ORF73-Null Murine Gammaherpesvirus 68 Reveals Roles for mLANA and p53 in Virus Replication

Abstract

Gammaherpesviruses establish lifelong, latent infections in host lymphocytesMurine gammaherpesvirus 68 (MHV68) is a member of the Gammaherpesvirinae subfamily of viruses that naturally infects rodents. Following infection of inbred mice by various routes, MHV68 undergoes acute replication at the primary site of inoculation, a process that facilitates the seeding of several cell types, including macrophages, dendritic cells, epithelial cells, and B cells, for latent or persistent infection (24,64,67,78,80). In latent infections, the viral genomes are maintained in a quiescent state for the lifetime of the host but retain the capacity to reactivate the lytic replication cycle and produce progeny virions. Like all gammaherpesviruses, MHV68 is defined by its capacity to establish latent infections in host lymphocytes.During latent gammaherpesvirus infections, viral transcription is restricted, and only those genes proposed to influence maintenance and immune evasion are transcribed. For MHV68, the latent transcription program varies by cell type (45), likely reflecting cell-type-specific needs for viral maintenance and demonstrating the dynamic nature of gammaherpesvirus latency. Of the viral transcripts expressed during latent infections, mLANA (a homologue of the Kaposi's sarcoma-associated herpesvirus [KSHV] latency-associated nuclear antigen [LANA]) is critically required, as mLANAnull viruses are unable to establish and maintain latent infections following intranasal inoculation of mice (26,48). This is consistent with a proposed role for mLANA in episome maintenance during latency. However, mLANA influences more than just the capacity of MHAV68 to persist during latency, as we and others observed acute replication deficits in the lungs of mice infected with mLANA-null mutants (48,63).Although a replication deficit was not originally borne out in cell culture growth assays, several lines of evidence suggest a role for mLANA in facilitating acute-phase viral replication in addition to the demonstrated role in regulating viral latency. First, in global and gene-specific MHV68 transcription analyses, orf73 was detected at very early times after infections in culture, and orf73 transcription occurred in the presence of cycloheximide, classifying orf73 as an immediate-early transcript (2,15,46). Further, herpesvirus saimiri (HVS) and rhesus rhadinovirus (RRV) LANAs suppress the progression of lytic replication in culture by inhibiting transcription of the * Corresponding author. Mailing address:

Use of a Virus-Encoded Enzymatic Marker Reveals that a Stable Fraction of Memory B Cells Expresses Latency-Associated Nuclear Antigen throughout Chronic Gammaherpesvirus Infection

“…In multistep growth curves, although the peak viral titers were nearly identical for MHV68 and MHV68.ORF73␤la, MHV68.ORF73␤la titers were decreased at 24 and 48 h postinoculation. This finding is consistent with the reported lytic replication defect of a mutant MHV68 lacking the expression of mLANA (19,40), suggest- ing that the C-terminal fusion of ␤-lactamase alters lytic replication functions of mLANA. The discrepancy between single-step (no defect) and multistep (delayed replication) growth of MHV68.ORF73␤la suggests that the C-terminal fusion of ␤-lactamase to mLANA causes a modest delay in virus spread.…”

“…For example, KSHV LANA binds to viral DNA and blocks the expression of Rta, the key transcriptional activator of reactivation from latency (32), induces its own expression (45), and modulates the transcription of multiple cellular genes (47). Both KSHV LANA (21) and MHV68 mLANA (19) dysregulate the activity of the tumor suppressor p53, perhaps in part as a means to promote virus growth and prevent cell death (19). Additionally, recent reports have demonstrated the mLANA-mediated proteosomal degradation of the NF-B family member p65/RelA (47) and the activation of G 1 /S cyclin promoters via interaction with cellular bromodomain-containing BET proteins (42).…”

Establishment of B-Cell Lines Latently Infected with Reactivation-Competent Murine Gammaherpesvirus 68 Provides Evidence for Viral Alteration of a DNA Damage-Signaling Cascade

“…Importantly, however, it should be noted that orf73 transcripts are not detected in all latently infected cell types in vivo, suggesting that such a function may not be an absolute requirement for long-term latency (3,41,49,71). Indeed, the PMA-inducible nature of orf73 transcription may reflect a prominent role in facilitating reactivation in a manner analogous to that described during lytic replication in fibroblasts (21).…”

“…Primers for orf6, orf8, orf9, mK3, orf72, orf73, M1, M3, M4, M6, M8/orf57, and M9 were previously described (inner primer sets [71]). Primers for orf50 and GAPDH were previously described (21). Primers for orf25 were 25-1, 5Ј-CCCATGGCAGTATCTAAAG AGG-3Ј, and 25-2, 5Ј-CGGAGTTGCTCTAACAGTTGTG-3Ј.…”

“…Reverse transcription-PCR (RT-PCR) for spliced orf50 or orf73 transcripts was performed as previously described (3,37). RNA was isolated from A20-HE1 or A20-HE2 cells prior to or at 8 h and 24 h after treatment with 20 ng/ml PMA, contaminating DNA was removed by DNase I digestion (Invitrogen), and RNA was reverse transcribed using SuperScript II (Invitrogen) as previously described (21). No-RT controls were carried out in parallel.…”