Novel cell type-specific antiviral mechanism of interferon gamma action in macrophages.

Interferon (IFN)-gamma and macrophages (Mphi) play key roles in acute, persistent, and latent murine cytomegalovirus (MCMV) infection. IFN-gamma mechanisms were compared in embryonic fibroblasts (MEFs) and bone marrow Mphi (BMMphi). IFN-gamma inhibited MCMV replication in a signal transducer and activator of transcription (STAT)-1alpha-dependent manner much more effectively in BMMphi (approximately 100-fold) than MEF (5-10-fold). Although initial STAT-1alpha activation by IFN-gamma was equivalent in MEF and BMMphi, microarray analysis demonstrated that IFN-gamma regulates different sets of genes in BMMphi compared with MEFs. IFN-gamma inhibition of MCMV growth was independent of known mechanisms involving IFN-alpha/beta, tumor necrosis factor alpha, inducible nitric oxide synthase, protein kinase RNA activated (PKR), RNaseL, and Mx1, and did not involve IFN-gamma-induced soluble mediators. To characterize this novel mechanism, we identified the viral targets of IFN-gamma action, which differed in MEF and BMMphi. In BMMphi, IFN-gamma reduced immediate early 1 (IE1) mRNA during the first 3 h of infection, and significantly reduced IE1 protein expression for 96 h. Effects of IFN-gamma on IE1 protein expression were independent of RNaseL and PKR. In contrast, IFN-gamma had no significant effects on IE1 protein or mRNA expression in MEFs, but did decrease late gene mRNA expression. These studies in primary cells define a novel mechanism of IFN-gamma action restricted to Mphi, a cell type key for MCMV pathogenesis and latency.

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 (gammaHV68) latency is undefined, as is the relationship between gammaHV68 latency and latency of primate gammaherpesviruses. We used a nested reverse transcriptase PCR strategy (sensitive to approximately one copy of gammaHV68 genome for each genomic region tested) to screen for the presence of viral transcripts in latently infected mice. Based on the positions of known latency-associated genes in other gammaherpesviruses, we screened for the presence of transcripts corresponding to 11 open reading frames (ORFs) in the gammaHV68 genome in RNA from spleens and peritoneal cells of latently infected B-cell-deficient (MuMT) mice which have been shown contain high levels of reactivable latent gammaHV68 (K. E. Weck, M. L. Barkon, L. I. Yoo, S. H. Speck, and H. W. Virgin, J. Virol. 70:6775-6780, 1996). To control for the possible presence of viral lytic activity, we determined that RNA from latently infected peritoneal and spleen cells contained few or no detectable transcripts corresponding to seven ORFs known to encode viral gene products associated with lytic replication. However, we did detect low-level expression of transcripts arising from the region of gene 50 (encoding the putative homolog of the Epstein-Barr virus BRLF1 transactivator) in peritoneal but not spleen cells. Latently infected peritoneal cells consistently scored for expression of RNA derived from 4 of the 11 candidate latency-associated ORFs examined, including the regions of ORF M2, ORF M11 (encoding v-bcl-2), gene 73 (a homolog of the Kaposi's sarcoma-associated herpesvirus [human herpesvirus 8] gene encoding latency-associated nuclear antigen), and gene 74 (encoding a G-protein coupled receptor homolog, v-GCR). Latently infected spleen cells consistently scored positive for RNA derived from 3 of the 11 candidate latency-associated ORFs examined, including ORF M2, ORF M3, and ORF M9. To further characterize transcription of these candidate latency-associated ORFs, we examined their transcription in lytically infected fibroblasts by Northern analysis. We detected abundant transcription from 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, consistent with a role of these viral genes during latent infection. We conclude that (i) we have identified several candidate latency genes of murine gammaHV68, (ii) expression of genes during latency may be different in different organs, consistent with multiple latency programs and/or multiple cellular sites 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 gammaHV68 and primate gammaherpesviruses. The implications of these findings for replacing previous operational definitions of gammaHV68 latency with a molecular definition are discussed.

Interferon gamma regulates acute and latent murine cytomegalovirus infection and chronic disease of the great vessels.

To define immune mechanisms that regulate chronic and latent herpesvirus infection, we analyzed the role of interferon gamma (IFN-gamma) during murine cytomegalovirus (MCMV) infection. Lethality studies demonstrated a net protective role for IFN-gamma, independent of IFN-alpha/beta, during acute MCMV infection. Mice lacking the IFN-gamma receptor (IFN-gammaR-/-) developed and maintained striking chronic aortic inflammation. Arteritis was associated with inclusion bodies and MCMV antigen in the aortic media. To understand how lack of IFN-gamma responses could lead to chronic vascular disease, we evaluated the role of IFN-gamma in MCMV latency. MCMV-infected IFN-gammaR-/- mice shed preformed infectious MCMV in spleen, peritoneal exudate cells, and salivary gland for up to 6 mo after infection, whereas the majority of congenic control animals cleared chronic productive infection. However, the IFN-gammaR was not required for establishment of latency. Using an in vitro explant reactivation model, we showed that IFN-gamma reversibly inhibited MCMV reactivation from latency. This was at least partly explained by IFN-gamma- mediated blockade of growth of low levels of MCMV in tissue explants. These in vivo and in vitro data suggest that IFN-gamma regulation of reactivation from latency contributes to control of chronic vascular disease caused by MCMV. These studies are the first to demonstrate that a component of the immune system (IFN-gamma) is necessary to regulate MCMV-associated elastic arteritis and latency in vivo and reactivation of a herpesvirus from latency in vitro. This provides a new model for analysis of the interrelationships among herpesvirus latency, the immune system, and chronic disease of the great vessels.

Latent murine cytomegalovirus infection in macrophages.

In this study we show that macrophages (Mphi) are latently infected with murine cytomegalovirus (MCMV). After clearance of acute MCMV infection, the predominant form of chronic infection in Balb mice is latency rather than persistence. Peritoneal exudate cells (PECs) from latently infected Balb mice (3-9 months postinfection) contained MCMV genome and reactivatable virus. Adherent cells from both resident and thioglycollate-elicited PECs carried more MCMV DNA (measured by PCR) than nonadherent cells, and were selectively enriched for Mphi. FACS sorted F4/80(+) Mphi contained MCMV DNA, while other FACS sorted cell populations from PECs were never positive for MCMV DNA. MCMV reactivated from FACS sorted F4/80(+) Mphi in 32% of cocultures with murine embryonic fibroblasts (MEFs). Since Mphi carry MCMV genome and reactivatable virus, but not lytic virus, they are latently infected with MCMV. We determined the frequency of Mphi carrying MCMV genome in PECs (about 1/50,000) using a limiting dilution PCR assay. Using this frequency and estimates of the total amount of MCMV genome in populations, we estimate that latently infected Mphi carry 1-10 copies of MCMV genome. To evaluate the origin of latently infected Mphi, we compared the frequency of cells carrying MCMV genome in the resident and elicited PECs. The frequency of Mphi carrying MCMV DNA was the same in resident and thioglycollate-elicited PECs, despite the fact that there was a ninefold increase in the number of Mphi recovered after thioglycollate elicitation. This argued for recruitment of bone marrow-derived Mphi (BMMphi) carrying MCMV genome into the peritoneum during inflammatory responses. Consistent with this hypothesis, MCMV genome, but not persistent virus, was detected in bone marrow cells from latently infected mice.