Human cytomegalovirus UL83-coded pp65 virion protein inhibits antiviral gene expression in infected cells

Abstract

The initial interaction of human cytomegalovirus with fibroblasts triggers, and then partially blocks, an innate immune response pathway that leads to the induction of IFN-responsive genes and proinflammatory chemokines. Infection of fibroblasts with human cytomegalovirus inhibited their ability to respond to exogenous IFN. Consistent with the observation that the block did not depend on de novo viral protein synthesis, ectopic expression of the viral UL83-coded pp65, an abundant virion protein, inhibited IFN signaling. Furthermore, DNA array analysis showed that infection with a pp65-deficient mutant virus caused a much stronger induction of many IFN-response and proinflammatory chemokine RNAs than infection with wild-type virus. The nuclear DNA-binding activities of transcription factors NF-kappaB and IRF1 were induced to a much greater extent after infection with the pp65-deficient mutant than with wild-type virus. IFN-stimulated gene factor 3 DNA-binding was modestly enhanced, whereas IRF3 activity was not affected by mutation of pp65. Together, these results imply that pp65, which is delivered to newly infected cells in the virion, antagonizes a pathway that affects NF-kappaB and IRF1 and prevents the accumulation of mRNAs encoded by numerous cellular antiviral genes.

Fig. 1.

HCMV blocks IFN signaling. (A) Untreated fibroblasts (Left) or fibroblasts continuously treated with 100 μg/ml cycloheximide beginning 1 h before infection (Right) were mock-infected or infected with wild-type HCMV. At 4 h after infection, IFN-α (2,000 units/ml) was added to the cultures, and at 6 h after infection, RNA was harvested and assayed for the IFN-sensitive gene MxA by Northern blot. The level of 28S ribosomal RNA (rRNA) was monitored by ethidium bromide fluorescence as a loading control. (B) Fibroblasts were pretreated with 100 μg/ml cycloheximide beginning 1 h before mock-infection or infection with wild-type HCMV. At 4 h after infection, 2,000 units/ml IFN-α was added to infected and mock-infected cells. At 6 h after infection, cells were lysed and extracts were analyzed by Western blot assay for the indicated proteins by using specific antibodies. Stat1 and Stat2 designate total Stat protein; p-Stat1 and p-Stat2 designate phosphorylated Stat proteins.

Fig. 2.

IFN-α signaling is antagonized by pp65. (A) Fibroblasts were infected with recombinant adenoviruses expressing hemagglutinin (HA)-tagged pp65 (Ad-pp65), HA-tagged pp71 (Ad-pp71), or GFP (Ad-GFP) at a multiplicity of 10⁴ particles per cell. At 24 h after infection, 2,000 units/ml IFN-α was added for 1 h. RNA was then prepared, and the level of MxA mRNA was assayed by Northern blot (Upper). Protein samples also were assayed by using an antibody specific for the HA epitope (α-HA) (Lower) to confirm the expression of pp65 and pp71. (B) Fibroblasts were infected with wild-type HCMV or a pp65-deficient mutant (Δpp65). After 6 h, cells were harvested and RNA was extracted and analyzed by Northern blot using ³²P-labeled probes specific for the indicated IFN-responsive genes.

Fig. 3.

Global effect of pp65 on cellular gene expression. DNA array analysis was carried out on duplicate samples of RNA prepared from fibroblasts at 6 h after infection with wild-type HCMV or Δpp65. (A) The total number of RNAs up- or down-regulated by a factor of ≥3 in two replicates was determined and graphed. (B) The number of RNAs induced by a factor ≥3 in both replicates, which were previously shown to be induced by IFN (10), was determined and graphed.

Fig. 4.

The activation of IRF1 and NF-κB is inhibited by pp65. Fibroblasts were infected with wild-type HCMV or Δpp65, and nuclear protein extracts were prepared 6 h later. The extracts were incubated with ³²P-labeled oligonucleotides containing an ISRE motif (A Left) or an NF-κB-binding site (B Left) and resolved on a nondenaturing polyacrylamide gel. To confirm the identity of the complexes binding the ISRE, supershift assays were performed on a Δpp65-infected nuclear extract by using antibodies to the indicated polypeptide constituents of ISRE-binding activities (A Right). To confirm the identity of the complex binding the NF-κB probe, a competition assay was carried out (B Right). Nuclear extracts from cells treated with tumor necrosis factor α were incubated with the ³²P-labeled NF-κB binding site probe plus either an unlabeled competitor DNA with the wild-type motif or a motif with a mutation that abolishes NF-κB binding.

Fig. 5.

Western blot assays of factors known to regulate IFN-responsive genes. Fibroblasts were infected with wild-type HCMV or Δpp65. At 6 h after infection, cells were lysed and extracts of total cell protein (Left) and nuclear protein (Right) were prepared. The protein samples were then analyzed by Western blot assays using antibodies specific for the indicated proteins.

Fig. 6.

Immunofluorescent localization of factors known to regulate IFN-responsive genes. Fibroblasts grown on coverslips were infected with wild-type HCMV or Δpp65. At 6 h after infection, the cells were fixed, permeabilized, and stained with antibodies to the proteins indicated (red). Nuclei were stained with DAPI (blue).