Characterization of the elements and proteins responsible for interferon-stimulated gene induction by human cytomegalovirus

Abstract

Human cytomegalovirus (HCMV) infection of human fibroblast cells activates a large number of interferon-stimulated genes (ISGs) in a viral envelope-cell membrane fusion-dependent mechanism. In this study, we identified two interferon response elements, the interferon-stimulated response element (ISRE) and the gamma interferon-activated site (GAS), which act as HCMV response sites (VRS). Gel mobility shift assays showed that cellular proteins form specific and identical complexes with ISRE and GAS elements, and the binding of these complexes to ISRE and GAS is stimulated by HCMV infection. Point mutations in the consensus sequences of ISRE and GAS completely abolished their activities in response to HCMV-mediated transactivation, as well as their abilities to interact with HCMV-activated VRS-binding proteins. Interferon regulatory factor 3 does not appear to be present in the VRS-binding complexes or to be involved directly in HCMV-mediated ISG activation. Using ProteinChip technology, four potential proteins were identified, ranging from 20 to 42 kDa, in the VRS-binding complexes. The data suggest that HCMV infection activates VRS-binding proteins, which then bind to the VRS and stimulate ISG expression.

Figures

FIG. 1.

Construction of EBV-based luciferase reporter plasmids for mapping the HCMV response sites (VRS). (A) Map of pElu-Basic. A luciferase reporter gene was cloned into an EBV-based plasmid with an SV40 poly(A) signal (SV40 pA). The promoter and regulatory elements were inserted between BglII and HindIII sites. The construct also contained a hygromycin resistance gene (Hygr), an ampicillin resistance gene (Ampr), an EBNA-1 gene, and an EBV replication origin, oriP. (B) pElu-ISRE. A 560-bp promoter region of isg54K gene (Pisg54K) was cloned upstream of a luciferase gene. The promoter contains an ISRE site, as shown in bolded sequences in the box; pElu-NRS and pElu-GAS are similar to pElu-ISRE except that the ISRE site was replaced by either an NRS or two GAS (bolded sequences in the boxes).

FIG. 2.

Luciferase assays demonstrated that ISRE and GAS elements were responsible for HCMV induction. The pElu-ISRE, pElu-NRS, and pElu-GAS plasmids (as indicated at the bottom of each panel) were transfected into HFF cells. After 1 week of transfection, the cells were split into six-well dishes and cultured an additional 3 days. The cells then were mock infected (mock), infected with live HCMV or UV-inactivated HCMV (UVC) for 8 h, or treated with IFN-α (shaded bar) for 4 h, followed by luciferase assays. Relative luciferase activities were obtained from the averages of triplicate points. Fold activation is indicated above each bar.

FIG. 3.

EMSA showing that the nuclear proteins specifically formed complexes with ISRE and GAS elements. HFF cells were mock infected (Mock) or infected with UV-HCMV (UVC) for 8 h. Nuclear extracts (NE) were prepared and used for EMSA. A 120-bp PCR fragment containing ISRE, NRS, or GAS was used as probe (PB) or competitor (CP). A few HCMV-activated complexes were observed.

FIG. 4.

Mutagenesis of the VRS. (A) Two point mutations were introduced into the consensus sequences (bold) of ISRE and GAS as indicated to generate two mutant luciferase reporter plasmids, pElu-ISREm and pElu-GASm. (B) The mutant plasmids, along with the wild-type (pElu-ISRE and pElu-GAS) and ISRE deletion (pElu-NRS) plasmids, were transfected into HFF cells. Cells were mock infected or infected with HCMV or UV-HCMV for 8 h, and luciferase assays were performed. HCMV-induced luciferase activity (white bars) was set as 100% for each reporter plasmid. Luciferase activity in the mock-infected cells and UV-HCMV-infected cells is shown as black bars and gray bars, respectively. (C) The protein-binding activities of mutant ISRE and GAS were tested in EMSA as described in the legend for Fig. 3. The 120-bp fragments were generated from the plasmids by PCR and labeled for use as probes. The probes (PB, indicated on the top) were incubated with nuclear extracts (NE) from mock infected (M) or 8 h-HCMV-infected (c) HFF cells.

FIG. 5.

The IRF-3 is not in the VRS-binding complexes. (A) Immunofluorescent staining showed that IRF-3 was translocated from the cytoplasm to the nucleus after HCMV infection. HFF cells were infected with HCMV for 2, 4, 6, or 8 h (b to e, as indicated), mock infected for 2 h (a), or infected with HCMV for 2 h in the presence of the PKC inhibitor H7 (f). After infection, the cells were fixed and the cellular localization of IRF-3 was determined using an anti-IRF-3 monoclonal antibody. (B) An in vivo phosphorylation assay showed that HCMV infection causes rapid phosphorylation of IRF-3. HFF cells were labeled with [32P]phosphate and mock infected or infected with HCMV for 1 or 4 h. The cells then were lysed, and IRF-3 protein was immunoprecipitated using an anti-IRF-3 monoclonal antibody. Phosphorylated IRF-3 is indicated by an arrow. (C) EMSA was performed as described in the legend for Fig. 3. Anti-IRF-3 antibody (α-IRF-3) was included in the reaction mixture. Antibody against IE1 (α-IE1) was used as a negative control. The amounts of antibodies (in microliters) used in this assay are indicated. (D) HCMV-activated VRS-binding proteins were partially purified on a heparin column. VRS-binding activity was measured in an EMSA (left panel), and IRF-3 was detected by immunoblot analysis (right panel). Analysis of the column fractions showed that the VRS-binding activity was detected in the 1 M NaCl fraction, whereas IRF-3 protein was detected in the 0.1 M NaCl fraction.

FIG. 6.

Characterization of VRS-binding proteins. (A) The 120-bp biotinylated DNA fragments containing ISRE (middle panel) or ISRE with two point mutations (ISREm, lower panel) or minus ISRE (NRS, upper panel) were bound to streptavidin on the protein chips. The 8-h HCMV-infected nuclear extracts were incubated with the protein chips for 2 h under gel shift conditions. After washing with the gel shift buffer, the bound proteins were analyzed by SELDI. Four peaks were detected with the ISRE probe, as indicated by the numbers, with molecular masses of 19.6, 24.6, 32.8, and 41.7 kDa. These proteins bound much more poorly and/or were not detectable with probes without an ISRE or with a mutant ISRE. (B) The biotinylated ISRE and GAS probes (PB) on the protein chips were incubated with the nuclear extracts (NE) of mock-infected cells or HCMV-infected cells. Binding of a 41.7-kDa protein to ISRE and GAS elements was stimulated five- to sixfold by HCMV infection.