Herpes simplex virus 1 has multiple mechanisms for blocking virus-induced interferon production

Abstract

In response to viral infection, host cells elicit a number of responses, including the expression of alpha/beta interferon (IFN-alpha/beta). In these cells, IFN regulatory factor-3 (IRF-3) undergoes a sequence of posttranslational modifications that allow it to act as a potent transcriptional coactivator of specific IFN genes, including IFN-beta. We investigated the mechanisms by which herpes simplex virus 1 (HSV-1) inhibits the production of IFN-beta mediated by the IRF-3 signaling pathway. Here, we show that HSV-1 infection can block the accumulation of IFN-beta triggered by Sendai virus (SeV) infection. Our results indicate that HSV-1 infection blocks the nuclear accumulation of activated IRF-3 but does not block the initial virus-induced phosphorylation of IRF-3. The former effect was at least partly mediated by increased turnover of IRF-3 in HSV-1-infected cells. Using mutant viruses, we determined that the immediate-early protein ICP0 was necessary for the inhibition of IRF-3 nuclear accumulation. Expression of ICP0 also had the ability to reduce IFN-beta production induced by SeV infection. ICP0 has been shown previously to play a role in HSV-1 sensitivity to IFN and in the inhibition of antiviral gene production. However, we observed that an ICP0 mutant virus still retained the ability to inhibit the production of IFN-beta. These results argue that HSV-1 has multiple mechanisms to inhibit the production of IFN-beta, providing additional ways in which HSV-1 can block the IFN-mediated host response.

FIG. 1.

Production of IFN is reduced in the presence of HSV-1. (A) Total RNA was isolated from infected HEC-1-B cells at 5 hpi with SeV alone (lanes 2 and 6), HSV-1 alone (lanes 3 and 7), or a combination of the two viruses (lanes 4 and 8). Infections were carried out in the presence (lanes 5 to 8) or absence (lanes 1 to 4) of CHX added at the time of infection. The harvested RNA was analyzed by RNase protection assay for the presence of IFN-β mRNA and the control GAPDH mRNA. (B) IFN-β levels in media from the same infected cells were determined by ELISA as described in Materials and Methods. Amounts of secreted IFN-β were not determined for the cells infected in the presence of CHX due to the fact that no protein expression was expected to take place. The data shown are from one experiment representative of several replicate experiments.

FIG. 2.

Inhibition of IRF-3 nuclear accumulation in cells infected with HSV-1. Cells were mock infected (A) or infected with SeV (B), HSV-1 (C), or a combination of the two viruses (D). Indirect immunofluorescence was used to determine the localization of IRF-3 at 6 hpi. After fixation, the cells were incubated with the monoclonal SL12.1 antibody to IRF-3, followed by a secondary rhodamine-conjugated anti-mouse immunoglobulin G (IgG). Image exposure time for IRF-3 was set by using the SeV-infected cells and was not changed in the other samples. (E) A sample (200 to 300) of cells was counted under the different conditions in two independent experiments. The average percentages of cells staining positive for IRF-3 nuclear accumulation are shown in the bar graph.

FIG. 3.

IRF-3 levels are reduced in HSV-1-infected cells. (A) Western blot showing total levels of IRF-3 at different time points postinfection after infection with SeV in the presence or absence of HSV-1. Proteins in cell lysates were separated by SDS-PAGE and detected with anti-IRF-3 antibody SL-12.1 (1:750 dilution). (B) Individual bands were scanned, and the intensity was quantified by computer analysis. The amount of IRF-3 was normalized to the amount present at time zero. The data plotted are the average of three independent experiments ± the standard error of the mean.

FIG. 4.

ICP0 is required for inhibition of nuclear accumulation of IRF-3. (A) Cells were infected with SeV alone or a combination of SeV with HSV-1 or an IE mutant. Indirect immunofluorescence was used to determine the localization of IRF-3 at 6 hpi, and the average percentage of cells staining positive for IRF-3 nuclear accumulation of two independent experiments are shown. Localization of IRF-3 was also determined for the ICP0 mutant 7134 (B) and its rescued 7134R (D) virus. Also shown are HSV-1 replication compartments formed after infection with 7134 (C) and 7134R (E). After fixation, the cells were incubated with the monoclonal SL-12.1 antibody to IRF-3 and the polyclonal 3-83 antibody to ICP8. The secondary antibodies were rhodamine-conjugated anti-mouse IgG and fluorescein-conjugated anti-rabbit IgG. (F) IRF-3 levels are not reduced with an ICP0 mutant. A Western blot shows total levels of IRF-3 at different times postinfection after virus infection. Proteins in the cell lysates were separated by SDS-PAGE and detected with anti-IRF-3 antibody SL-12.1 (1:750 dilution). (G) Combined IRF-3 bands were scanned, and the intensity was quantified by computer analysis. The amount of IRF-3 was normalized to the amount present at time zero.

FIG. 5.

Infection with d106 inhibits the activation of IRF-3. (A) The d106 and d109 viruses were tested for their ability to inhibit IRF-3 nuclear accumulation. Cells were infected with SeV alone or a combination of SeV and HSV-1 viruses. Indirect immunofluorescence was used to determine the localization of IRF-3 at 6 hpi, and the percentages of cells staining positive for IRF-3 nuclear accumulation are shown. (B) Degradation of IRF-3 is enhanced in the presence of d106. A Western blot shows total levels of IRF-3 at 5 hpi. Proteins in cell lysates were separated by SDS-PAGE and detected with anti-IRF-3 antibody SL-12.1 (1:750 dilution). Combined IRF-3 bands were scanned, and the intensity was quantified by computer analysis. The amount of IRF-3 was normalized to the amount present in the mock-infected samples.

FIG. 6.

Effect of HSV-1 and viral mutants on the production of IFN. (A) Total RNA was isolated from HEC-1-B cells at 5 hpi with SeV alone or a combination of SeV with different HSV-1 viruses. The harvested RNA was analyzed by RNase protection assay for the presence of IFN-β mRNA and control GAPDH mRNA. (B) Medium from the same infected cells was isolated and analyzed by ELISA for IFN-β as described in Materials and Methods. The data are from one experiment representative of replicate experiments.

FIG. 7.

Production of IFN in cells lacking DNA-PK. M059J and M059K cells were mock infected or infected with SeV. Total RNA was isolated at 5 hpi and analyzed by RNase protection assay for the presence of IFN-β mRNA and the control GAPDH mRNA.