to identify a novel regulatory element for a specific gene, to identify latency-associated transcripts not previously recognized, and to define the genome-wide effects of a specific gene...

Human gammaherpesviruses, Epstein-Barr virus, and human herpesvirus 8/Kaposi's sarcoma-associated herpesvirus are important pathogens associated with diseases, including lymphomas and other malignancies. Murine gammaherpesvirus 68 (MHV-68) is used as an experimental model system to study the host immune control of infection and explore novel vaccine strategies based on latency-deficient live viruses. We studied the properties and the potential of a recombinant MHV-68 (AC-RTA) in which the genes required for persistent infection were replaced by a constitutively expressed viral transcription activator, RTA, which dictates the virus to lytic replication. After intranasal infection of mice, replication of AC-RTA in the lung was attenuated, and no AC-RTA virus or viral DNA was detected in the isolated splenocytes, indicating a lack of latency in the spleen. Infection of the AC-RTA virus elicited both cellular immune responses and virus-specific IgG at a level comparable to that elicited by infection of the wild-type virus. Importantly, vaccination of AC-RTA was able to protect mice against subsequent challenge by the wild-type MHV-68. AC-RTA provides a vaccine strategy for preventing infection of human gammaherpesviruses. Furthermore, our results suggest that immunity to the major latent antigens is not required for protection.