Herpes Simplex Virus Virion Host Shutoff Attenuates Establishment of the Antiviral State

Herpes simplex virus mutants lacking the vhs protein are severelyattenuated in animal models of pathogenesis and exhibit reducedgrowth in primary cell culture. As a result of these properties,viruses with vhs deleted have been proposed as live-attenuatedvaccines. Despite these findings and their implications forvaccines, the mechanisms by which vhs promotes infection incell culture and in vivo are not understood. In this study wedemonstrate that vhs-deficient viruses replicate to reducedlevels in interferon (IFN)-primed cells and that this deficithas both IFN-dependent and IFN-independent components. Furthermore,vhs-defective viruses induce increased and physiologically activelevels of IFN, increased amounts of IFN-stimulated transcripts,and more phosphorylated eIF2 ${alpha}$ . In addition, we demonstrate greateraccumulation of viral RNAs following infection with a vhs-deficientvirus. This leads to the hypothesis that attenuation of viruseslacking vhs may be attributed to increased levels of double-strandedRNA, a potent pathogen-associated molecular pattern. Togetherthese data show that vhs likely functions to reduce innate immuneresponses and thereby acts as a critical determinant of viralpathogenesis.

INTRODUCTION

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INTRODUCTION

Herpes simplex virus type (HSV) is a ubiquitous human pathogenresponsible for a variety of conditions, including cold sores,genital sores, and keratitis in immunocompetent hosts. In neonates,or in hosts lacking a competent immune system, HSV can becomedisseminated and cause severe and life-threatening diseases,including hepatitis and encephalitis. The outcome of infectionis determined largely by the status of the host's immune responseand its interaction with the many viral genes that act to counterit. Of these, the virion host shutoff protein (vhs), while beingnonessential for replication cell in culture, is a crucial factorfor both HSV type 1 (HSV-1) and HSV-2 replication, virulence,and pathogenesis in vivo (35, 39). Viruses lacking vhs havean increased susceptibility to interferon (IFN), an increasedability to activate dendritic cells, and a decreased abilityto down-regulate major histocompatibility complex class I incell culture (31, 40, 43). These observations highlight themultifactorial impact of vhs on the innate and adaptive immunesystem (32). In addition, the reduced replication of vhs-deficientviruses relative to wild-type virus is observed rapidly, within24 h of infection in the cornea or vaginal epithelium (35, 39),suggesting that an inability to counter innate immunity, ratherthan adaptive immunity, contributes most significantly to theattenuated phenotype. In addition, vhs of HSV-2 plays an importantrole early in HSV-2 pathogenesis in vivo by interfering withthe IFN-mediated antiviral response (5). It has been proposedthat HSVs with vhs deleted might be useful as live attenuatedvaccines in both prophylactic and therapeutic settings and assuch have proved effective in animal models (11, 15, 44, 45).Together these data point to a critical role for vhs in thecounteraction of the innate immune system, but the precise mechanismshave yet to be determined.

Biochemically, the function of vhs has been well characterized.vhs, a 58-kDa phosphoprotein, is a virally encoded RNase withRNase activity that degrades both host and viral mRNA and therebycontributes to the marked decrease in host protein synthesisthat follows HSV infection (16). As a component of the HSV tegument,vhs targets host mRNAs before de novo viral gene expressionby association with cellular translation initiation factors(8, 9). vhs is believed to promote viral infection by depletingcellular mRNA, thereby decreasing its competition with viralmRNA for cellular translation machinery and perhaps by decreasingcertain specific genes important for the host response to infection(7). vhs also contributes to infection by regulating the stabilityof viral RNA as infection progresses and through stabilizationof the gE/gI complex, a complex necessary for cell-to-cell spreadin vitro and in vivo (14).

In order to sense infection and to rapidly inhibit the growthand spread of pathogens, mammalian cells have evolved a numberof innate defenses, many of which are triggered by the recognitionof a variety of pathogen-associated molecular patterns (PAMPs).For viruses, these PAMPs include envelope proteins and nucleicacids, especially double-stranded RNA (dsRNA) and DNA (30).All three of these species of PAMP are implicated in the recognitionof HSV and the initiation of the cellular antiviral responsepathway. In brief, nuclear translocation and activation of NF- ${kappa}$ Bas well as interferon regulatory factors 3 and 7 (IRF-3 andIRF-7) result in the increased expression of hundreds of genes,including the type I beta interferon (IFN-β) (3, 4). IFN-βfurther amplifies the response and signals the infection toadjacent cells through JAK/STAT signaling. In the infected cell,the dsRNA-dependent protein kinase R (PKR) is activated by dsRNAand phosphorylates the translation initiation factor eIF2 ${alpha}$ , promotingthe shutdown of viral and host protein synthesis. While PKRis recognized as a major kinase mediating the phosphorylationof eIF2 ${alpha}$ , other kinases also contribute to the phosphorylationof eIF2 ${alpha}$ (47).

To date, four HSV genes are known to play major roles in IFNresistance (vhs and the ICP0, ICP34.5, and US11 genes) (2, 6,12, 18, 21, 23), but the mechanism of vhs in IFN resistanceremains obscure. In addition, the profound in vivo attenuationof HSV strains lacking vhs remains poorly understood. In continuouscell lines, viruses lacking vhs grow with wild-type kineticsin one-step and multiple-step growth curves. In primary cells,however, a distinct pattern of multiple-step growth emerges.We have shown previously that HSV-1 vhs mutants show wild-typevirus growth up to 24 h, but thereafter their growth is significantlyreduced (37). For HSV-2 vhs mutants, the attenuation occurseven sooner under these culture and infection conditions (5).Therefore, we have hypothesized that vhs plays a crucial rolein maintaining the susceptibility of cells to HSV beyond theinitial round of infection via suppression of innate immuneresponses.

To test this hypothesis, we measured cellular events downstreamof viral recognition, including host gene expression, eIF2 ${alpha}$ phosphorylation,and IFN-β expression, comparing the cellular response toa wild-type (KOS) virus to that of a vhs-null mutant ( ${Delta}$ vhs₁).We report herein that infection of mouse embryo fibroblasts(MEFs) with a virus lacking a functional vhs protein leads toan accumulation of viral RNAs, increased phosphorylation ofeIF2 ${alpha}$ , and increased production of IFN-β. Taken together,these findings imply that accumulated viral RNAs induce expressionof antiviral genes that attenuate vhs-deficient viruses.

MATERIALS AND METHODS

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MATERIALS AND METHODS

Cells and viruses. Viral stocks were grown and titers were determined on Vero cellsas previously described (27). The HSV-1 wild-type strain KOSwas the background strain for most of this study (34). Constructionof the KOS-derived vhs-null HSV-1 strain NHB (referred to inthis paper as the ${Delta}$ vhs₁ mutant) has been described previously(39). For studies with HSV-2, strain 333 was the backgroundstrain from which the vhs-null virus strain 333d41 was constructed,and this null strain is referred to as the ${Delta}$ vhs₂ mutant (35).MEF cultures were generated from 129 Sv/Ev mice at embryonicday 15 and passaged once before being plated for infection.MEFs were cultured in Dulbecco's modified Eagle's medium supplementedwith 10% fetal calf serum, 0.1 mM sodium pyruvate, 250 U/mlpenicillin, 250 µg/ml streptomycin, and 250 ng/ml amphotericinB. Isogenic IFN- ${alpha}$ β ${gamma}$ R^–/– (AG129) (24) and PKR^–/–(129PK) (48) MEFs were also utilized. For multiple-step growthcurves, cells were pretreated overnight with 100 U/ml IFN- ${alpha}$ (Sigma,St. Louis, MO) where appropriate, and cells were infected ata multiplicity of infection (MOI) of 0.01.

HSV-1 microarray production. ArrayOligoSelector was used to select 182 70-mer oligonucleotidesspecific for HSV-1 transcripts (1). The oligonucleotides werecompared to the HSV-1 strain 17 viral genome (GenBank accessionno. NC_001806) and the mouse RefSeq transcriptome. When possible,three nonoverlapping oligonucleotides were chosen for each HSV-1transcript. In addition, 68 HSV-1-specific oligonucleotidesidentical to those generated previously were synthesized (36).Selected cellular genes were also represented on the array.To facilitate normalization, Arabidopsis thaliana ready-to-spotoligonucleotides (Stratagene, La Jolla, CA) were also included.Each oligonucleotide probe was synthesized by Illumina, Inc.(San Diego, CA). Oligonucleotides were solubilized at 20 µMin 2x SSC (1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate)and 0.75 M betaine for printing. The oligonucleotides were spottedin triplicate onto expoxide-coated glass slides (Corning, Corning,NY) by use of a linear servo arrayer (17). The printed slideswere placed in an oven at room temperature under 70% humidityfor 12 to 14 h. After incubation, the slides were cross-linkedusing the UV Stratalinker 2400 (Stratagene, La Jolla, CA) at150 µJ/cm².

RNA preparation. Cultures of 2.5 x 10⁶ MEFs in 100-mm dishes were infected atan MOI of 5 with KOS or the ${Delta}$ vhs₁ mutant for 30 min followedby removal of the inoculum and addition of complete medium.At the times postinfection indicated in the figures, RNA washarvested as previously described (26). RNA was purified andDNase treated with the RNeasy minikit (Qiagen, Valencia, CA).Total RNA quality was determined with an Agilent 2100 bioanalyzer(Agilent Technologies, Santa Clara, CA) according to the manufacturer'srecommendations.

cDNA synthesis and labeling. First-strand cDNA was generated by random and oligo(dT)-primedreverse transcription (Superscript II; Invitrogen, Carlsbad,CA) utilizing the 3DNA Array 900MPX kit (Genisphere, Hatfield,PA), according to the manufacturer's protocol. The cDNAs werequantified via spectrophotometry to confirm recovery. Sampleswere paired and balanced by mass.

Hybridization. Each sample pair was suspended in sodium dodecyl sulfate-basedhybridization buffer (Genisphere) and Array 50dT blocker (Genisphere).Two hybridizations were carried out in a sequential manner.The primary hybridization was performed under a supported glasscoverslip (Erie Scientific, Portsmouth, NH) at 62°C for16 to 20 h. Prior to the secondary hybridization, the slideswere washed as described in the manufacturer's protocol. Secondaryhybridization was carried out using the 3DNA Array 350 kit (Genisphere)according to the manufacturer's protocol, washed, and treatedwith Dyesaver (Genisphere) before scanning.

Data analysis. Slides were scanned immediately following hybridization on aScanArray Express HT scanner (PerkinElmer, Waltham, MA). Laserpower was kept constant, and photomultiplier tube values wereset for optimal intensity with minimal background. Griddingand analysis of images were performed with Scanarray softwareexpress v3.0 (PerkinElmer). The resulting median pixel valueswere imported into GeneSpring v7.3 software (Agilent), and localbackground intensities were subtracted from individual spotintensities. To account for dye swap, the "signal" channel and"control" channel measurements were reversed in such samplesso that signal derived from ${Delta}$ vhs₁ mutant-infected MEF RNA occupiedthe signal channel and that of the KOS-infected MEF RNA occupiedthe control channel. The mean signal and control intensitiesof the on-slide duplicate spots were calculated, and the controlvalues were normalized with GeneSpring's "normalize to positivecontrol genes" function using four SpotReport oligonucleotidearray validation system spike-in controls. If the control channelsignal was lower than 10 relative fluorescence units (rfu),then 10 rfu was used instead. Signal-to-normalized-control ratioswere calculated, and the cross-chip averages were derived fromthe antilog of the mean of the natural log ratios across allbiologic replicates. Oligonucleotide elements that receiveda detectable call (intensity of >200 rfu or local signal-to-backgroundratio of >2 in at least one channel) by Scanarray softwarein either the Cy3 or Cy5 in at least two of four samples ofat least one time point were identified, and all others wereexcluded from the analysis. Genes were not filtered based onsignificance in order to be able to visualize global trends.Differences in dye incorporation and quantum yield were normalizedby the addition of A. thaliana mRNA spikes into the reversetranscription reaction in equal molar amounts along with theexperimental mRNA. Data represent two independent biologicalexperiments with dye swaps such that two technical experimentswere performed for each independent biological sample.

Real-time reverse transcription (RT)-PCR. Total RNA was harvested from MEFs in 35-mm wells that were mocktreated or infected at an MOI of 5 utilizing the Aurum totalRNA minikit as described in the kit protocol (Bio-Rad, Hercules,CA). cDNA was generated using the iScript cDNA synthesis kitas described in the kit protocol (Bio-Rad). PCRs were preparedwith iQ SYBR green supermix (Bio-Rad), 5% acetamide, primers(IDT, Coralville, IA), and 2 µl of cDNA. All PCRs wereperformed in duplicate with the Bio-Rad iCycler. Primers specificfor 18S rRNA were used as a reference gene for normalizationbetween samples.

IFN-β ELISA. Confluent MEFs plated in 35-mm wells were mock treated or infectedat an MOI of 5 for 3, 6, 9, or 12 h with KOS or the ${Delta}$ vhs₁ mutantand cultured in 1 ml of medium. Culture supernatants were harvestedat the indicated times and stored at –20°C until assayed.IFN-β in the medium was measured using 50 µl of harvestedmedium in a mouse IFN-β enzyme-linked immunosorbent assay(ELISA) as described in the kit protocol (PBL Biomedical Laboratories,Piscataway, NJ).

VSV and HSV challenge assay. MEF monolayers were mock treated or infected at MOI of 5 withKOS or the ${Delta}$ vhs₁ mutant for 12 h, at which time the medium (1ml) was collected and clarified by low-speed centrifugation(300 rpm, 5 min) to remove cellular debris. To neutralize infectiousHSV, the medium was divided and treated with either 50 µlof Dulbecco's modified Eagle's medium or HSV-1 neutralizingantibody (Sigma, St. Louis, MO) for 1 h at 37°C. Treatedmedium was used as a pretreatment for fresh secondary MEF monolayers,with 150 µl of medium used per 35-mm well. After 18 hof pretreatment, the medium was removed and the MEFs were infectedwith either vesicular stomatitis virus (VSV) or HSV at an MOIof 0.01. Some pretreated wells were left uninfected in orderto assay the effectiveness of the neutralizing antibody treatment.After 2 days postinfection (VSV) or 3 days postinfection (HSV),the medium and cells were harvested, and the titers of infectiousparticles were determined on Vero cells.

Western blot analysis. Protein lysates were prepared, processed, and analyzed as previouslydescribed (26). The antibodies used in this study included totaleIF2 ${alpha}$ (FL-315) (Santa Cruz Biotechnology, Santa Cruz, CA), phospho-specificeIF2 ${alpha}$ (pS52) (BioSource International Inc, Camarillo, CA), andthe secondary antibody goat anti-rabbit-horseradish peroxidaseconjugate (Bio-Rad).

RESULTS

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RESULTS

Growth of the ${Delta}$ vhs₁ mutant is reduced in IFN-treated MEFs. vhs-null viruses exhibit near-normal growth in continuous celllines such as Vero and Hep-2 (28), with only slight growth defectsin terms of plaque size and burst size (28, 29, 39). Studiesof primary explant cultures of human and murine corneal buttons,however, showed that late in infection ( $≥$ 28 to 72 h), growthdifferences between a vhs-null virus and KOS became apparent(37). No differences, however, were apparent at high MOIs. Inthis study we compared the growth of the ${Delta}$ vhs₁ mutant with thatof KOS in primary MEF cultures infected at a low MOI that weremock treated or pretreated with recombinant alpha IFN (rIFN- ${alpha}$ ).As previously reported, the vhs-null virus showed about 1-loglower growth than the wild-type KOS (Fig. 1A). When cells werepretreated overnight with rIFN- ${alpha}$ , significant differences betweenthe growth of KOS and the ${Delta}$ vhs₁ mutant were apparent at 24 h(>10-fold, P < 0.01), with the difference continuing toincrease up to 48 h (80 to 100-fold, P < 0.01) (Fig. 1B).These data were consistent with previous data (5) and with thehypothesis that the vhs function is important for maintainingthe susceptibility of IFN-primed cells to HSV infection.

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FIG. 1. In vitro replication in MEFs. Confluent primary MEF monolayers were infected with KOS or the ${Delta}$ vhs₁ mutant at an MOI of 0.01. (A) Multiple-step growth curve analysis of untreated cells. (B) Multiple-step growth curve analysis of cells pretreated overnight with 100 U/ml of rIFN- ${alpha}$ . At the indicated times postinfection, cells and supernatants were harvested and viral titers were measured on Vero cells. Results shown are the mean titers of the results for three independent experiments.

${Delta}$ vhs₁ virus-infected MEFs produce increased IFN-β and ISG RNA. IFN-β transcript expression and IFN protein synthesis andsecretion are induced in response to virus infection and recognitionof pathogen-associated molecular patterns (PAMPs). In turn,IFN-stimulated genes (ISGs) are up-regulated and activated.We therefore assessed whether infection of primary culturedcells with the ${Delta}$ vhs₁ mutant resulted in an increase in IFN orISG synthesis relative to KOS-infected cells. Analysis of IFN-βRNA by real-time RT-PCR revealed that cells infected with bothKOS and the ${Delta}$ vhs₁ mutant expressed increased amounts of IFN-βRNA relative to mock-infected cells, although the increase ininduction for the ${Delta}$ vhs₁ mutant-infected cells relative to themock-treated cells was higher than that for KOS-infected cells(Fig. 2A). When comparing the ratios of IFN-β expression( ${Delta}$ vhs₁ to KOS), ${Delta}$ vhs₁ mutant-infected cells expressed 8- to 13-foldmore IFN-β RNA than KOS-infected cells from 6 to 12 h postinfection(hpi) (Fig. 2B). To assess the downstream effect of this induction,we subsequently examined the expression of the IFIT1 (interferon-inducedprotein with tetratricopeptide repeats 1, also known as ISG56)gene, which is rapidly induced by type 1 IFN (33, 41). Real-timeRT-PCR revealed that as for IFN-β expression, IFIT expressionwas induced by both the ${Delta}$ vhs₁ mutant and KOS relative to mock-infectedcells, but the induction was higher for the ${Delta}$ vhs₁ mutant (Fig.2C). When the ratios of IFIT1 expression ( ${Delta}$ vhs₁ to KOS) are compared,the ${Delta}$ vhs₁ virus-infected cells express 5- to 10-fold more IFIT1RNA than KOS-infected cells from 6 to 12 hpi (Fig. 2D).

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FIG. 2. Real-time RT-PCR analysis of IFN-β and IFIT1 mRNA expression. Confluent MEF monolayers were mock treated or infected with KOS or the ${Delta}$ vhs₁ mutant at an MOI of 5. At the indicated times postinfection, RNA was harvested and used for real-time RT-PCR. (A) Change in expression for IFN-β RNA relative to expression in mock-treated cells. (B) IFN-β RNA expression as a ratio of the ${Delta}$ vhs₁ mutant to KOS at each time point indicated. (C) Change in expression for IFIT1 RNA relative to mock-treated cells. (D) IFIT1 RNA expression as a ratio of the ${Delta}$ vhs₁ mutant to KOS at each time point indicated. All data shown are the mean changes from the results for three independent experiments.

Having shown increases in the levels of IFN-β and IFIT1RNA expressed by ${Delta}$ vhs₁ mutant-infected cells compared to thoseexpressed by KOS, we next wanted to assess the levels of IFN-βprotein produced in these infected cells. We therefore measuredIFN-β secreted into the culture medium over the first 12h of infection by use of an IFN-β ELISA (Fig. 3). We foundthat while the levels of IFN-β secreted by KOS-infectedcells increased from 0 to 6 hpi, they subsequently leveled off.Cells infected with the ${Delta}$ vhs₁ mutant also showed an increasein IFN-β synthesis 0 to 6 hpi, but in contrast to KOS-infectedcells, the secretion of IFN-β did not level off but rathercontinued to increase up to 12 h. In general, the data for theIFN-β protein expression mirrored that for its RNA expression.

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FIG. 3. IFN-β secretion by infected cells. Confluent MEF monolayers were mock treated or infected with KOS or the ${Delta}$ vhs₁ mutant at an MOI of 5. At the indicated times postinfection, the supernatants were harvested and IFN-β secretion was measured by ELISA. Shown are the results from three independent experiments.

${Delta}$ vhs₁ virus infection in MEFs produces functional IFN. The real-time RT-PCR and ELISA results described above indicatedhigher IFN-β production from MEFs infected with the ${Delta}$ vhs₁mutant than from MEFs infected with KOS. To test the physiologicalrelevance of the observed difference and whether the levelsof secreted IFN were antiviral, culture medium was collectedfrom MEFs treated with mock lysate or infected with KOS or the ${Delta}$ vhs₁ mutant and used to pretreat secondary wild-type and IFN- ${alpha}$ β ${gamma}$ R^–/–MEF cultures. These MEFs were then challenged with VSV, KOS,or the ${Delta}$ vhs₁ mutant and the titers of the resultant viruses weredetermined. Pretreatment with medium from KOS-infected cellsreduced the VSV titer on MEFs an average of 100-fold, and mediumfrom ${Delta}$ vhs₁ mutant-infected cells reduced the VSV titer 400-fold.Changes are relative to titers on IFN- ${alpha}$ β ${gamma}$ R^–/–MEFs and are from two experiments. It should also be noted thatthe VSV titers on IFN- ${alpha}$ β ${gamma}$ R^–/– MEFs were comparablefor KOS-infected supernatant- and ${Delta}$ vhs₁ mutant-infected supernatant-treatedgroups.

Pretreatment with medium from KOS-infected cells reduced theKOS titer on MEFs an average of 6-fold and reduced ${Delta}$ vhs₁ mutanttiters an average of 50-fold, reflecting the higher sensitivityof the ${Delta}$ vhs₁ mutant to IFN. Pretreatment with medium from ${Delta}$ vhs₁mutant-infected cells reduced the KOS titer on MEFs an averageof 10-fold and reduced ${Delta}$ vhs₁ mutant titers an average of 100-fold.As described above for the VSV challenge assays, changes arerelative to titers on IFN- ${alpha}$ β ${gamma}$ R^–/– MEFs and arefrom two experiments, and HSV titers on IFN- ${alpha}$ β ${gamma}$ R^–/–MEFs were comparable for KOS-infected supernatant-treated and ${Delta}$ vhs₁ mutant-infected supernatant-treated groups. Taken together,these data show that the released IFN is antiviral and thatcells infected with an HSV-1 mutant that lacks vhs secrete moreantiviral IFN than a KOS-infected cell.

HSV-1 ${Delta}$ vhs₁ virus-infected MEFs have increased levels of viral transcripts. The open reading frames of HSV, especially those of the lategene class, are located on opposing DNA strands and producelarge amount of mRNAs with regions of partial complementarity.Previous studies have identified the presence of dsRNA in HSV-infectedcells (13, 46). HSV-1 vhs degrades viral mRNAs during infection,and in its absence, viral immediate-early gene expression isprolonged, while early and late gene expression is delayed,compared to a wild-type virus (28). That said, the precise kineticsand preference of this degradation activity have not been examined.We therefore wished to compare viral RNA expression levels in ${Delta}$ vhs₁ mutant- and KOS-infected MEFs, in order to evaluate thepotential for overexpression of dsRNA in vhs-deficient virusinfections. The assessment was performed using two concomitantapproaches, gene array and real-time RT-PCR.

Viral gene array analysis. Our custom viral microarray as described in Materials and Methodswas designed to examine all known HSV genes and selected cellulargenes. The results are shown by a line graph, with each linerepresenting the ratio ( ${Delta}$ vhs₁ mutant to KOS) of gene expressionfor each viral (Fig. 4A) and cellular (Fig. 4B) gene. The linegraphs also indicate the significance of the expression ratioat 12 hpi. A y-axis value of 1 indicates no difference betweenthe KOS and ${Delta}$ vhs₁ mutant results. The data showed the same trendsover two independent experiments. At the 3-h time point, therewas expression of most classes of viral genes by both virusesbut a generalized underexpression of many genes by the ${Delta}$ vhs₁mutant relative to KOS (Fig. 4C). At the 6-, 9-, and 12-h timepoints, however, there was overexpression of most genes in allclasses for the ${Delta}$ vhs₁ mutant relative to KOS (Fig. 4C). Similarly,there was overexpression of cellular genes at these time pointsin ${Delta}$ vhs₁ mutant-infected cells. Together these data suggest thatthere is global degradation of viral and cellular RNAs mediatedby vhs that occurs between 3 and 12 hpi. Further details ofthese data are available in Table S1 in the supplemental material.

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FIG. 4. Microarray analysis of viral and cellular mRNA expression. (A) Line graph depicting the expression of detectable signal from 231 oligonucleotide spots designed to detect all known viral genes. The approximately 90 viral genes were represented in a redundant fashion by at least three oligonucleotides. (B) Line graph depicting the expression of the 92 detectable cellular genes. The time in hours is represented on the x axis, and the change in expression is represented on the y axis (log scale). Each line represents the mean of the ratio of intensity signals for a particular transcript from ${Delta}$ vhs₁ mutant-infected MEFs to that from KOS-infected MEFs. The lines are colored to indicate the significance of the under- or overexpression at 12 hpi, by standard error of measurement, ranging from red (+3 standard deviations) to yellow (0 standard deviations) to blue (–3 standard deviations). (C) Within each viral gene kinetic class and cellular genes, the ratio ( ${Delta}$ vhs₁ mutant to KOS) of the gene expression levels was averaged and converted to a log₂ ratio to visualize trends in gene class expression over time.

Real-time RT-PCR analysis. Validation of the viral gene array data was performed via real-timeRT-PCR for the mRNAs encoding ICP4, -22, and -27 (immediate-earlygenes); ICP6, ICP8, and thymidine kinase (early genes); andvhs, ICP34.5, US11, and gC (leaky-late and true-late genes).The accumulation of these transcripts in KOS- and ${Delta}$ vhs₁ mutant-infectedMEFs over time is shown relative to their levels at 1 hpi. Asexpected, viral transcripts in KOS-infected cells accumulatedover the 12-h time course, broadly according to their kineticclasses (Fig. 5A). Similarly, mRNA levels in ${Delta}$ vhs₁ mutant-infectedcells increased over time, although to a greater extent thanseen in KOS-infected MEFs (Fig. 5B). For both KOS and the ${Delta}$ vhs₁mutant, the greatest changes were seen in early and late genes.When the levels of viral mRNA were compared as a ratio ( ${Delta}$ vhs₁mutant to KOS), there was a notable underexpression of almostall genes analyzed at 3 hpi by the ${Delta}$ vhs₁ mutant (Fig. 5C), consistentwith the gene arrays (Fig. 4). In contrast, from 6 to 12 hpi,the ${Delta}$ vhs₁ mutant-infected cells exhibited a marked accumulationof all viral genes examined, compared to KOS-infected cells.To summarize, when absolute values of viral mRNA were assessed,both viruses accumulated viral transcripts throughout infection,but ${Delta}$ vhs₁ mutant-infected cells accumulated them at a greaterrate than did KOS-infected cells. When the expression levelsof actin mRNA for the two viruses were compared (Fig. 5D), weobserved evidence of strong RNA degradation mediated by KOS,but not by the ${Delta}$ vhs₁ mutant, between 6 and 12 hpi. Taken together,these data are consistent with the gene array studies and alsowith the hypothesis that a vhs-deficient virus can express moredsRNA than a wild-type virus.

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FIG. 5. Real-time RT-PCR analysis of selected viral and cellular genes. Confluent MEF monolayers were mock treated or infected with KOS or the ${Delta}$ vhs₁ mutant at an MOI of 5, and RNA was extracted for RT-PCR analysis. Viral genes are color coded by kinetic class, and genes are shown in the graphs in the same order as they appear in the key at the bottom of the figure. (A) Change in mRNA expression for selected viral genes relative to that for KOS at the 1-h time point. (B) Change in mRNA expression for selected viral genes relative to that for the ${Delta}$ vhs₁ mutant at the 1-h time point. (C) Ratio of gene expression for each time point for selected genes reported as a log₂ ratio of ${Delta}$ vhs₁ mutant to KOS. (D) Actin mRNA expression reported as a log₂ ratio of ${Delta}$ vhs₁ mutant to KOS. Results shown are the means for two independent experiments.

Phosphorylation of eIF2 ${alpha}$ is increased in HSV ${Delta}$ vhs mutant-infected MEFs. Activation of the PKR pathway heralds the presence of dsRNA,stimulates IFN expression, and is a pivotal marker of the cellularantiviral state. Once activated by dsRNA binding, PKR phosphorylatesthe translation initiation factor eIF2 ${alpha}$ on serine 51. In thisstate, eIF2 ${alpha}$ is unable to recycle GDP for GTP and translationinitiation is shut down. The HSV-1 gene products ICP34.5 andUS11 are known to directly counter this translational shutoffresponse by inactivating or reversing the effects of PKR, therebyallowing viral replication to continue. No other HSV proteinshave been shown to have activity in this regard, but given therole for vhs in countering IFN production (Fig. 3) (5) and controllingviral mRNA levels (Fig. 4 and 5), we hypothesized that the presenceof vhs activity might impact PKR-dependent eIF2 ${alpha}$ phosphorylation.Because of the demonstrated ability of HSV-2 vhs to stronglycounter IFN production and responses in vitro and in vivo (5),we also wanted to compare the impacts of HSV-2 vhs on the phosphorylationof eIF2 ${alpha}$ . We therefore assayed for phosphorylated eIF2 ${alpha}$ by Westernblotting lysates of MEFs that were mock treated or infectedwith KOS, ${Delta}$ vhs₁ mutant, HSV-2 333, or ${Delta}$ vhs₂ mutant (333d41) viruses(Fig. 6A and B). At 6 and 12 hpi, increases were seen in phosphorylatedeIF2 ${alpha}$ in ${Delta}$ vhs₁ and ${Delta}$ vhs₂ mutant-infected cells compared with therespective wild-type-infected MEFs. The increases, however,in eIF2 ${alpha}$ phosphorylation for the ${Delta}$ vhs₂ mutant-infected cells (eightfoldat 6 hpi) relative to those infected with HSV-2 333 were greaterthan those seen for the ${Delta}$ vhs₁ mutant (twofold at 6 hpi) relativeto KOS.

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FIG. 6. Western blot analysis of eIF2 ${alpha}$ phosphorylation in infected cells. Confluent MEF monolayers were mock treated or infected at an MOI of 5. At the indicated times postinfection, cell lysates were prepared and eIF2 ${alpha}$ phosphorylation was assessed by Western blotting. (A) Total and phosphorylated eIF2 ${alpha}$ assessed over time following infection with KOS or the ${Delta}$ vhs₁ mutant. (B) Total and phosphorylated eIF2 ${alpha}$ assessed over time following infection with HSV-2 333 or the ${Delta}$ vhs₂ mutant.

Partial restoration of ${Delta}$ vhs₁ mutant growth in IFN- ${alpha}$ β ${gamma}$ R^–/– MEFs but not PKR^–/– MEFs. In order to further determine the role of IFNs and PKR in controlling ${Delta}$ vhs₁ virus replication, we performed a multiple-step in vitrogrowth analysis of primary MEFs derived from either IFN- ${alpha}$ β ${gamma}$ R^–/–or PKR^–/– mice (Fig. 7). Compared to the resultwith control MEFs, in IFN signaling-deficient IFN- ${alpha}$ β ${gamma}$ R^–/–MEFs, we found that the growth of the ${Delta}$ vhs₁ virus was only partiallyrestored and still slightly and reproducibly attenuated relativeto that of KOS (Fig. 1). As expected, pretreatment with rIFN- ${alpha}$ did not attenuate ${Delta}$ vhs₁ virus replication (data not shown), incontrast to what was observed in the control MEFs (Fig. 1B).These results indicated that IFN-dependent signaling was mostly,but not wholly, responsible for the attenuation of ${Delta}$ vhs₁ virusobserved in control MEFs. In PKR^–/– MEFs, ${Delta}$ vhs₁ virusreplication was reduced 10-fold compared to replication of KOS(Fig. 7B), comparable to the attenuation observed for controlMEFs (Fig. 1). Furthermore, the phosphorylation of eIF2 ${alpha}$ inducedby ${Delta}$ vhs₁ virus was not significantly increased over levels formock infection in PKR^–/– MEFs (data not shown).These data show that despite the induction of increased eIF2 ${alpha}$ phosphorylation, the loss of PKR does not restore the IFN-dependentgrowth defect to vhs-deficient HSV-1.

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FIG. 7. In vitro replication in IFN- ${alpha}$ β ${gamma}$ R^–/– and PKR^–/– MEFs. Confluent primary MEF monolayers were infected with KOS (solid lines) or the ${Delta}$ vhs₁ (dashed lines) mutant at an MOI of 0.01. (A) Multiple-step growth curve analysis of IFN- ${alpha}$ β ${gamma}$ R^–/– MEFs. (B) Multiple-step growth curve analysis of PKR^–/– MEFs. At the indicated times postinfection, cells and supernatants were harvested and viral titers were measured on Vero cells.

DISCUSSION

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DISCUSSION

The vhs function of HSV has been extensively studied in vitroand in vivo, and a clear dichotomy has emerged. In most continuouscell culture systems, lack of vhs activity has little impacton viral growth at any MOI. In primary cells, however, lossof vhs has a far greater impact, especially at a low MOI, andespecially for HSV-2 (5). For HSV-2, vhs is a major generalmediator of viral resistance to IFN- ${alpha}$ β and to the antiviraleffects of PKR, and the virulence of a vhs-deficient HSV-2 islargely restored in IFN- ${alpha}$ βR^–/– mice (5, 25).Perhaps the most different result between HSV-1 and HSV-2 isthat while vhs deletion from both HSV types induces higher levelsof phosphorylated eIF2 ${alpha}$ , only HSV-2 ${Delta}$ vhs virus replication isrestored in the absence of PKRs. The IFN-dependent attenuationof an HSV-1 ${Delta}$ vhs mutant is therefore independent of PKR and likelyindependent of eIF2 ${alpha}$ phosphorylation. This may explain, at leastin part, the greater impact of vhs on the IFN resistance ofHSV-2 than on that of HSV-1. While ICP0, ICP34.5, and US11 allplay roles in modulating aspects of the IFN response to HSV-1,viruses lacking vhs are not hypersensitive to IFN in culture,and virulence is not restored to vhs-deficient viruses in IFN- ${alpha}$ β ${gamma}$ R^–/–mice (19, 23). While vhs of HSV-2 is more critical than itsHSV-1 counterpart for resistance to the IFN response, paradoxically,the in vivo attenuation of HSV-1 mutants lacking vhs is fargreater than that of an equivalent vhs-deficient HSV-2 strain(35, 38, 39). The rapidity with which HSV-1 vhs mutants areso attenuated, coupled with the inability of mouse strains lackingadaptive immunity to restore any growth or virulence to vhs-negativeHSV-1 strains (25), nevertheless underscores the role for vhsin countering the IFN response.

In this study we have shown that IFN significantly reduces thereplication of the ${Delta}$ vhs₁ mutant but that the replication of KOSis almost completely unaffected. Moreover, in the absence ofexogenous rIFN- ${alpha}$ , the growth defect in primary cells of a viruslacking vhs is more apparent following a secondary round ofreplication. Together these data are consistent with the ideathat vhs is important for maintaining cellular permissivenessto HSV infection following priming with IFN. These data formouse cells are consistent with those seen previously for humancells, showing that this is not a mouse-specific phenomenon(37). The results of other studies have differed with this workand with each other regarding the IFN sensitivity of viruseslacking vhs (5, 23, 37, 40). It seems very likely that thesedifferences arise from the use of differing cell lines, andit is notable that results obtained with primary cells of eitherhuman or mouse origin are consistent with each other.

We show herein that vhs significantly affects IFN-β expression.The pattern of IFN-β RNA and protein induction by KOS wasconsistent with previous studies, with a rapid induction ofan antiviral response followed by its shutoff (22). In contrast, ${Delta}$ vhs₁ virus infection caused an induction that was followed byan inexorable increase in IFN-β expression over time. Thistoo is consistent with previous studies of HSV-1 and HSV-2 (5,40). Moreover, the VSV and HSV challenge assays showed thatthe IFN secreted into supernatants of infected cells is activelyantiviral, despite the fact that the IFN produced is significantlydiluted by the culture media and is likely unstable in culture.Some contrasts between these studies and others, however, areseen when the expression of the downstream IFIT1 (ISG56) geneis examined. IFIT1 inhibits the initiation of translation bybinding to various subunits of the translation initiation factoreIF3 and has been suggested to be a major antiviral mechanismfor blocking viral replication (10). Our current work showsstrong induction of IFIT1 by KOS but significantly strongerinduction in the absence of vhs. Previous work has shown IFIT1to be undetectable following infection with KOS or with a vhs-nullmutant (20) and that IFIT1 was detectable only when ICP0, amajor factor in the shutoff of IFN expression and of ISGs bya proteasome-dependent mechanism, was mutated (6). Consistentwith our work, however, was that IFIT1 induction by an ICP0gene-null virus was further enhanced following deletion of vhs.Yet both vhs-null viruses in both studies were ICP0 competent,used the KOS strain, employed similar MOIs, and were similarotherwise (37). It seems unlikely therefore that different virusesor infection methods can account for this discrepancy. It ispossible that technical variances such as primer optimization,assay sensitivity, and quantification methods for RT-PCR contributeto the difference in IFIT1 results. In addition, the use ofprimary cells (MEFs) in this study, compared with continuouscells (human embryonic lung fibroblasts) in the previous studies,is likely to have an impact, especially in light of recent datashowing that expression of IFIT1 in vivo is tissue specific(42). Ongoing studies in our laboratory investigating the expressionprofiles and phenotypes of vhs-deficient viruses in vivo arelikely to further define the impact of vhs on IFN and ISG expression.

A key issue in this study is the mechanism by which vhs dampensthe innate immune response to HSV. This mechanism has been difficultto define in part because the pathways by which HSV infectionis recognized that lead to the antiviral state are also poorlyunderstood. Many potential mechanisms exist, but it is knownthat efficient recognition of viral dsRNA and signal transductionare required to induce host gene expression. Data in this studysuggest that vhs may be operating on both of these requirements.First, vhs may serve to minimize the accumulation of viral RNAthroughout infection to levels optimal for efficient viral replicationbut below the levels detected by RNA sensing molecules suchas RIG-I and below those that trigger dsRNA-inducible antiviralpathways such as PKR and RNase L. Second, vhs may serve to reducethe accumulation of RNA expressed by pivotal genes involvedin the antiviral state, such as IFN genes and ISGs, as evidencedby the altered expression of IFN-β and IFIT1 in this study.vhs also likely enhances, quantitatively or temporally, theexpression of the many viral genes involved in the counteringof the IFN response. This may occur in a selective or a nonselectivefashion, but either way represents a powerful general mechanismby which HSV can serve to dampen many host response pathwayssimultaneously. Together, all these activities of vhs serveas a versatile and powerful counter to the various arms of thehost's innate immune response. Current in vivo functional genomicanalysis ongoing in our laboratory using vhs mutants will likelyelucidate this possibility further.

ACKNOWLEDGMENTS

This work was supported by NIH grants to David A. Leib (EY10707),to Lynda A. Morrison (AI57573), and to the Department of Ophthalmologyand Visual Sciences (P30EY02687). Support for the Departmentfrom Research to Prevent Blindness and a Lew Wasserman Scholarshipto David A. Leib are gratefully acknowledged.

We acknowledge David Wang for help with selection of oligonucleotidesand Michael Heinz and Eric Tycksen of the Washington UniversityMicroarray Core for helpful design, construction, and implementationof microarray technology.

FOOTNOTES

* Corresponding author. Mailing address: Washington University School of Medicine, Department of Ophthalmology and Visual Sciences, 660 South Euclid Ave., Box 8096, St. Louis, MO 63110. Phone: (314) 362-2689. Fax: (314) 362-3638. E-mail: leib{at}vision.wustl.edu

Published ahead of print on 26 March 2008.

Supplemental material for this article may be found at http://jvi.asm.org/.

REFERENCES

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