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Regulation of orf73 (LANA) gene expression is critical to the establishment and maintenance of latency following infection by members of the gamma-2 herpesvirus (rhadinovirus) family. Previous studies of murine gammaherpesvirus 68 (gammaHV68) have demonstrated that loss of LANA function results in a complete failure to establish virus latency in the spleens of laboratory mice. Here we report the characterization of alternatively spliced LANA and v-cyclin (orf72) transcripts encoded by gammaHV68. Similar to other rhadinoviruses, alternative splicing, coupled with alternative 3' processing, of a ca. 16-kb transcriptional unit can lead to expression of either LANA or v-cyclin during gammaHV68 infection. Spliced LANA and v-cyclin transcripts were initially identified from an analysis of the gammaHV68 latently infected B-cell lymphoma cell line S11E, but were also detected during lytic infection of NIH 3T12 fibroblasts. 5' Random amplification of cDNA ends (RACE) analyses identified two distinct promoters, p1 and p2, that drive expression of spliced LANA transcripts. Analysis of p1 and p2, using transiently transfected reporter constructs, mapped the minimal sequences required for promoter activity and demonstrated that both promoters are active in the absence of any viral antigens. Analysis of spliced LANA and v-cyclin transcripts in spleens recovered from latently infected mice at days 16 and 42 postinfection revealed that spliced v-cyclin transcripts can only be detected sporadically, suggesting that these may be associated with cells reactivating from latency. In contrast, spliced LANA transcripts were detected in ca. 1 in 4,000 splenocytes harvested at day 16 postinfection. Notably, based on the frequency of viral genome-positive splenocytes at day 16 postinfection (ca. 1 in 200), only 5 to 10% of viral genome-positive splenocytes express LANA. The failure of the majority of infected splenocytes at day 16 postinfection to express LANA may contribute to the contraction in the frequency of latently infected splenocytes as chronic infection is established, due to failure to maintain the viral episome in proliferating B cells.

Murine gammaherpesvirus 68 (gammaHV68) provides a tractable small animal model with which to study the mechanisms involved in the establishment and maintenance of latency by gammaherpesviruses. Similar to the human gammaherpesvirus Epstein-Barr virus (EBV), gammaHV68 establishes and maintains latency in the memory B-cell compartment following intranasal infection. Here we have sought to determine whether, like EBV infection, gammaHV68 infection in vivo is associated with B-cell proliferation during the establishment of chronic infection. We show that gammaHV68 infection leads to significant splenic B-cell proliferation as late as day 42 postinfection. Notably, gammaHV68 latency was found predominantly in the proliferating B-cell population in the spleen on both days 16 and 42 postinfection. Furthermore, virus reactivation upon ex vivo culture was heavily biased toward the proliferating B-cell population. DNA methyltransferase 1 (Dnmt1) is a critical maintenance methyltransferase which, during DNA replication, maintains the DNA methylation patterns of the cellular genome, a process that is essential for the survival of proliferating cells. To assess whether the establishment of gammaHV68 latency requires B-cell proliferation, we characterized infections of conditional Dnmt1 knockout mice by utilizing a recombinant gammaHV68 that expresses Cre-recombinase (gammaHV68-Cre). In C57BL/6 mice, the gammaHV68-Cre virus exhibited normal acute virus replication in the lungs as well as normal establishment and reactivation from latency. Furthermore, the gammaHV68-Cre virus also replicated normally during the acute phase of infection in the lungs of Dnmt1 conditional mice. However, deletion of the Dnmt1 alleles from gammaHV68-infected cells in vivo led to a severe ablation of viral latency, as assessed on both days 16 and 42 postinfection. Thus, the studies provide direct evidence that the proliferation of latently infected B cells is critical for the establishment of chronic gammaHV68 infection.