ORF73 of murine herpesvirus-68 is critical for the establishment and maintenance of latency

Abstract

In vitro studies have established that the latency-associated nuclear antigen encoded by human Kaposi's sarcoma-associated herpesvirus and the related ORF73 gene product of herpesvirus saimiri interact with virus origins of replication to facilitate maintenance of episomal DNA. Such a function implies a critical role for ORF73 in the establishment and maintenance of latency in vivo. To determine the role of ORF73 in virus pathogenesis, the ORF73 gene product encoded by murine herpesvirus-68 (MHV-68) was disrupted by making an ORF73 deletion mutant, D73, and an independent ORF73 frameshift mutant, FS73. The effect of the mutations introduced in ORF73 on MHV-68 pathogenesis was analysed in vivo using a well-characterized murine model system. These studies have revealed that ORF73 is not required for efficient lytic replication either in vitro or in vivo. In contrast, a severe latency deficit is observed in splenocytes of animals infected with an ORF73 mutant, as assessed by infectious centre reactivation assay or by in situ hybridization detection of latent virus. Assessment of viral genome-positive cells in sorted splenocyte populations confirmed the absence of ORF73 mutant virus from splenic latency reservoirs, including germinal centre B cells. These data indicate a crucial role for ORF73 in the establishment of latency and for virus persistence in the host. INTRODUCTIONGammaherpesviruses characteristically establish lifelong latency in lymphoid cell populations and are commonly associated with malignant lymphoproliferative disorders. Critical for the maintenance of latent gammaherpesviral genomes in dividing cells are episome maintenance functions, which are required for the replication of viral genomes during mitosis and their accurate segregation into daughter cells . In the case of Epstein-Barr virus (EBV), the virus-encoded EBNA-1 protein facilitates episome maintenance via interaction with the origin of plasmid replication (Yates et al., 1984(Yates et al., , 2000Lupton & Levine, 1985), and in vitro studies have demonstrated that EBNA-1 is essential for the immortalization of B cells and the establishment of latency (Lee et al., 1999). Similarly, Kaposi's sarcoma-associated herpesvirus (KSHV) expresses a multifunctional latencyassociated nuclear antigen (LANA-1) encoded by ORF73, which binds directly to the terminal repeats of the viral genome to mediate stable episome replication and segregation (Ballestas et al., 1999;Cotter & Robertson, 1999;Ballestas & Kaye, 2001;Grundhoff & Ganem, 2003). A similar function has also been demonstrated for the ORF73 gene product encoded by the related gamma-2 herpesvirus herpesvirus saimiri (HVS) (Smith et al., 2001;.From in vitro studies, it is clear that the trans-acting plasmid maintenance proteins encoded by gammaherpesviruses are essential for the stable maintenance of viral genomes during latency. However, the contribution of virus-encoded maintenance functions to virus persistence in vivo is not known. To directly address this question, we have focussed our studie...

Bovine herpesvirus 4 ORF73 is dispensable for virus growth in vitro, but is essential for virus persistence in vivo

“…Also, one cannot exclude that the deletion of the entire ORF73 could have disrupted neighbouring virus functions. Although the disruption of other virus functions could in theory have explained the phenotype observed for the ORF73 BoHV-4 recombinant, this phenotype was consistent with less dramatic mutations of MHV-68 ORF73 (Fowler et al, 2003;Moorman et al, 2003).…”

“…These studies revealed that the expression product of ORF73 (pORF73) is a multifunctional protein essential for latency (Calderwood et al, 2005;Fowler et al, 2003;Renne et al, 2001). The best-characterized pORF73 is the latency-associated nuclear antigen 1 (LANA-1) of HHV-8.…”

Latent herpesvirus infection arms NK cells

“…Our experiments examining the arming of NK cells, as well as previous experiments that uncovered heightened resistance against Listeria monocytogenes during herpesvirus latency, 18 made use of the latency-defective MuHV-4 mutant O73.stop. This virus replicates to normal 22,28 or near-normal levels 23 during acute infection but is severely defective in its ability to persist in the latent state. NK-cell activation is equivalent for wild-type MuHV-4 and O73.stop during acute infection.…”

“…22,23 O73.stop undergoes productive acute replication, elicits a normal humoral immune response (data not shown and Barton et al 18 ), and activates NK cells during acute infection indistinguishably from wild-type MuHV-4 (supplemental Figure 3). However, 28 days after infection with O73.stop, there was no increase in GzmB protein expression or degranulation by NK cells (Figures 1-2).…”

CD4+ T cells specific for a model latency‐associated antigen fail to control a gammaherpesvirus in vivo

“…MHV-68 also evades CD8 + T cells during episome maintenance. Its episome maintenance protein, ORF73 [32,33], lacks the glycine/alanine-rich motif of EBNA-1, but incorporates the same cis-acting inhibition of protein synthesis and degradation [5]. The MHV-68 ORF73 promoters are also remarkably analogous to those of EBNA-1, with one promoter just upstream of the ORF and two more that splice across multiple viral repeat units to generate an extensive 5 0 untranslated region [34].…”