Rhesus cytomegalovirus particles prevent activation of interferon regulatory factor 3.

An essential and evolutionarily conserved component of the innate host response to viral infection is transcriptional induction of interferon (IFN)-stimulated genes (ISGs). Important ISGs include components of the double-stranded RNA (dsRNA)-dependent protein kinase (PKR), 2-5A oligoadenylate synthetase (2-5A), and Mx pathways. Intracellular inhibition of virus replication takes place through translation inhibition (PKR), single-stranded RNA cleavage (2-5A), and modulation of viral polymerase and protein trafficking (Mx). Viruses induce ISG expression via two separate but related mechanisms. First, the IFN-dependent pathway involves activation of the IFN receptor by exogenous IFN-α/β secreted from infected cells. Binding of IFN-α or -β to its receptor triggers JAK/STAT signal transduction that ultimately leads to the transcription of ISGs (24, 36). However, in recent years it has become increasingly clear that viral infection also triggers a subset of ISGs independent from secreted IFN. This takes place in response to specific molecular components of pathogens, called pathogen-associated molecular patterns (e.g., bacterial lipopolysaccharide or dsRNA and glycoproteins) that activate Toll-like receptors (TLRs) (16). TLR signaling activates nuclear factor κB (NF-κB) via the I kappa kinase (IKK) complex, thereby upregulating proinflammatory genes (19). Specific TLRs are also known to activate IFN regulatory factor 3 (IRF3). IRF3 is a constitutively expressed protein that normally shuttles freely between the cytoplasm and nucleus. It becomes activated via phosphorylation of carboxy-terminal serine and threonine residues, resulting in homodimerization and association with histone acetyltransferase nuclear proteins p300 and CREB-binding protein (CBP) (39, 42). The proteins act as transcriptional coactivators and cause IRF3 to become localized to the nucleus (23, 25, 39, 42). This complex stimulates transcription of a subset of ISGs and, together with NF-κB, IFN-β itself (16). Recent studies show that phosphorylation of IRF3 is performed by virus-induced IKK-related kinases (IKK-related IKKɛ and TANK-binding kinase 1 [TBK1]) (12, 35). Thus, viral infection results in NF-κB-dependent and IRF3-dependent induction of proinflammatory genes and a subset of ISGs, respectively. In addition, the induction of IFN-β gene in turn activates the IFN-dependent pathways and a second wave of ISG induction. Evolutionary pressure exerted by ISG antiviral effects has given rise to viral countermeasures against either the induction or function of ISGs or both (13). In fact, recent data suggest that all enveloped DNA viruses, as well as diverse RNA viruses, trigger ISG expression through IRF3 when viral gene expression is inhibited (10). However, IRF3 was generally inactivated when cells were infected with live, translationally active virus. For instance, the alphaherpesvirus herpes simplex virus triggers a strong induction of ISGs when viral gene expression is inhibited by either UV treatment or in the presence of cycloheximide (CHX) (29, 33). Live virus, however, interferes with ISG induction through the activities of the viral proteins ICP0 and vhs (26). Similarly, the poxvirus protein E3L blocks IRF3-dependent ISG expression (38) and the gammaherpesvirus Kaposi's sarcoma associated virus encodes three IRF homologous that interfere with both in IFN-dependent and -independent pathways (18). In contrast to these other large DNA viruses, however, the betaherpesvirus human cytomegalovirus (HCMV) triggers a strong induction of ISGs upon infection of cells in culture (45, 46) and activates IRF3 (30, 33). HCMV-mediated ISG induction requires membrane fusion events and is to a large extent triggered by the major viral glycoprotein gB (5). In fact, treatment of fibroblasts with purified gB triggers a strong ISG response similar to, but faster than, intact virus (5, 37), which is mediated through CD14/TLR-2 and IRF3 (4, 11). UV inactivation of virus or infection of cells in the presence of CHX exacerbates induction of ISGs and proinflammatory genes, suggesting that HCMV inhibits ISG induction, at least partially (8, 33). Indeed, the viral tegument protein pp65 was implicated in this partial inhibition of ISG induction, but the molecular mechanism is controversial, either involving interference with the nuclear translocation of NF-κB, IRF1 (7), or IRF3 (1). Rhesus CMV (RhCMV) is an emerging animal model for HCMV that recapitulates many aspects of HCMV pathogenesis and epidemiology (28, 40). Similar to HCMV, RhCMV is highly prevalent in animals of a given colony of rhesus macaques. Moreover, RhCMV is an opportunistic infection during simian AIDS (34) making this model ideal for study of the interaction between the two viruses. The recent completion of the genomic sequence of RhCMV further revealed a high conservation of both genes that are essential for growth in tissue culture and nonessential genes, including many known or predicted immunomodulatory genes (15). In addition, RhCMV encodes its own unique genes that are of unknown function. During an initial evaluation of the transcriptional reprogramming that occurs in rhesus fibroblasts during in vitro infection with RhCMV, we observed a complete absence of ISG induction during all stages of viral infection. In contrast, HCMV induced a strong ISG and proinflammatory gene response in rhesus fibroblasts, as observed previously in human fibroblasts. The lack of ISG induction was consistent with a lack of IRF3 activation in RhCMV-infected cells. Moreover, neither IRF3 activation nor ISG induction was observed upon treatment of infected cells with CHX or after UV inactivation of RhCMV. Thus, unlike all other enveloped viruses examined to date, RhCMV does not trigger ISG induction even in the absence of viral gene expression. Since it is unlikely that the virus lacks molecular signals that activate IRF3, we hypothesize that structural components of the RhCMV virion prevent ISG induction. This hypothesis is supported by the finding that RhCMV inhibits IRF3 activation by live or UV-inactivated HCMV.