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Journal of Virology logoLink to Journal of Virology
. 2016 Aug 26;90(18):8351–8359. doi: 10.1128/JVI.00939-16

Relative Contributions of Herpes Simplex Virus 1 ICP0 and vhs to Loss of Cellular IFI16 Vary in Different Human Cell Types

Megan H Orzalli 1,*, Nicole M Broekema 1,*, David M Knipe 1,✉
Editor: R M Longnecker2
PMCID: PMC5008076  PMID: 27412599

ABSTRACT

The herpes simplex virus 1 (HSV-1) ICP0 protein is an E3 ubiquitin ligase that promotes the degradation of several host cell proteins. Most studies have found that ICP0 promotes the loss of IFI16 in infected cells, but one study reported that ICP0 was not necessary or sufficient for loss of IFI16 in a tumor-derived cell line. Therefore, in this study, we examined the requirement for ICP0 in promoting the loss of IFI16 in several normal and tumor-derived cell lines. HSV-1 infection resulted in an observable decrease of IFI16 protein levels in normal human foreskin fibroblasts (HFFs), normal oral keratinocytes (NOKs), and HeLa cells but not in U2OS cells. During infection with an ICP0-null virus, we observed a reduced loss of IFI16 in HFFs and NOKs but not in HeLa cells. Ectopic expression of ICP0 from a transfected plasmid was sufficient to promote the loss of IFI16 in HFFs and NOKs. In the absence of ICP0, we observed a delayed reduction of IFI16 protein that correlated with a reduction in the steady-state levels of IFI16 mRNA. In addition, we show that the ICP0-independent loss of IFI16 in HeLa cells is dependent in part on the activity of the viral virion host shutoff (vhs) tegument protein. Together, these results demonstrate that HSV-1 promotes the loss of IFI16 through at least two mechanisms: (i) by ICP0-dependent degradation of IFI16 and (ii) by vhs-dependent turnover of IFI16 mRNA. In addition, this study highlights a potential intrinsic difference between normal and tumor-derived cells for the activities of IFI16 and HSV-1 ICP0.

IMPORTANCE HSV-1 is a ubiquitous virus that establishes a lifetime persistent infection in humans. The relative success of HSV-1 as a pathogen is, in part, dependent on the expression of viral proteins that counteract host intrinsic defense mechanisms and that modulate immune responses during viral infection. In this study, we examined the relative roles of two viral gene products for the ability to promote loss of the antiviral IFI16 DNA sensor. We demonstrate that the viral immediate early ICP0 protein plays a dominant role in the loss of IFI16 in normal, but not tumor-derived, human cell lines. In contrast, viral vhs-mediated loss of IFI16 by mRNA destabilization is revealed to be dominant in tumor-derived cells in which ICP0 is nonfunctional. Together, these results contribute to our understanding of how HSV-1 modulates IFI16 protein levels and highlight cell-type-dependent differences between normal and tumor-derived cells.

INTRODUCTION

The innate immune response plays a critical role in limiting viral replication and dissemination. Initially, the host responds to the presence of an invading virus through the secretion of interferons and proinflammatory cytokines. These effector molecules act in an autocrine or paracrine manner to induce antiviral genes that block virus replication, and they promote viral clearance through the recruitment of specialized immune cells. Production of these effector molecules is mediated by cellular signaling pathways, which are initiated by the recognition of viral pathogen-associated molecular patterns (PAMPs) by cellular pattern recognition receptors (PRRs). Viral PAMPs can include structural proteins such as glycoproteins or capsid, viral RNAs, or even viral DNAs (1), which are recognized by a growing number of PRRs, including Toll-like receptor 9 (TLR9) (2), interferon-inducible protein 16 (IFI16) (3), and cyclic GMP-AMP synthase (cGAS) (4).

A large body of evidence shows that IFI16, a member of the AIM2-like receptor family of DNA sensors, plays an important role in the host response to DNA viruses, particularly against those that replicate in the nuclei of infected cells. IFI16 has been implicated in the initiation of DNA virus-induced inflammasome signaling (5–8), the transcription of antiviral cytokines and interferon-stimulated genes (ISGs) (9–11), and the restriction of DNA virus replication (12–16). The importance of IFI16 in antiviral immunity is underscored by the identification of viral proteins that inhibit its antiviral activities. Two distinct members of the herpesvirus family, human cytomegalovirus (HCMV) and herpes simplex virus 1 (HSV-1), encode proteins that modulate the antiviral activities of IFI16. The HCMV pUL83 tegument protein inhibits antiviral transcription within infected cells by preventing the oligomerization of IFI16 by blocking intermolecular pyrin-pyrin interactions (11). In addition, we and others have demonstrated that the viral ICP0 immediate early protein modulates antiviral gene expression and chromatinization of the HSV-1 genome by promoting the degradation of IFI16 in primary human fibroblasts (6, 10, 13, 14, 17).

Recently, it has been suggested that ICP0 is neither sufficient nor necessary to promote the loss of IFI16 (15). This conclusion was based on virus infections and the inability of a tetracycline-inducible ICP0 expression system to promote the loss of endogenous IFI16 in tumor-derived cells (15). Cell-type-dependent differences for ICP0 functions have been described before (18–20). Therefore, we set out to clarify the role of ICP0 in promoting IFI16 loss during HSV-1 infection by examining ICP0-dependent and -independent effects on IFI16 in a panel of human cell lines from normal and tumor-derived origins.

In this study, we demonstrated that ICP0 is sufficient to promote the degradation of IFI16 in normal human cells but does not target IFI16 for degradation in two tumor-derived cell lines. Furthermore, we observed that under conditions in which ICP0 is not functional, the virion host shutoff (vhs) tegument protein can promote the loss of IFI16 protein by reducing IFI16 mRNA levels.

MATERIALS AND METHODS

Cells.

HeLa cells, U2OS cells, and primary foreskin fibroblasts (HFFs) cells were obtained from the American Type Cell Culture (ATCC). HeLa and U2OS cells were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 5% fetal bovine serum (FBS), 5% bovine calf serum (BCS), and 2 mM glutamine. HFFs were cultured in DMEM supplemented with 10% FBS and 2 mM glutamine (DMEM-10). Human telomerase (htert)-immortalized keratinocytes (normal oral keratinocytes [NOKs]) were a kind gift from Karl Munger (Tufts Medical School) and cultured in keratinocyte-SFM (Gibco) supplemented with penicillin-streptomycin. For cycloheximide treatment, HFFs were plated at a density of 1 × 105 for 24 h prior to treatment. Overlay medium was replaced with DMEM-10 containing 100 μg/ml of cycloheximide or diluent (H2O) for the desired times.

Viruses and viral infections.

HSV-1 wild-type strains KOS (21), 17 (22), and F (23) and the HSV-1 KOS vhs1 mutant virus (24) were grown and titrated on Vero cells. HSV-1 KOS 7134 (25), KOS 7134R (25), F R7910 (26), F R7911 (27), and17 dl1403 (28) were grown and titrated on U2OS cells (29). HSV-1 KOS d109 was grown and titrated on FO6 cells (30). Cell monolayers were incubated with virus diluted in phosphate-buffered saline (PBS)-ABC (PBS with CaCl2 and MgCl2) supplemented with 1% heat-inactivated calf serum and 0.1% glucose in a 37°C shaking incubator for 1 h. Following viral adsorption, the monolayers were replaced with DMEM supplemented with 1% heat-inactivated calf serum and incubated at 37°C for the desired time.

Plasmids and DNA transfections.

Cells were plated at a density of 5 × 104 in 24-well plates 24 h prior to transfection. Cells were transfected with 500 ng of plasmid DNA expressing wild-type ICP0 (pICP0) or an ICP0 RING finger mutant (pICP0.Rfm) (10) using the Lipofectamine LTX reagent (Invitrogen) according to the manufacturer's instructions. Cells were fixed and stained for immunofluorescence at 24 h posttransfection as described below.

Western blotting.

Cells were harvested in lithium dodecyl sulfate (LDS) sample buffer and run on NuPAGE NOVEX 4 to 12% bis-Tris protein gels (Life Technologies). Proteins were transferred to polyvinylidene difluoride (PVDF) membranes, and the membranes were blocked in 5% powdered skim milk-PBS (wt/vol) prior to incubation with primary antibody. Membranes were washed 3 times with PBS containing 0.05% Tween 20 (PBST), incubated in secondary antibody for 1 h at room temperature, and washed twice with PBST and once with PBS. Membranes were incubated with SuperSignal West Pico chemiluminescent substrate (Thermo Scientific) and exposed to film. The antibodies used in this study were anti-IFI16 (1:2,000; Abcam), anti-ICP0 (1:2,000; EastCoast Bio), anti-ICP4 (1:1,000; 58S [31]), anti-glyceraldehyde-3-phosphate dehydrogenase (anti-GAPDH; 1:5,000; Abcam), and secondary goat anti-mouse and goat anti-rabbit antibodies (1:5,000; Santa Cruz).

Immunofluorescence.

Transfected or infected cells were fixed with 2% formaldehyde, permeabilized with 0.5% NP-40, and blocked in 5% normal goat serum overnight at 4°C. Fixed cells were incubated with primary antibodies for 30 min at 37°C and washed twice with PBST, followed by one wash with PBS. Alexa Fluor 488- and 594-conjugated secondary antibodies (1:500; Jackson ImmunoResearch) were incubated with cells for 2 h at 25°C. The coverslips were washed as described above and mounted on slides with ProLong Gold antifade reagent (Invitrogen). Images were acquired using an Axioplan 2 microscope (Zeiss) with a 63× objective and Hamamatsu charge-coupled-device (CCD) camera (model C4742-95). Primary antibodies used for immunofluorescence were anti-IFI16 (1:200; Abcam) and anti-ICP0 (1:150; CLU7p) (32).

Quantitative reverse transcription-PCR.

Total cellular RNA was harvested from infected cells using an RNeasy kit (Qiagen) and quantified with a NanoDrop spectrophotometer (Thermo Scientific). An aliquot of 1 μg of RNA was incubated with DNase (DNA free; Ambion) for 1 h at 37°C. DNase-treated RNAs were reverse transcribed (high-capacity cDNA reverse transcription kit; Life Technologies), and cDNA was analyzed (Step One Plus; Applied Biosystems) using Fast SYBR green master mixes (Applied Biosystems). Primers used in this study were as follows: for IFI16, 5′-ACTGAGTACAACAAAGCCATTTGA-3′ and 5′-TTGTGACATTGTCCTGTCCCCAC-3′; for 18S RNA, 5′-GCATTCGTATTGCGCCGCTA-3′ and 5′-AGCTGCCCGGCGGGT-3′; and for beta interferon (IFN-β), 5′-AAACTCATGAGCAGTCTGCA-3′ and 5′-AGGAGATCTTCAGTTTCGGAGG-3′.

RESULTS

HSV infection promotes the loss of cellular IFI16 in a cell-type-dependent manner.

We and others have demonstrated that HSV-1 infection results in decreased levels of IFI16 in normal human fibroblasts in an ICP0-dependent manner (6, 10). However, it has also been reported that ICP0 is not necessary for the loss of IFI16 in other cell types (15). We therefore compared the losses of IFI16 in several cell lines that are either from normal tissues, including HFFs and NOKs, or from human cancers (HeLa and U2OS cells). Consistent with our previous study (10), HSV-1 KOS wild-type (WT) virus infection resulted in decreased IFI16 protein levels as early as 2 h postinfection (hpi) in HFFs (Fig. 1A). Similarly, HSV-1 infection resulted in an early loss of IFI16 in NOKs (Fig. 1B). In contrast, HSV infection in HeLa cells resulted in only a slight decrease in IFI16 protein levels as early as 4 hpi (Fig. 1C), while we observed no decrease of IFI16 in infected U2OS cells (Fig. 1D). The last observation was consistent with our previous study (13), which demonstrated that endogenous IFI16 protein levels in U2OS cells are unaffected by HSV-1 infection. These results suggested that HSV-1 infection reduces IFI16 protein levels in a cell-type-dependent manner.

FIG 1.

FIG 1

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HSV-1 infection promotes cell-type-dependent loss of IFI16. HFFs (A), NOKs (B), HeLa cells (C), and U2OS cells (D) were infected with KOS strain HSV-1 at an MOI of 10. Protein lysates were isolated from infected cells at 2, 4, 6, or 8 hpi, separated by SDS-PAGE, and analyzed by Western blotting. Progression of infection was monitored by the detection of the viral ICP4 immediate early protein, and GAPDH levels were used as a loading control.

Prior to the loss of IFI16 in HSV-1-infected HFFs, diffuse nuclear and nucleolar IFI16 relocalizes to foci that contain the viral ICP0 protein (Fig. 2A) (10). This relocalization is consistent with the ability of ICP0 to promote the degradation of IFI16 during infection. To determine whether the relocalization of IFI16 occurs in other cell types during HSV-1 infection, we compared the localization of IFI16 in HSV-1-infected HFFs, NOKs, and HeLa and U2OS cells by indirect immunofluorescence microscopy. HSV-1 KOS infection of HFFs and NOKs resulted in an early intranuclear relocalization of IFI16, which colocalized with the HSV-1 ICP0 protein (Fig. 2A and B, 3 hpi). By 6 hpi, IFI16 was barely detectable in the infected cells (Fig. 2A and B, 6 hpi), consistent with the loss of IFI16 observed by Western blotting (Fig. 1A and B). In contrast to the phenotype in HFFs and NOKs, we did not observe an intranuclear relocalization of IFI16 in either HeLa or U2OS cells infected with WT HSV-1 (Fig. 2C and D). Together, these results demonstrated that HSV-1 promotes the redistribution and loss of IFI16 during infection in some, but not all, cell types.

FIG 2.

FIG 2

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HSV-1 infection promotes cell-type-dependent intranuclear relocalization of IFI16. HFFs (A), NOKs (B), HeLa cells (C), and U2OS cells (D) grown on coverslips were infected with KOS strain HSV-1 at an MOI of 10. HSV-1-infected or mock-infected cells were fixed and stained for IFI16 and HSV-1 ICP0 protein at 3 or 6 hpi.

HSV-1 infection promotes the loss of IFI16 in an ICP0-dependent and -independent manner.

Given that the kinetics of IFI16 loss during infection differed between HFFs and NOKs and HeLa cells, we next asked whether the loss of IFI16 in each of these cell lines was dependent on ICP0. Cells were infected with the HSV-1 7134 ICP0-null virus or the corresponding 7134R rescued virus at a multiplicity of infection (MOI) of 10, and whole-cell lysates were examined for IFI16 protein at 2-h intervals for a period of 8 h. In HFFs and NOKs, 7134R virus infection resulted in a loss of IFI16 similar to that observed during WT HSV-1 infection in Fig. 1 (Fig. 3A and B). In contrast, in the absence of ICP0, we observed a transient decrease and recovery in IFI16 protein levels in infected HFFs and NOKs (Fig. 3A and B). IFI16 losses in HeLa cells appeared similar in the presence and absence of ICP0 (Fig. 2C), indicating that the HSV-1-mediated loss of IFI16 in HeLa cells is independent of ICP0. Interestingly, in all three cell types examined, IFI16 protein levels rebounded by 8 hpi in the absence of ICP0. The ICP0-dependent loss of IFI16 in HFF cells described above was not specific to the KOS virus strain examined, because ICP0-null viruses constructed in both strain 17 (dl1403) and F strain (R7910) of HSV-1 were unable to promote the loss of IFI16 compared to their WT counterparts, strain 17 and the R7911 ICP0+ virus, respectively (Fig. 3D).

FIG 3.

FIG 3

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ICP0 is required for efficient IFI16 loss in normal human cells. HFFs (A), NOKs (B), and HeLa cells (C) were infected with a KOS strain ICP0-null (7134) or rescue virus (7134R) at an MOI of 10. Protein lysates were isolated from infected cells at 2, 4, 6, or 8 hpi, separated by SDS-PAGE, and analyzed by Western blotting for IFI16 protein. HSV-1 ICP4 and ICP0 protein levels were examined as markers of viral infection. (D) HFFs were infected with HSV-1 strain 17 or F strain viruses (R7911) or their corresponding ICP0 null viruses (dl1403 and R7910, respectively) at an MOI of 10. Protein lysates were isolated from infected cells at 3 or 6 hpi, separated by SDS-PAGE, and analyzed by Western blotting as for panels A to C.

Transfected ICP0 is sufficient to promote the loss of IFI16 in certain cell types.

We and others have observed that expression of ICP0 from the replication-defective d106 virus was sufficient to reduce IFI16 protein levels compared to a virus lacking ICP0 (6, 10). However, based on those experiments, we could not rule out that ICP0 acts in concert with a tegument protein to mediate the ICP0-dependent loss of IFI16 during infection. To test whether ICP0 is sufficient as a viral protein to reduce IFI16 protein levels, we exogenously expressed ICP0 from a plasmid in the absence of other viral genes. In HFFs and NOKs that expressed ICP0 from the transfected plasmid (pICP0), we observed in a decrease in IFI16 by immunofluorescence (Fig. 4A and B); however, expression of ICP0 defective for E3 ubiquitin ligase activity (pICP0.Rfm) did not promote the loss of IFI16 in either cell type (Fig. 4A and B). Furthermore, plasmid expression of WT ICP0 in either HeLa or U2OS cells did not result in detectable changes in IFI16 protein levels (Fig. 4C and D), confirming that ICP0 is not capable of promoting the loss of endogenous IFI16 in these tumor-derived cell lines.

FIG 4.

FIG 4

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Transient ICP0 expression is sufficient to promote loss of IFI16. HFFs (A), NOKs (B), HeLa cells (C), and U2OS cells (D) grown on coverslips were mock transfected (LTX) or transfected with plasmids expressing WT ICP0 (pICP0) or an ICP0 RING finger mutant (pICP0.RFm). Cells were fixed and stained for IFI16 and ICP0 at 24 h posttransfection.

Cancer-derived cells are less responsive to DNA virus infection.

ICP0 promotes the loss of IFI16 during HSV-1 infection to overcome the host innate immune response and promote viral replication (6, 10, 13). The importance of ICP0 in promoting virus replication is cell type dependent, with primary fibroblasts being the most restrictive for ICP0-null virus replication (33). The inability of ICP0 to promote the loss of IFI16 in cancer-derived cells suggested that innate immune signaling may be defective in these cells. We therefore compared the innate immune responses to HSV-1 viral DNA in our panel of normal and cancer-derived cells. We infected cells with d109 recombinant HSV-1, which lacks viral gene expression and potently activates DNA-dependent signaling pathways (10, 30, 34), and assayed for IFN-β RNA expression by quantitative reverse transcription-PCR (qRT-PCR) at 6 hpi. The ability of ICP0 to promote the loss of IFI16 appeared to correlate with the antiviral responsiveness of a cell type to HSV-1 d109 virus infection (Fig. 5 and Table 1). HFFs and NOKs were the most responsive to HSV-1 d109 virus infection, while we observed very little or no IFN-β induction in infected cancer-derived cells (U2OS and HeLa) or cell lines immortalized with viral gene products (HEK293 and HEK293T) (Fig. 5). Together, these results demonstrated that certain cancer-derived cells have reduced innate immune responses to DNA virus infection and that this reduced response correlates with the inability of ICP0 to promote the degradation of IFI16 in these cell lines.

FIG 5.

FIG 5

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HSV-1 infection induces cell-type-dependent antiviral transcriptional responses. HFFs, NOKs, and HEK293, HEK293T, U2OS, and HeLa cells were infected with HSV-1 d109 virus at an MOI of 10. Total RNA was isolated from infected cells at 6 hpi, DNase treated, and reverse transcribed, and IFN-β transcripts were analyzed by qPCR. Transcripts were normalized to 18S rRNA and are plotted as a fold induction over mock infection. Results are averages from three independent experiments.

TABLE 1.

Summary of IFI16 properties in different cell lines

Cell type ICP0-dependent degradation of IFI16 (reference) Innate immune response to d109
HFF Yes ++++
NOK Yes +++
HeLa No +
U2OS No −
HEK293 No (13) −
HEK293T No (50) −
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HSV-1 infection results in decreased IFI16 mRNA levels.

The ICP0-independent loss of IFI16 prompted us to examine whether HSV-1 modulates the steady-state levels of IFI16 protein by inhibiting cellular gene expression. To test this hypothesis, we examined whether IFI16 transcript levels changed during infection with the 7134 and 7134R viruses. In HFFs and HeLa cells, we observed a 60 to 80% decrease in IFI16 transcripts by 4 hpi regardless of whether the viruses expressed ICP0 (Fig. 6A and B), although we consistently observed that 7134R infection of HFFs resulted in slightly less IFI16 mRNA than 7134 virus infection. At later time points postinfection (6 and 8 h), we observed a rebound in IFI16 transcripts in 7134-infected cells, consistent with the rebound in IFI16 protein levels shown in Fig. 3. These results indicated that HSV-1 infection can reduce IFI16 transcript levels through a mechanism independent of ICP0; nevertheless, ICP0 does play a role in limiting the rebound in IFI16 transcripts late in infection—likely through ICP0-dependent inhibition of expression of interferon and ISGs, which includes the gene for IFI16 (10).

FIG 6.

FIG 6

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HSV-1 infection reduces IFI16 transcripts in an ICP0-independent manner. HFFs (A) or HeLa cells (B) were infected with HSV-1 7134 (ICP0−) or 7134R (ICP0+) viruses at an MOI of 10. Total RNA was isolated from mock-infected or HSV-1-infected cells at 2, 4, 6, or 8 hpi and analyzed by qRT-PCR. IFI16 transcripts were normalized to 18S rRNA and plotted as a percentage of transcripts quantified in a mock-infected sample. Results are averages from three independent experiments. (C) HFFs were treated with 100 μg/ml of cycloheximide (CHX), and protein lysates were isolated at 0, 1, 2, and 3 h posttreatment. IFI16 and GAPDH protein levels were examined by Western blotting and quantified by ImageJ using a protein dilution series as a standard curve; they are plotted relative to IFI16 protein levels at 0 h after cycloheximide treatment (D). A dashed line demarks the time point at which 50% of the IFI16 protein has been lost in CHX-treated cells (154 min). Results in panels C and D are representative data from three independent experiments.

To determine whether decreased IFI16 transcripts during infection could be associated with changes in IFI16 protein levels, we examined the half-life of IFI16 in uninfected HFFs. Cells were treated with the translation inhibitor cycloheximide, and cell lysates were harvested hourly and examined by Western blotting. We observed decreased IFI16 protein levels in the presence of cycloheximide compared to those in vehicle-treated samples (Fig. 6C). When IFI16 band intensities were quantified and a line of best fit was modeled to the data set, the half-life of IFI16 was estimated to be 154 min (Fig. 6D). Given the short half-life of IFI16 in these cells, it was conceivable that the decrease in IFI16 transcripts during HSV-1 infection could account for the ICP0-independent loss of IFI16 protein shown in Fig. 3.

Viral vhs activity is important for promoting IFI16 mRNA loss during HSV-1 infection.

HSV-1 infection results in a general suppression of host protein synthesis in part through virus-mediated destabilization of host mRNAs. The ability of the virus to mediate this host shutoff response is dependent on the UL41 (vhs) tegument protein (24, 35–37). The endoribonuclease activity of vhs is important for the turnover of both host and viral mRNAs during infection (reviewed in reference 38), and viruses that lack vhs are attenuated in vivo (39). The replication defect of a vhs mutant can be partially rescued in mice deficient for type I interferon signaling (40), linking vhs activity to the evasion of the host innate immune response.

To determine whether vhs plays a role in reducing the steady-state levels of IFI16 during HSV-1 infection, we examined IFI16 transcript levels during infection with a vhs mutant virus (vhs1) that harbors a point mutation in the nuclease domain of the UL41 protein (24) or a WT virus (KOS). In both HFFs and HeLa cells, HSV-1 KOS virus infection decreased IFI16 transcripts as expected (Fig. 7A and B); however, in the absence of functional vhs endoribonuclease activity, there was a less dramatic reduction in IFI16 mRNA, indicating that the endoribonuclease activity of vhs is required for maximal loss of IFI16 transcripts during HSV-1 infection. Interestingly, infection with the HSV-1 vhs1 mutant virus did not fully rescue the decrease in IFI16 mRNA, suggesting that additional viral gene products affect IFI16 mRNA levels during infection.

FIG 7.

FIG 7

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HSV-1 infection reduces IFI16 transcripts and protein in a vhs-dependent manner. HFFs (A and C) or HeLa cells (B and D) were infected with HSV-1 vhs1 or WT HSV-1 at an MOI of 10. Total RNA (A and B) or protein lysates (C and D) were isolated at 2, 4, 6, or 8 hpi. IFI16 transcripts were normalized to 18S rRNA and plotted as a percentage of transcripts quantified in a mock-infected sample. Protein lysates were separated by SDS-PAGE, and IFI16 protein levels were examined by Western blotting. Progression of infection was monitored by the detection of the viral ICP4 immediate early protein, and GAPDH levels were examined as a loading control. *, P < 0.05, Student's t test.

When we examined IFI16 protein levels in HFFs infected with the vhs1 or WT KOS viruses, we observed little or no difference in the loss of IFI16 in the presence or absence of a functional vhs protein (Fig. 7C). This result was surprising, given the vhs-dependent effect on IFI16 mRNA levels described above (Fig. 7A); however, it is possible that ICP0 in these cells masks the vhs-dependent phenotype. Indeed, when we examined IFI16 protein levels in infected HeLa cells, in which we have not observed an ICP0-dependent effect on IFI16, we observed a vhs-dependent loss in IFI16 compared to WT HSV-1 infection (Fig. 7D). Furthermore, disruption of vhs activity resulted in a striking transient increase in IFI16 protein levels at 6 hpi, which contrasts with the reduced IFI16 protein levels in KOS virus-infected cells. Because we did not observe a corresponding increase in IFI16 transcripts above the levels in mock-infected samples during infection with the vhs1 virus (Fig. 7B), this suggested that HSV infection stabilizes IFI16 or increases IFI16 protein synthesis in the absence of vhs. Together, these results strongly indicated that vhs plays a role in promoting the loss of IFI16 during infection and its phenotype is revealed only in the absence of ICP0.

DISCUSSION

We and others have shown that the reduction in IFI16 protein levels in HSV-1-infected human fibroblasts is promoted by ICP0 in a proteasome- and E3 ligase domain-dependent manner (6, 10, 17). However, Cuchet-Lourenço et al. (15) observed that the losses of IFI16 were equivalent in HepaRG and U2OS cells infected with ICP0− and ICP0+ viruses. They concluded that ICP0 was not required for degradation of IFI16. We therefore studied in more depth two normal cell types and two cell lines derived from tumors. We confirmed that HFFs and NOKs show ICP0-dependent loss of IFI16 in HSV-1 KOS infection, while HeLa cells showed equal losses of IFI16 when infected with ICP0+ or ICP0− viruses, and we observed little loss of IFI16 in HSV-1 KOS-infected U2OS cells. The loss of IFI16 in HeLa cells was at least in part due to HSV-1 vhs-mediated degradation of IFI16 transcripts. Therefore, at least two aspects of HSV-1 infection, ICP0 and vhs, can reduce IFI16 protein levels, and their relative contributions in reducing IFI16 protein levels differ depending on the cell type examined.

Potential mechanisms of variability in ICP0-dependent degradation of IFI16 in different cell lines.

We observed that ICP0 was required to promote the loss of IFI16 in certain cell lines but not in other cell lines. Furthermore, in support of cell-type-dependent loss of IFI16 by ICP0, Cuchet-Lourenço et al. reported that ICP0 was not required for loss of IFI16 in U2OS and HepaRG cells, both tumor-derived cells (15). What are the possible mechanisms for these differences? First, we observed that the degradation of IFI16 by ICP0 correlated with the ability of individual cell lines to induce IFN-β in response to d109 virus infection. It is known that binding to DNA causes a conformational change in IFI16 (41), and it has been proposed that this conformational change may promote downstream signaling responses. Therefore, it is conceivable that when IFI16 binds to DNA, it undergoes a conformational change that allows ICP0 or a cellular adaptor protein to bind and promote ubiquitination of IFI16 and its subsequent degradation. The inability of ICP0 to target IFI16 for degradation in tumor-derived cell lines may therefore reflect an intrinsic inability of IFI16 in these cell lines to bind to viral DNA or undergo a conformational change. Differences in ICP0 and IFI16 localization within the nucleus may also explain the cell-type-dependent loss of IFI16. Prior to its degradation in normal human cells, diffuse nuclear IFI16 relocalizes to ICP0-containing foci through an unidentified mechanism (10; this study). In contrast, we observed that IFI16 remains diffuse in HSV-1-infected HeLa and U2OS cells. Therefore, the inability of IFI16 to accumulate in ICP0 foci during HSV-1 infection of tumor-derived cells may prevent ICP0 from promoting IFI16 degradation. IFI16 inhibits cell growth (42); therefore, there may be a selection for mutational or other means of inactivation of IFI16 in tumor cells for optimal growth. We observed no difference in the sequences of IFI16 cDNA from HFFs and HeLa cells (M. H. Orzalli and D. M. Knipe, unpublished results), indicating that the inability of ICP0 to promote the degradation of IFI16 in tumor-derived cells is not due to differences in protein sequence. Interestingly, Lau et al. (43) have reported that human papillomavirus E7 binds to STING and inhibits the cGAS-STING pathway. This reported effect is downstream of IFI16, but it is conceivable that HPV proteins expressed in HeLa cells may also inhibit IFI16 function and/or prevent its degradation promoted by ICP0. The effects of HPV E7 and E6 on IFI16 function should be tested in HFFs, in which IFI16 is known to be functional. Alternatively, the ability of ICP0 to target IFI16 for degradation may rely on a posttranslational modification that is absent in HeLa cells, IFI16 may be sequestered from ICP0 by another interaction, or another cellular protein may bridge ICP0 and IFI16 to promote degradation of the latter. Interestingly, it was recently observed that HSV-1 infection is able to promote the degradation of IFI16 in BRCA1-expressing (184B5) but not BRCA1-deficient (HCC1937) mammary epithelial cells (44). BRCA1 is best characterized as a tumor suppressor and is mutated or dysfunctional in a variety of tumor-derived cells (45). Future studies will be necessary to define the role of BRCA1 in promoting ICP0-dependent loss of IFI16 in additional cell types, including those used in this study.

ICP0 is sufficient for IFI16 degradation in transfected cells.

We observed that exogenous overexpression of WT ICP0, but not an ICP0 E3 ubiquitin ligase mutant, from a transfected plasmid clearly results in decreased IFI16 protein levels in normal cells, arguing that ICP0 is capable of inducing the degradation of IFI16 independently of other viral proteins. However, Cuchet-Lourenço et al. (15) reported that tetracycline-induced expression of ICP0 expression was not sufficient to induce loss of IFI16 in a HepaRG cell line. It is conceivable that HepaRG is missing a cellular function needed for ICP0 to promote degradation of IFI16. Alternatively, ICP0 may require a conformational change in IFI16 that occurs in the presence of foreign DNA, an event that would be observed during transient transfection but not when ICP0 is induced from an integrated gene in stable cell lines.

Diner et al. (17) concluded that ICP0 was not sufficient for degradation of IFI16 because they reported no change in total IFI16 levels in cells transfected with a plasmid expressing ICP0. However, the efficiency of transfection may not have been high enough to observe a change in the total population of IFI16 in all cells. Instead, it may be necessary to look at individual ICP0-expressing cells to see a change in IFI16 levels, as we have observed in this study by immunofluorescence.

Role of vhs in loss of IFI16.

Viruses are controlled at the cellular level by both constitutively expressed intrinsic immune responses and the induction of innate immune signaling pathways. Successful viruses must overcome these cellular responses to efficiently replicate and spread. Large DNA viruses, including members of the Herpesviridae family, are particularly adept at inhibiting intrinsic and innate immune responses through the expression of immunomodulatory proteins. In addition to directly targeting antiviral proteins and cellular innate signaling components to counteract intrinsic and innate immune responses, viral gene products can also have indirect effects on the antiviral host response to infection by targeting broad cellular pathways. HSV-1 infection results in a general suppression of host protein synthesis, in part through virus-mediated destabilization of host mRNAs. This vhs-mediated response attenuates the establishment of an antiviral state by reducing the expression of interferons and ISGs (46, 47), which are induced upon viral infection. Less clear is the effect of vhs on the steady-state levels of constitutively expressed host proteins. HSV can reduce tumor necrosis factor receptor (TNFR) replenishment on the cell surface in a vhs-dependent manner (48). The relative short half-life of TNFR (30 min) makes this protein particularly sensitive to vhs-dependent downregulation because TNFR must be continuously resynthesized to maintain its expression on the cell surface. Similarly, we observed that the half-life of IFI16 is also relatively short (154 min), and IFI16 is thus subject to vhs-mediated downregulation. Interestingly, we observed that the RNase enzymatic activity of vhs was not required for the full loss of IFI16 transcripts following HSV-1 infection, suggesting that additional viral proteins may regulate IFI16 transcript and protein levels. In addition to vhs, the HSV-1 immediate early ICP27 protein contributes to the host shutoff response by decreasing cellular RNAs (49). Therefore, it is conceivable that ICP27 contributes to the observed vhs-independent loss of IFI16 transcripts. Further studies are needed to determine whether ICP27 and/or other viral proteins contribute to the downregulation of IFI16 transcripts during HSV-1 infection.

Together, these studies suggest that HSV-1 vhs and likely other viral proteins that regulate host protein synthesis play a role in promoting the loss of cellular proteins with short half-lives and that this ability to regulate constitutively expressed protein levels may have important implications for the progression of viral infection.

ACKNOWLEDGMENTS

We thank Jeho Shin for technical assistance and Patrick T. Waters for assistance with the manuscript.

REFERENCES

  • 1.Takeuchi O, Akira S. 2010. Pattern recognition receptors and inflammation. Cell 140:805–820. doi: 10.1016/j.cell.2010.01.022. [DOI] [PubMed] [Google Scholar]
  • 2.Lund J, Sato A, Akira S, Medzhitov R, Iwasaki A. 2003. Toll-like receptor 9-mediated recognition of Herpes simplex virus-2 by plasmacytoid dendritic cells. J Exp Med 198:513–520. doi: 10.1084/jem.20030162. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Unterholzner L, Keating SE, Baran M, Horan KA, Jensen SB, Sharma S, Sirois CM, Jin T, Latz E, Xiao TS, Fitzgerald KA, Paludan SR, Bowie AG. 2010. IFI16 is an innate immune sensor for intracellular DNA. Nat Immunol 11:997–1004. doi: 10.1038/ni.1932. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Sun L, Wu J, Du F, Chen X, Chen ZJ. 2013. Cyclic GMP-AMP synthase is a cytosolic DNA sensor that activates the type I interferon pathway. Science 339:786–791. doi: 10.1126/science.1232458. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Kerur N, Veettil MV, Sharma-Walia N, Bottero V, Sadagopan S, Otageri P, Chandran B. 2011. IFI16 acts as a nuclear pathogen sensor to induce the inflammasome in response to Kaposi sarcoma-associated herpesvirus infection. Cell Host Microbe 9:363–375. doi: 10.1016/j.chom.2011.04.008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Johnson KE, Chikoti L, Chandran B. 2013. Herpes simplex virus 1 infection induces activation and subsequent inhibition of the IFI16 and NLRP3 inflammasomes. J Virol 87:5005–5018. doi: 10.1128/JVI.00082-13. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Singh VV, Kerur N, Bottero V, Dutta S, Chakraborty S, Ansari MA, Paudel N, Chikoti L, Chandran B. 2013. Kaposi's sarcoma-associated herpesvirus latency in endothelial and B cells activates gamma interferon-inducible protein 16-mediated inflammasomes. J Virol 87:4417–4431. doi: 10.1128/JVI.03282-12. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Ansari MA, Singh VV, Dutta S, Veettil MV, Dutta D, Chikoti L, Lu J, Everly D, Chandran B. 2013. Constitutive interferon-inducible protein 16-inflammasome activation during Epstein-Barr virus latency I, II, and III in B and epithelial cells. J Virol 87:8606–8623. doi: 10.1128/JVI.00805-13. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Li T, Diner BA, Chen J, Cristea IM. 2012. Acetylation modulates cellular distribution and DNA sensing ability of interferon-inducible protein IFI16. Proc Natl Acad Sci U S A 109:10558–10563. doi: 10.1073/pnas.1203447109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Orzalli MH, DeLuca NA, Knipe DM. 2012. Nuclear IFI16 induction of IRF-3 signaling during herpesviral infection and degradation of IFI16 by the viral ICP0 protein. Proc Natl Acad Sci U S A 109:E3008–E3017. doi: 10.1073/pnas.1211302109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Li T, Chen J, Cristea IM. 2013. Human cytomegalovirus tegument protein pUL83 inhibits IFI16-mediated DNA sensing for immune evasion. Cell Host Microbe 14:591–599. doi: 10.1016/j.chom.2013.10.007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Gariano GR, Dell'Oste V, Bronzini M, Gatti D, Luganini A, De Andrea M, Gribaudo G, Gariglio M, Landolfo S. 2012. The intracellular DNA sensor IFI16 gene acts as restriction factor for human cytomegalovirus replication. PLoS Pathog 8:e1002498. doi: 10.1371/journal.ppat.1002498. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Orzalli MH, Conwell SE, Berrios C, Decaprio JA, Knipe DM. 2013. Nuclear interferon-inducible protein 16 promotes silencing of herpesviral and transfected DNA. Proc Natl Acad Sci U S A 110:E4492–E4501. doi: 10.1073/pnas.1316194110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Johnson KE, Bottero V, Flaherty S, Dutta S, Singh VV, Chandran B. 2014. IFI16 restricts HSV-1 replication by accumulating on the HSV-1 genome, repressing HSV-1 gene expression, and directly or indirectly modulating histone modifications. PLoS Pathog 10:e1004503. doi: 10.1371/journal.ppat.1004503. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Cuchet-Lourenço D, Anderson G, Sloan E, Orr A, Everett RD. 2013. The viral ubiquitin ligase ICP0 is neither sufficient nor necessary for degradation of the cellular DNA sensor IFI16 during herpes simplex virus 1 infection. J Virol 87:13422–13432. doi: 10.1128/JVI.02474-13. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Lo Cigno I, De Andrea M, Borgogna C, Albertini S, Landini MM, Peretti A, Johnson KE, Chandran B, Landolfo S, Gariglio M. 2015. The nuclear DNA sensor IFI16 acts as a restriction factor for human papillomavirus replication through epigenetic modifications of the viral promoters. J Virol 89:7506–7520. doi: 10.1128/JVI.00013-15. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Diner BA, Lum KK, Javitt A, Cristea IM. 2015. Interactions of the antiviral factor interferon gamma-inducible protein 16 (IFI16) mediate immune signaling and herpes simplex virus-1 immunosuppression. Mol Cell Proteomics 14:2341–2356. doi: 10.1074/mcp.M114.047068. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Everett RD, Boutell C, Orr A. 2004. Phenotype of a herpes simplex virus type 1 mutant that fails to express immediate-early regulatory protein ICP0. J Virol 78:1763–1774. doi: 10.1128/JVI.78.4.1763-1774.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Yao F, Schaffer PA. 1995. An activity specified by the osteosarcoma line U2OS can substitute functionally for ICP0, a major regulatory protein of herpes simplex virus type 1. J Virol 69:6249–6258. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Kalamvoki M, Roizman B. 2014. HSV-1 degrades, stabilizes, requires, or is stung by STING depending on ICP0, the US3 protein kinase, and cell derivation. Proc Natl Acad Sci U S A 111:E611–E617. doi: 10.1073/pnas.1323414111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Schaffer P, Vonka V, Lewis R, Benyesh-Melnick M. 1970. Temperature-sensitive mutants of herpes simplex virus. Virology 42:1144–1146. doi: 10.1016/0042-6822(70)90364-8. [DOI] [PubMed] [Google Scholar]
  • 22.Brown SM, Ritchie DA, Subak-Sharpe JH. 1973. Genetic studies with herpes simplex virus type 1. The isolation of temperature-sensitive mutants, their arrangement into complementation groups and recombination analysis leading to a linkage map. J Gen Virol 18:329–346. [DOI] [PubMed] [Google Scholar]
  • 23.Ejercito PM, Kieff ED, Roizman B. 1968. Characterization of herpes simplex virus strains differing in their effect on social behavior of infected cells. J Gen Virol 2:357–364. doi: 10.1099/0022-1317-2-3-357. [DOI] [PubMed] [Google Scholar]
  • 24.Read GS, Frenkel N. 1983. Herpes simplex virus mutants defective in the virion-associated shutoff of host polypeptide synthesis and exhibiting abnormal synthesis of alpha (immediate early) viral polypeptides. J Virol 46:498–512. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Cai WZ, Schaffer PA. 1989. Herpes simplex virus type 1 ICP0 plays a critical role in the de novo synthesis of infectious virus following transfection of viral DNA. J Virol 63:4579–4589. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Kawaguchi Y, Van Sant C, Roizman B. 1997. Herpes simplex virus 1 alpha regulatory protein ICP0 interacts with and stabilizes the cell cycle regulator cyclin D3. J Virol 71:7328–7336. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Van Sant C, Kawaguchi Y, Roizman B. 1999. A single amino acid substitution in the cyclin D binding domain of the infected cell protein no. 0 abrogates the neuroinvasivesness of herpes simplex virus without affecting its ability to replicate. Proc Natl Acad Sci U S A 96:8184–8189. doi: 10.1073/pnas.96.14.8184. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Stow ND, Stow EC. 1986. Isolation and characterization of a herpes simplex virus type 1 mutant containing a deletion within the gene encoding the immediate early polypeptide Vmw110. J Gen Virol 67:2571–2585. doi: 10.1099/0022-1317-67-12-2571. [DOI] [PubMed] [Google Scholar]
  • 29.Cai W, Schaffer PA. 1992. Herpes simplex virus type 1 ICP0 regulates expression of immediate-early, early, and late genes in productively infected cells. J Virol 66:2904–2915. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Samaniego LA, Neiderhiser L, DeLuca NA. 1998. Persistence and expression of the herpes simplex virus genome in the absence of immediate-early proteins. J Virol 72:3307–3320. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Showalter SD, Zweig M, Hampar B. 1981. Monoclonal antibodies to herpes simplex virus type 1 proteins, including the immediate-early protein ICP 4. Infect Immun 34:684–692. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Lium EK, Panagiotidis CA, Wen X, Silverstein S. 1996. Repression of the alpha0 gene by ICP4 during a productive herpes simplex virus infection. J Virol 70:3488–3496. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Boutell C, Everett RD. 2013. Regulation of alphaherpesvirus infections by the ICP0 family of proteins. J Gen Virol 94:465–481. doi: 10.1099/vir.0.048900-0. [DOI] [PubMed] [Google Scholar]
  • 34.Eidson KM, Hobbs WE, Manning BJ, Carlson P, DeLuca NA. 2002. Expression of herpes simplex virus ICP0 inhibits the induction of interferon-stimulated genes by viral infection. J Virol 76:2180–2191. doi: 10.1128/jvi.76.5.2180-2191.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Strom T, Frenkel N. 1987. Effects of herpes simplex virus on mRNA stability. J Virol 61:2198–2207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Kwong AD, Kruper JA, Frenkel N. 1988. Herpes simplex virus virion host shutoff function. J Virol 62:912–921. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Oroskar AA, Read GS. 1989. Control of mRNA stability by the virion host shutoff function of herpes simplex virus. J Virol 63:1897–1906. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Smiley JR. 2004. Herpes simplex virus virion host shutoff protein: immune evasion mediated by a viral RNase? J Virol 78:1063–1068. doi: 10.1128/JVI.78.3.1063-1068.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Strelow LI, Leib DA. 1995. Role of the virion host shutoff (vhs) of herpes simplex virus type 1 in latency and pathogenesis. J Virol 69:6779–6786. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Leib DA, Harrison TE, Laslo KM, Machalek MA, Moorman NJ, Virgin HW. 1999. Interferons regulate the phenotype of wild-type and mutant herpes simplex viruses in vivo. J Exp Med 189:663–672. doi: 10.1084/jem.189.4.663. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Jin T, Perry A, Jiang J, Smith P, Curry JA, Unterholzner L, Jiang Z, Horvath G, Rathinam VA, Johnstone RW, Hornung V, Latz E, Bowie AG, Fitzgerald KA, Xiao TS. 2012. Structures of the HIN domain:DNA complexes reveal ligand binding and activation mechanisms of the AIM2 inflammasome and IFI16 receptor. Immunity 36:561–571. doi: 10.1016/j.immuni.2012.02.014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Xin H, Curry J, Johnstone RW, Nickoloff BJ, Choubey D. 2003. Role of IFI 16, a member of the interferon-inducible p200-protein family, in prostate epithelial cellular senescence. Oncogene 22:4831–4840. doi: 10.1038/sj.onc.1206754. [DOI] [PubMed] [Google Scholar]
  • 43.Lau L, Gray EE, Brunette RL, Stetson DB. 2015. DNA tumor virus oncogenes antagonize the cGAS-STING DNA-sensing pathway. Science 350:568–571. doi: 10.1126/science.aab3291. [DOI] [PubMed] [Google Scholar]
  • 44.Dutta D, Dutta S, Veettil MV, Roy A, Ansari MA, Iqbal J, Chikoti L, Kumar B, Johnson KE, Chandran B. 2015. BRCA1 regulates IFI16 mediated nuclear innate sensing of herpes viral DNA and subsequent induction of the innate inflammasome and interferon-beta responses. PLoS Pathog 11:e1005030. doi: 10.1371/journal.ppat.1005030. [DOI] [PMC free article] [PubMed] [Google Scholar] [Retracted]
  • 45.O'Connor MJ. 2015. Targeting the DNA damage response in cancer. Mol Cell 60:547–560. doi: 10.1016/j.molcel.2015.10.040. [DOI] [PubMed] [Google Scholar]
  • 46.Duerst RJ, Morrison LA. 2004. Herpes simplex virus 2 virion host shutoff protein interferes with type I interferon production and responsiveness. Virology 322:158–167. doi: 10.1016/j.virol.2004.01.019. [DOI] [PubMed] [Google Scholar]
  • 47.Pasieka TJ, Lu B, Crosby SD, Wylie KM, Morrison LA, Alexander DE, Menachery VD, Leib DA. 2008. Herpes simplex virus virion host shutoff attenuates establishment of the antiviral state. J Virol 82:5527–5535. doi: 10.1128/JVI.02047-07. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48.Liang L, Roizman B. 2006. Herpes simplex virus 1 precludes replenishment of the short-lived receptor of tumor necrosis factor alpha by virion host shutoff-dependent degradation of its mRNA. J Virol 80:7756–7759. doi: 10.1128/JVI.00587-06. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 49.Hardwicke MA, Sandri-Goldin RM. 1994. The herpes simplex virus regulatory protein ICP27 contributes to the decrease in cellular mRNA levels during infection. J Virol 68:4797–4810. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 50.Orzalli MH, Broekema NM, Diner BA, Hancks DC, Elde NC, Cristea IM, Knipe DM. 2015. cGAS-mediated stabilization of IFI16 promotes innate signaling during herpes simplex virus infection. Proc Natl Acad Sci U S A 112:E1773–E1781. doi: 10.1073/pnas.1424637112. [DOI] [PMC free article] [PubMed] [Google Scholar]

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