Human cytomegalovirus elicits a coordinated cellular antiviral response via envelope glycoprotein B

Abstract

Previous studies have shown that human cytomegalovirus (CMV) is a potent elicitor of interferon-stimulated gene (ISG) expression. Induction of the interferon pathway does not require replication-competent virus, and envelope glycoprotein B (gB) from CMV is a viral structural component that can directly induce transcription of ISGs. Here we extend these earlier findings by defining the consequences of inducing the interferon pathway. We found that cells respond to CMV or soluble gB by establishing a functional antiviral state within cell types critical in CMV biology, such as fibroblasts and endothelial cells. We have also discovered new insights into the mechanism by which the pathway is initiated. Interferon regulatory factor 3 (IRF3), a key transcriptional regulator of cellular interferon responses, is activated by CMV virions and soluble gB. Thus, IRF3 becomes activated via "outside-in" signal transduction events. This is a novel mechanism of activation of this key transcription factor by viruses. In comparison to soluble gB (gB(1-750)), which comprises the entire ectodomain of gB, a truncation mutant encompassing only the amino-terminal region of gB (gB(1-460)) was markedly less effective at inducing antiviral responses. This indicates that the region of gB from residues 461 to 750 is important for initiation of the antiviral response. In addition, CMV and gB establish an antiviral state in alpha/beta interferon null cells, illustrating that primary induction of ISGs by CMV and gB is sufficient to establish the antiviral response and that interferon secretion is not necessary for the antiviral effect. Taken together, our findings reveal that CMV initiates a coordinated antiviral response through contact between gB and an as-yet-unidentified cell surface receptor(s).

FIG. 1.

Schematic diagram of gB constructs showing the region of wild-type gB represented by each truncation mutant. Constructs are shown with the amino terminus at the left, and prominent structural features are labeled with the corresponding amino acid residue number. The carboxy-terminal fragment comprises amino acid residues 461 to 906. gB_1-750 comprises the entire ectodomain, while gB_1-460 is truncated at the site of furin cleavage. Each truncation mutant has a six-histidine tag at its carboxy terminus. MSD, membrane-spanning domain; Cyto, cytoplasmic tail.

FIG. 2.

Cell response to treatment with CMV, gB, and gB_1-460. (A and B) CMV and gB trigger a functional antiviral state in cells. Human fibroblasts were stimulated as indicated (interferon, 100 U/ml). At 6 h poststimulation, the treatments were removed and the monolayers were challenged with approximately 100 PFU of VSV per well. The mock-treated cells were not infected with VSV. Plaque formation was visualized by crystal violet staining at 48 h postinfection. CMV replicates with much slower kinetics than VSV; thus, any visible plaque formation is the result of VSV, not CMV, growth. (C) gB_1-460 minimally induces ISG activation. Human fibroblasts were mock treated or treated with gB_1-750 or gB_1-460 (1 μg/ml). At 8 h posttreatment, total RNA was harvested from cells and subjected to RNase protection analysis with ISG54- and actin-specific probes.

FIG. 3.

Effects of CMV and gB on IRF3. (A) IRF3 becomes phosphorylated in response to CMV and gB. Human fibroblasts were mock or CMV infected (MOI = 0.1 PFU/cell) or treated with soluble gB (1 μg/ml) in the presence of cycloheximide for 6 h. Cell lysates were prepared and analyzed via SDS-8% PAGE followed by immunoblotting with an IRF3-specific antibody. The different phosphorylated forms of IRF3 are indicated. (B) CMV and gB induce IRF3 nuclear localization. Human fibroblasts were mock infected, infected with CMV or UV-inactivated CMV, or treated with soluble gB or the gB_1-460 truncation in the presence of cycloheximide. At 6 h posttreatment, the cells were fixed and the cellular localization of IRF3 was determined by indirect immunofluorescence.

FIG. 4.

CMV and gB induce beta interferon secretion. Human fibroblasts were mock infected, infected with CMV or UV-inactivated CMV, or treated with soluble gB. At 12 h posttreatment, cell supernatants were assayed for beta interferon by ELISA.

FIG. 5.

Primary induction of ISGs by CMV and gB contributes directly to the antiviral response. GRE cells were treated as indicated (interferon, 100 U/ml). At 6 h posttreatment, the inoculum was removed and the monolayers were challenged with approximately 100 PFU of VSV per well. Plaque formation was visualized by crystal violet staining at 48 h postinfection.

FIG. 6.

Effects of CMV in endothelial cells. (A) CMV induces ISG expression in endothelial cells. Human fibroblasts or endothelial cells were mock infected (M) or infected with CMV (strain AD169 or VHL/E) at an MOI of 2 PFU/cell. At the indicated times postinfection, total cellular RNA was harvested and subjected to RNase protection analysis with OAS- and actin-specific probes. (B) CMV triggers an antiviral state in endothelial cells. HUVEC were stimulated as indicated (interferon, 100 U/ml). At 6 h poststimulation, the treatments were removed and the monolayers were challenged with approximately 100 PFU of VSV per well. Plaque formation was visualized by crystal violet staining at 48 h postinfection.

FIG. 7.

Soluble gB induces ISG expression in endothelial cells. Endothelial cells from three different sources were mock treated (M) or stimulated with soluble gB. At the indicated times posttreatment, total cellular RNA was harvested and subjected to RNase protection analysis with OAS-, ISG54-, and actin-specific probes.