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. 2004 Feb;78(4):1675-84.
doi: 10.1128/jvi.78.4.1675-1684.2004.

The herpes simplex virus ICP0 RING finger domain inhibits IRF3- and IRF7-mediated activation of interferon-stimulated genes

Rongtuan Lin  1 , Ryan S Noyce, Susan E Collins, Roger D Everett, Karen L Mossman
Affiliations

The herpes simplex virus ICP0 RING finger domain inhibits IRF3- and IRF7-mediated activation of interferon-stimulated genes

Rongtuan Lin et al. J Virol. 2004 Feb.

Abstract

Virus infection induces a rapid cellular response in cells characterized by the induction of interferon. While interferon itself does not induce an antiviral response, it activates a number of interferon-stimulated genes that collectively function to inhibit virus replication and spread. Previously, we and others reported that herpes simplex virus type 1 (HSV-1) induces an interferon -independent antiviral response in the absence of virus replication. Here, we report that the HSV-1 proteins ICP0 and vhs function in concert to disable the host antiviral response. In particular, we show that ICP0 blocks interferon regulatory factor IRF3- and IRF7-mediated activation of interferon-stimulated genes and that the RING finger domain of ICP0 is essential for this activity. Furthermore, we demonstrate that HSV-1 modifies the IRF3 pathway in a manner different from that of the small RNA viruses most commonly studied.

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Figures

FIG. 1.
FIG. 1.
Expression of ISG56 is only observed in the absence of ICP0 expression. (A) Western blot analysis of whole-cell extracts (40 μg) of HEL fibroblasts harvested 12 h postinfection with the indicated viruses. The ISG56 protein is only visible following infection with UV-inactivated wild-type virus (strain KOS, WT-UV) or an ICP0 null mutant (n212). (B) RT-PCR analysis of total RNA harvested from HEL fibroblasts at various times postinfection with the indicated viruses. In the absence of ICP0, transient ISG56 mRNA accumulation is seen, whereas in the absence of HSV-1 replication (UV-inactivated KOS or genetically inactivated KM110), sustained ISG56 mRNA accumulation is observed. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
FIG. 2.
FIG. 2.
Deletion of vhs and ICP0 results in sustained ISG56 mRNA accumulation. (A) Construction of a double mutant virus deleted for vhs and ICP0. U2OS cells were coinfected with Δsma and 7134 to create a double mutant virus as outlined in Materials and Methods. (Left panel) PCR analysis was used to confirm the internal SmaI-SmaI fragment deletion that defines the vhs Δsma mutation. BstEII-cut lambda DNA was used as a marker. (Right panel) Southern blot analysis with a radiolabeled ICP0 probe was used to distinguish wild-type ICP0 and 7134 ICP0 sequences based on the presence of a unique SstI site within the lacZ gene that replaces the ICP0 sequence in the 7134 mutation. (B) RT-PCR analysis of total RNA harvested from HEL fibroblasts at various times postinfection with the indicated viruses. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
FIG. 3.
FIG. 3.
ICP0 blocks virus-, IRF3-, and IRF7-mediated activation of ISG-specific promoters. Luciferase assays in 293 cells were used to measure activation of the RANTES (A) or IFN-β (B) promoters in response to infection with the paramyxovirus Sendai virus or expression of constitutively activated forms of IRF3 [IRF-3(5D)] and IRF7 [IRF-7(Δ247-467)] in the presence or absence of full-length ICP0. Results represent the mean ± standard deviation of three independent experiments.
FIG. 4.
FIG. 4.
Schematic representation of ICP0 functional domains and mutations used in this study. The uppermost line depicts the 775-amino-acid ICP0 open reading frame (thick line) with the positions of the introns (thin angled lines) and the amino acid coordinates at the intron-exon junctions marked. These sequences are contained within the ICP0 expression plasmid p111 (19). Also marked are the positions of the first and last cysteine residues of the RING finger, the nuclear localization signal (nls) (residues 501 to 510) (17), the USP7 interaction domain (residues 594 to 633) (58), and the sequences required for self-multimerization and efficient localization at ND10 (residues 633 to 720) (11, 55, 58). Deletion mutants 1-519, 1-388, and 1-312 express C-terminally truncated proteins and were constructed by utilizing the MluI, XmaI, and NruI restriction sites at the positions of these 3′ truncations. Mutant 105-519 expresses the coding region between the XhoI and MluI sites. Mutant ins150 contains a 4-amino-acid insertion in codon 150 (p110F1) (19), mutant K144E has a lysine to glutamic acid substitution in the helix region of the RING finger (16), and deletion mutants Δ594-632, Δ106-149, Δ162-188, and Δ9-76 have the stated deletions (p110D12, p110FXE, p110D22, and p110D1, respectively) (17). Truncation mutant 1-593 has stop codons inserted into codon 594 (p110E52X) (58). Finally, mutant 1-241 contains only the sequences encoded in exons 1 and 2 (p110262) (25). Mutants Δ106-149, Δ162-188, and ins150 have lost ubiquitin E3 ligase activity both in vitro and in vivo, while mutant K144E retains in vitro activity but appears to have reduced activity in vivo (4, 20).
FIG. 5.
FIG. 5.
ICP0 RING finger is both necessary and sufficient to block virus-, IRF7-, and IRF3-mediated ISG promoter activation. Luciferase assays in 293 cells were used to measure the activation of the IFN-β promoter following Sendai virus infection or expression of constitutively activated IRF3 [IRF-3(5D)] and IRF7 [IRF-7(Δ247-467)] in the presence or absence of full-length and mutant forms of ICP0 (refer to Fig. 4). Results represent the mean ± standard deviation of three independent experiments.
FIG. 6.
FIG. 6.
ICP0 fails to degrade known components of the IRF3 pathway. Western blot analysis of whole-cell extracts (40 μg) or nuclear extracts (25 μg, for DNA-PK) of HEL fibroblasts harvested 10 h postinfection with the indicated viruses. Wild-type ICP0 fails to induce the degradation of IRF3, CBP, or TBK-1, components of the cellular antiviral pathway, whereas DNA-PK is specifically degraded by wild-type but not mutant ICP0. In the absence of wild-type ICP0, ISG56 protein is observed, whereas expression of IRF7 is only detected with a recombinant adenovirus expressing human IRF7 (AdIRF7). Actin was used as an internal loading control.
FIG. 7.
FIG. 7.
HSV-1 fails to hyperphosphorylate IRF3. Western blot analysis of whole-cell extracts (40 μg) of A549 cells harvested 6 and 12 h postinfection with the indicated viruses. Endogenous IRF3 is present as two species and becomes hyperphosphorylated and subsequently degraded following infection with the paramyxovirus Newcastle disease virus (NDV). A shift from the lower to the upper form of IRF3 is readily observed following infection with wild-type and ICP0 null viruses but not in the absence of virus gene expression.
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