Three distinct regions of the murine gammaherpesvirus 68 genome are transcriptionally active in latently infected mice

The program(s) of gene expression operating during murine gammaherpesvirus 68 (gamma HV68) latency is undefined, as is the relationship between gamma HV68 latency and latency of primate gammaherpesviruses. We used a nested re verse transcriptase PCR strategy (sensitive to approximately one copy of ga mma HV68 genome for each genomic region tested) to screen for the presence of viral transcripts in latently infected mice. Based on the positions of k nown latency-associated genes in other gammaherpesviruses, we screened for the presence of transcripts corresponding to 11 open reading frames (ORFs) in the gamma HV68 genome in RNA from spleens and peritoneal cells of latent ly infected B-cell-deficient (MuMT) mice which have been shown contain high levels of reactivable latent gamma HV68 (K. E. Week, M, L. Barkon, L, I. Y oo, S, H. Speck, and H. W. Virgin, J, Virol, 70: 6775-6780, 1996). To contr ol for the possible presence of viral lytic activity, we determined that RN A from latently infected peritoneal and spleen cells contained few or no de tectable transcripts corresponding to seven ORFs known to encode viral gene products associated with lytic replication. However, we did detect low-lev el expression of transcripts arising from the region of gene 50 (encoding t he putative homolog of the Epstein-Barr virus BRLF1 transactivator) in peri toneal but not spleen cells. Latently infected peritoneal cells consistentl y scored for expression of RNA derived from 4 of the 11 candidate latency-a ssociated ORFs examined, including the regions of ORF M2, ORF M11 (encoding v-bcl-2), gene 73 (a homolog of the Kaposi's sarcoma-associated herpesviru s [human herpesvirus 8] gene encoding latency-associated nuclear antigen), and gene 74 (encoding a G-protein coupled receptor homolog, v-GCR), Latentl y infected spleen cells consistently scored positive for RNA derived from 3 of the 11 candidate latency-associated ORFs examined, including ORF M2, OR F M3, and ORF M9, To further characterize transcription of these candidate latency-associated ORFs, we examined their transcription in lytically infec ted fibroblasts by Northern analysis. We detected abundant transcription fr om regions of the genome containing ORF M3 and ORF M9, as well as the known lytic-cycle genes. However, transcription of ORF M2, ORF M11, gene 73, and gene 74 was barely detectable in lytically infected fibroblasts, consisten t with a role of these viral genes during latent infection. We conclude tha t (i) we have identified several candidate latency genes of murine gamma HV 68, (ii) expression of genes during latency may be different in different o rgans, consistent with multiple latency programs and/or multiple cellular s ites of latency, and (iii) regions of the viral genome (v-bcl-2 gene, v-GCR gene, and gene 73) are transcribed during latency with both gamma HV68 and primate gammaherpesviruses, The implications of these findings for replaci ng previous operational definitions of gamma HV68 latency with a molecular definition are discussed.