Postattachment Events Associated with Viral Entry Are Necessary for Induction of Interferon-Stimulated Genes by Human Cytomegalovirus

Abstract

Utilizing a human cytomegalovirus-specific fusion inhibitor and an antiglycoprotein H antibody, we studied the role of virion fusion in interferon-stimulated gene (ISG) induction. Our results indicate that ISG induction does not occur when virion-mediated, post-high-affinity attachment events are inhibited by either reagent. Thus, virion-mediated postattachment events, such as fusion, are required for ISG induction.A large number of interferon-stimulated genes (ISGs) are strongly activated following human cytomegalovirus (HCMV) infection (19). In early studies, UV-irradiated HCMV was shown to induce ISGs (19). Subsequent studies revealed that virion envelope proteins, specifically glycoprotein B (gB), were responsible for the induction of ISGs (3,17). Soluble gB applied to human fibroblasts resulted in the induction of ISGs. Because the latter experiment was done under nonphysiological conditions and because gB has been shown to have roles in both virion attachment and fusion, it was unclear whether virion attachment alone was sufficient to induce ISG expression. Alternatively, other HCMV envelope glycoproteins or perhaps the fusion process itself is required for ISG activation during natural infection, as is the case for ISG induction by herpes simplex virus (14). In this study, we examined the requirement of postattachment steps of HCMV entry for the induction of ISGs.To characterize the role of virion fusion in HCMV-mediated ISG induction, an HCMV-specific fusion inhibitor, CFI02, was used (2). It was previously shown that this inhibitor has no effect on the attachment of virus to cells, but that it efficiently blocks subsequent viral-cell fusion at a 2 M concentration (7). CFI02 was used to determine if the compound also blocks induction of ISGs by HCMV (Fig. 1A). Human foreskin fibroblast (HFF) cells were either mock infected or infected with HCMV in the presence of dimethyl sulfoxide (control) or CFI02 for 8 h. Induction of the ISGs isg54K and cig49 was determined by RNA blot analysis. A cellular pseudogene, 7SK (13), was used as an internal control. Induction of ISGs by HCMV was inhibited in the presence of CFI02 (Fig. 1A, compare lanes 2 and 3). These results indicated that a postattachment step of HCMV infection, presumably virion fusion with the cellular membrane, was required for the induction of ISG expression. The effect of CFI02 inhibition on HCMV fusion was specific because (i) it had no effect on CFI02-resistant, HCMV-mediated ISG induction (the resistance phenotype was mapped to gB) (7) (lanes 4 and 5); (ii) it did not block murine-CMV-mediated ISG induction (lanes 9 and 10); (iii) it had no effect on alpha interferon (IFN-␣)-induced ISG expression (lanes 6 and 7); and (iv) it was not able to block induction of ISGs when added 2 h after infection (lanes 3 and 8). Furthermore, inhibition of HCMV-induced ISG expression by CFI02 had no effect on IFN-␣-mediated induction of ISGs (lanes 11 and 12). In addition, as shown previously (19), HCMV is able to induce isg54K at 12 and 24 h postinfection (hpi)...

The IFN-Independent Response to Virus Particle Entry Provides a First Line of Antiviral Defense That Is Independent of TLRs and Retinoic Acid-Inducible Gene I

“…In contrast, ISG induction was not observed upon treatment with replicating DNA viruses because these viruses encode proteins that inhibit IRF3 activation and IFN production (59,(61)(62)(63)(64). Virus binding and entry are required for the IFN-independent antiviral response following HSV-1 (52,60) and HCMV (60,65) infection, which suggests that the interaction with a cell surface receptor is insufficient to trigger the response. However, replication of viral nucleic acid was not required for the induction of this response.…”

Rhesus Cytomegalovirus Particles Prevent Activation of Interferon Regulatory Factor 3

“…For HCMV it has been shown that the major surface gB is predominantly responsible for induction of ISGs (5, 37). Interestingly, an inhibitor of membrane fusion that binds specifically to gB inhibited ISG induction but did not prevent viral attachment to cells (31). It is thus likely that gB-mediated membrane fusion is the major event triggering ISG induction during entry by HCMV.…”

Differential Initiation of Innate Immune Responses Induced by Human Cytomegalovirus Entry into Fibroblast Cells

“…The ability of a soluble version of gB to activate IFN signaling suggests that gB binding to a cell surface receptor during virus entry is sufficient for antiviral responses. Interestingly, a small molecule HCMV entry inhibitor that targets gB, as well as neutralizing Abs to both gB and gH, inhibit ISG accumulation (36). These studies suggest that glycoprotein-cell interactions during the entry process are important for HCMV-induced IFN activation.…”