Herpes Simplex Virus Type 2 Virion Host Shutoff Protein Regulates Alpha/Beta Interferon but Not Adaptive Immune Responses during Primary Infection In Vivo

The herpes simplex virus (HSV) virion host shutoff (vhs) protein,the product of the UL41 (vhs) gene, is an important determinantof HSV virulence. vhs has been implicated in HSV interferencewith host antiviral immune responses, down-regulating expressionof major histocompatibility complex molecules to help HSV evadehost adaptive immunity. The severe attenuation of vhs-deficientviruses in vivo could reflect their inability to escape immunedetection. To test this hypothesis, BALB/c or congenic SCIDmice were infected intravaginally (i.vag.) with the HSV type2 (HSV-2) vhs null mutant 333d41 or the vhs rescue virus 333d41^R.vhs-deficient virus remained severely attenuated in SCID micecompared with rescue virus, indicating that vhs regulation ofadaptive immune responses does not influence HSV pathogenesisduring acute infection. Innate antiviral effectors remain intactin SCID mice; prominent among these is alpha/beta interferon(IFN- ${alpha}$ /ß). The attenuation of HSV-2 vhs mutants couldreflect their failure to suppress IFN- ${alpha}$ /ß-mediatedantiviral activity. To test this hypothesis, 129 and congenicIFN- ${alpha}$ /ß receptor-deficient (IFN- ${alpha}$ /ßR^-/-) micewere infected i.vag. with wild-type virus, vhs null mutants333-vhsB or 333d41, or the vhs rescue virus 333d41^R. Whereasvhs-deficient viruses showed greatly reduced replication inthe genital mucosa of 129 mice compared with wild-type or vhsrescue viruses, they were restored to nearly wild-type levelsof replication in IFN- ${alpha}$ /ßR^-/- mice over the first 2days postinfection. Only wild-type and vhs rescue viruses causedsevere genital disease and hind limb paralysis in 129 mice,but infection of IFN- ${alpha}$ /ßR^-/- mice restored the virulenceof vhs-deficient viruses. vhs-deficient viruses replicated asvigorously as wild-type and rescue viruses in the nervous systemsof IFN- ${alpha}$ /ßR^-/- mice. Restoration was specific for thevhs mutation, because thymidine kinase-deficient HSV-2 did notregain virulence or the capacity to replicate in the nervoussystems of IFN- ${alpha}$ /ßR^-/- mice. Furthermore, the defectin the IFN- ${alpha}$ /ß response was required for restorationof vhs-deficient virus replication and virulence, but the IFN- ${alpha}$ /ß-stimulatedprotein kinase R pathway was not involved. Finally, vhs of HSV-2has a unique capacity to interfere with the IFN- ${alpha}$ /ßresponse in vivo, because an HSV-1 vhs null mutant did not recoverreplication and virulence after i.vag. inoculation into IFN- ${alpha}$ /ßR^-/-mice. These results indicate that vhs plays an important roleearly in HSV-2 pathogenesis in vivo by interfering with theIFN- ${alpha}$ /ß-mediated antiviral response.

INTRODUCTION

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Introduction

Herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) are ubiquitoushuman pathogens that cause a variety of serious diseases. PrimaryHSV infections typically initiate at mucosal surfaces, wherelytic replication in epithelial cells generates mucosal lesions.Peripheral replication amplifies the virus load and increasesuptake of virus into sensory nerve termini. Establishment oflatent infections in sensory neurons secures shelter for thevirus throughout the life of the host. Periodic reactivationsresult in disease recurrence and provide an opportunity fortransmission to new hosts.

The virion host shutoff (vhs) protein of HSV plays a significantrole in promoting pathogenesis at the cell and organismal levels.In the infected cell, vhs possesses both endo- and exonucleaseactivity (8, 9, 57) and mediates the rapid shutoff of proteinsynthesis via degradation of both cellular and viral mRNAs (22,23, 39, 43). As a component of the virion tegument, vhs is releaseddirectly into the cytosol and can immediately exert its effectson the newly infected cell. Cellular mRNAs are almost completelydegraded within 6 h of infection with HSV-1, and within just2 h by HSV-2 (19). The activity of HSV-2 vhs is also approximately40-fold stronger than that of HSV-1 (10, 11). In vivo, vhs nullmutants of HSV-1 and HSV-2 are profoundly attenuated, implicatingvhs as a virulence factor that helps HSV establish robust infection.HSV-1 lacking vhs activity replicates to 1,000-fold-lower titersin the cornea, trigeminal ganglia, and brain of mice than wild-typevirus and has impaired capacity to enter the central nervoussystem, establish latency, and undergo reactivation (48-50).vhs-deficient HSV-2 strains (333-vhsB or 333d41) also replicatemuch less efficiently than wild-type virus in the genital mucosaand nervous tissue of mice and cause less disease (47). vhshas been implicated in down-regulating major histocompatibilitycomplex (MHC) class I (18, 54) and class II (55) molecules.This activity has functional implications because it is associatedwith reduced cytotoxic T-lymphocyte recognition of HSV-infectedcells (54). Thus, vhs-deficient viruses may be unable to evadecritical adaptive immune responses during acute infection, leadingto their attenuation.

Alternatively, the attenuation of HSV-1 and HSV-2 vhs null mutantswithin 24 to 48 h postinfection (47-50) suggests a pivotal rolefor mediators of innate immunity in controlling acute infection.A key mediator of innate antiviral immunity to virus infectionis the alpha/beta interferon (IFN- ${alpha}$ /ß) response. Themany subtypes of IFN- ${alpha}$ and IFN-ß are released frominfected cells and bind to a single IFN- ${alpha}$ /ß receptor(IFN- ${alpha}$ /ßR). Receptor-mediated signal transduction stimulatesexpression of numerous IFN-stimulated genes (44, 53) whose productscan interfere with viral replication by disrupting macromolecularsynthesis. HSV-1 in culture is relatively insensitive to theeffects of IFN- ${alpha}$ /ß, being reduced 2- to 5-fold forreplication, compared to a more than 10,000-fold reduction forvesicular stomatitis virus (16, 32). In addition, HSV-1 virulenceis not substantially increased in mice lacking the IFN- ${alpha}$ /ßR(IFN- ${alpha}$ /ßR^-/-) (25), suggesting that HSV has a meansto effectively counter the IFN- ${alpha}$ /ß response. Determinantsof HSV-1 IFN resistance have been mapped to ICP0, U_s11, and ${gamma}$ 34.5 (3, 4, 16, 17, 32, 33). Each of these proteins has beenconfirmed as a contributor to HSV-1 virulence (1, 2, 5; S. Wardand D. Leib, personal communication). Evidence for HSV-1 vhsas an additional inhibitor of the IFN- ${alpha}$ /ß responseis controversial (32, 52) and is not borne out in vivo (25).Replication of an HSV-1 vhs null mutant is slightly elevatedand prolonged in the corneas of IFN- ${alpha}$ /ßR^-/- mice comparedto wild-type mice. However, the titer of vhs-deficient HSV-1is 1,000-fold lower than that of wild-type virus at all timesin IFN- ${alpha}$ /ßR^-/- mice (25), indicating that HSV-1 vhsdoes not play a significant role in counteracting IFN- ${alpha}$ /ßRsignaling in vivo.

Mediators of IFN resistance in HSV-2 have not been well characterized,but several observations suggest that vhs may contribute inthis case. Genetic studies have mapped IFN resistance to a 7.4-kbregion of the HSV-2 genome that contains the vhs gene and portionsof two flanking genes (35). Another genetic locus influencingHSV-2 resistance to IFN- ${alpha}$ /ß has been mapped 60 kb distantfrom the vhs locus (51). This region contains the UL13 viralkinase, which influences either vhs synthesis or activity (36,41). In addition to genetic evidence, HSV-2 is even more resistantto IFN- ${alpha}$ /ß in vitro than HSV-1 (24), and HSV-2 vhsactivity is more potent than that of HSV-1 vhs (10-12, 19).These observations led us to investigate whether HSV-2 vhs interfereswith the host IFN- ${alpha}$ /ß response. Here we use a mousemodel of genital infection with HSV-2 to examine the role ofvhs in countering host adaptive immune and innate responsesto primary infection.

MATERIALS AND METHODS

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Materials and Methods

Cells and viruses. Vero cells were maintained in Dulbecco's modified Eagle's mediumsupplemented with 3% newborn and 3% fetal calf serum. SB5, aplaque-purified stock of HSV-2 strain 333, was obtained fromthe American Type Culture Collection (VR-2546). HSV-1 strainUL41NHB (49) and HSV-2 strain 333d41 (47) were obtained fromDavid Leib. The vhs open reading frame (ORF) in UL41NHB is disruptedby insertion of the Escherichia coli lacZ gene under the controlof the human cytomegalovirus immediate-early 1 (IE1) promoterat a ScaI site. 333d41 has a targeted deletion within the UL41gene between two XcmI sites, resulting in a 939-bp excisionand complete lack of vhs activity. 333d41^R was constructed bycotransfecting full-length 333d41 DNA with p41SB5-B, a plasmidcontaining a BglII genomic fragment of HSV-2 333 that includesthe UL41 gene (47). Briefly, 1 µg of each DNA mixed withLipofectamine (Invitrogen) was used to transfect Vero cells.Viral progeny were screened by PCR for the deletion within vhsby using the primers 5'-GCG TCC AAC CGA TAA ATC AAG C-3'(forward)and 5'-TTA CCC AAA AGT CCC TGT TCC C-3' (reverse), with 30 cyclesof 95°C for 30 s, 66.1°C for 30 s, and 72°C for60 s. PCR products were analyzed on agarose gels stained withethidium bromide. Isolates were plaque purified three times,and identity of the rescue virus was confirmed by Southern blotanalysis using a BamHI digest (data not shown). The RNase activityof 333d41^R was confirmed by Northern blot analysis in an mRNAdegradation assay described elsewhere (47) and was identicalto that of SB5 (data not shown). HSV-2 strains 333-vhsB and ${Delta}$ TK^- (27) were the generous gifts of Jim Smiley. 333-vhsB containsa cassette consisting of the ICP6 promoter driving the lacZgene (40). The cassette was inserted at codon 30 of the vhsORF, disrupting the UL41 gene. ${Delta}$ TK^- contains a 180-bp deletionin the thymidine kinase (tk) ORF of strain 333 that inhibitstk activity. Cell lysate stocks of each strain were preparedas previously described (31). Titers of virus stocks were determinedby simultaneous titration on Vero cell monolayers to ensureequivalent doses of virus in the inocula.

Animals and infections. BALB/c mice were purchased from the National Cancer Institute(Frederick, Md.). CB-17.SCID mice were purchased from Taconic(Germantown, N.Y.). IFN- ${alpha}$ /ßR^-/- (34) and congenic 129[129Sv(ev)] mice were generously provided by Michel Aguet andwere obtained from Skip Virgin. Protein kinase R-deficient (PKR^-/-)mice (56) on the 129 background were generously provided byBob Silverman and were obtained from Skip Virgin. 129, IFN- ${alpha}$ /ßR^-/-,and PKR^-/- mice were bred and housed in the Department of ComparativeMedicine, Saint Louis University School of Medicine. All micewere maintained in accordance with institutional and PublicHealth Service guidelines and were used at 6 weeks of age.

Vaginal swab and neural tissue titers. Vaginal vaults were swabbed twice at 10 h and days 1 through4 postinfection using type 1 calcium alginate swabs (Puritan).The swabs were placed together into vials containing 1 ml ofphosphate-buffered saline and stored at -80°C. At 6 dayspostinfection, mice were sacrificed and the spinal cord, brainstem,and brain were dissected, placed in microcentrifuge tubes containingphosphate-buffered saline and 1-mm glass beads, and stored at-80°C. Thawed tissues were disrupted using a Mini BeadBeater8 (Biospec Products). Viral titers in swab samples and tissueswere determined by standard plaque assay (21).

Disease scores. Severity of genital and neurologic disease was assessed on days2 through 6 postinfection using the following scale: 0, no signs;1, mild erythema and edema of the external genitalia; 2, moderateerythema and edema of the external genitalia; 3, severe erythemaand edema of the external genitalia with presence of lesions;4, bilateral hind limb paralysis; 5, death.

Statistics. Significance of difference in viral titer between groups onindividual days was determined by the t test. The nonparametricKruskall-Wallis test was used to determine significance of differencein disease scores between groups on individual days.

RESULTS

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Results

vhs-deficient mutants of HSV-1 and HSV-2 replicate poorly comparedto wild-type virus in the mouse cornea and genital mucosa, respectively.They also cause minimal disease and have reduced capacity toreplicate in the nervous system (47, 49). We reasoned that ifadaptive immune responses are responsible for the attenuationof vhs-deficient viruses, then replication and virulence shouldbe restored in mice lacking adaptive immune functions. To testthis hypothesis, BALB/c and congenic CB-17.SCID mice were infectedi.vag. with 2 x 10⁶ PFU of the vhs-deficient mutant 333d41 (27)or the vhs rescue virus, 333d41^R. Titers of vhs rescue viruscollected from daily vaginal swabs of BALB/c and SCID mice weresignificantly higher than those of 333d41 on days 2 to 4 postinfection(Fig. 1A).

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FIG.1. Pathogenesis of vhs-deficient HSV-2 is not restored in SCID mice. Groups of four BALB/c and congenic CB.17-SCID mice were infected i.vag. with 2 x 10⁶ PFU of vhs-deficient HSV-2, strain 333d41, or the vhs rescue virus 333d41^R. (A) Replication in the genital mucosa of BALB/c and SCID mice. The titer of virus in vaginal swab samples was determined by standard plaque assay. Data points represent the geometric mean ± standard error of the mean (SEM). (B) Severity of genital and neurological disease in BALB/c and SCID mice. Mice were scored for signs of disease as described in Materials and Methods. Values represent the arithmetic mean ± SEM. (C) Replication in the nervous systems of BALB/c and SCID mice. Mice were sacrificed 6 d postinfection. Brain, brainstem, and spinal cord tissues were disrupted by bead beating. Viral titers were determined by standard plaque assay. The dashed line indicates the limit of detection. Bars represent the geometric mean ± SEM.

Correspondingly, BALB/c mice infected with vhs rescue virusrapidly developed severe genital inflammation, and lesions werepresent by 6 days postinfection (Fig. 1B). Inflammation provokedby 333d41 infection was comparatively moderate. The same dichotomywas observed in SCID mice, where rescue virus caused pronouncedinflammation but vhs-deficient virus did not (Fig. 1B). Interestingly,genital inflammation was more severe in BALB/c mice than SCIDmice, suggesting an immunopathologic component to the inflammatoryresponse.

Virus titer in the nervous system was determined on day 6 postinfection.vhs rescue virus replicated to high titers in the spinal cordof BALB/c mice and, as anticipated, levels of 333d41 were muchlower (Fig. 1C). Brainstem and brain tissue of BALB/c mice containedless rescue virus, but 333d41 could not be detected. In SCIDmice, rescue virus replicated to high titer in the central nervoussystem, but titers of 333d41 remained low (Fig. 1C). A secondvhs-deficient virus, 333-vhsB, also remained attenuated in SCIDmice (data not shown). These results indicate that adaptiveimmune responses were not responsible for the reduced capacityof vhs-deficient virus to replicate or cause disease and, thus,that vhs does not play a significant role in counteracting theadaptive immune response during acute infection.

SCID mice lack T and B lymphocytes, but they possess normalinnate immune mechanisms. A key mediator of innate antiviralresponses is IFN- ${alpha}$ /ß. Although vhs of HSV-1 does notappear to influence IFN-mediated antiviral resistance in vivo(25), three observations suggested that HSV-2 may utilize vhsto counteract the IFN- ${alpha}$ /ß response. IFN sensitivityof a wild-type HSV-2 strain has been mapped to a region thatincludes the vhs locus (35), a UL13^- HSV-2 strain with reducedvhs activity permits more IFN production in vivo (46), and HSV-2vhs activity is much stronger and faster than that of HSV-1(10, 11, 19). We therefore examined the pathogenesis of vhs-deficientHSV-2 strains in mice genetically deficient in the receptorfor all IFN- ${alpha}$ /ß (IFN- ${alpha}$ /ßR^-/-).

IFN- ${alpha}$ /ßR^-/- and wild-type 129 mice were infected i.vag.with 2 x 10⁶ PFU of 333 (clone SB5), 333d41, or vhs rescue virus333d41^R. In 129 mice, 333d41 replicated approximately 100-foldless efficiently in the genital mucosa at all time points comparedto wild-type and vhs rescue viruses (Fig. 2A). In IFN- ${alpha}$ /ßR^-/-mice, replication profiles of SB5 and 333d41^R were similar tothose seen in wild-type 129 mice over the first 2 days postinfectionbut were maintained at higher levels thereafter (Fig. 2B). Strikingly,titers of vhs-deficient 333d41 were increased 20- to 50-foldat all time points in IFN- ${alpha}$ /ßR^-/- mice compared with129 mice (P < 0.0001). Levels of 333d41 replication in IFN- ${alpha}$ /ßR^-/-mice were not statistically different from levels of wild-typeand rescue viruses over the first 2 days postinfection (Fig.2B). These data indicate a significant recovery of replicationby vhs-deficient virus in the vaginal mucosa of mice lackingthe capacity to mount an IFN- ${alpha}$ /ß-mediated antiviralresponse. 333-vhsB was slightly more attenuated than 333d41in 129 mice but also showed significantly enhanced replicationin the genital mucosa of IFN- ${alpha}$ /ßR^-/- mice (data notshown).

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FIG. 2. Replication in the genital mucosa of 129 and IFN- ${alpha}$ /ßR^-/- mice. Groups of mice were infected i.vag. with 2 x 10⁶ PFU of the indicated HSV-2 strains. The titer of virus in vaginal swab samples of 129 mice (n = 10 to 12) (A) and IFN- ${alpha}$ /ßR^-/- mice (n = 6 to 8) (B) was determined by standard plaque assay; for ${Delta}$ TK^-, n = 5. Data points represent the geometric mean ± standard error of the mean. The dashed line indicates the limit of detection. *, P = 0.0178; **, P < 0.0001 (for 333d41 compared with 333d41^R).

Signs of genital disease in 129 mice infected with wild-typeand vhs rescue viruses became readily apparent by 4 days postinfectionand were severe 5 to 6 days postinfection (Fig. 3A). Most micedeveloped hind limb paralysis by day 6 postinfection. The diseasecourse in IFN- ${alpha}$ /ßR^-/- mice infected with SB5 or 333d41^Rwas similar to that in 129 mice, although accelerated by approximately1 day (Fig. 3B). Whereas infection of 129 mice with vhs-deficientvirus had resulted in mild genital inflammation (Fig. 3A), IFN- ${alpha}$ /ßR^-/-mice showed severe genital disease (P = 0.013 and 0.001 on days5 and 6, respectively) that developed with approximately thesame kinetics as disease caused by wild-type and vhs rescueviruses (Fig. 3B). Furthermore, signs of neurologic diseasewere equally apparent in IFN- ${alpha}$ /ßR^-/- mice infectedwith vhs-deficient, wild-type, or vhs rescue viruses. Thesedata indicate that the virulence of vhs-deficient virus is markedlyincreased in IFN- ${alpha}$ /ßR^-/- mice relative to that in wild-typevirus.

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FIG. 3. Severity of genital and neurological disease in 129 and IFN- ${alpha}$ /ßR^-/- mice. Mice as described in the legend for Fig. 2 were scored for signs of disease. Values for 129 (A) and IFN- ${alpha}$ /ßR^-/- (B) mice represent the arithmetic mean ± standard error of the mean. **, P < 0.001, as determined by Kruskall-Wallis nonparametric test for 333d41 compared with 333d41^R.

At 6 days postinfection, the titers of 333d41 were significantlylower than those of wild-type and vhs rescue viruses in spinalcord and brainstem and could not be detected in the brain of129 mice (Fig. 4A). In contrast, 333d41 replicated to levelsequivalent to wild-type and rescue viruses in the spinal cordand brainstem of IFN- ${alpha}$ /ßR^-/- mice (Fig. 4B; P <0.0001 compared to 129 mice for both tissues). Only the braincontained lower titers of 333d41 than the wild-type and rescueviruses. Thus, replication of vhs-deficient virus is largelyrestored in the nervous systems of IFN- ${alpha}$ /ßR^-/- mice.

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FIG. 4. Replication in the nervous systems of 129 and IFN- ${alpha}$ /ßR^-/- mice. Mice as described in the legend for Fig. 2 were sacrificed on day 6 postinfection. Brain, brainstem, and spinal cord tissues of 129 (A) and IFN- ${alpha}$ /ßR^-/- (B) mice were disrupted by bead beating. Viral titers were determined by standard plaque assay. The dashed line indicates the limit of detection. *, P = 0.032 to 0.012; **, P < 0.0001 for 333d41 compared with 333d41^R as determined by t test. n = 9 to 11 for 129 samples; n = 5 for IFN- ${alpha}$ /ßR^-/- samples due to deaths of mice. For ${Delta}$ TK^-, n = 5.

We observed in these experiments that the 333d41^R virus hada slightly less virulent phenotype than the wild-type 333 derivativeSB5. Specifically, development of genital disease was moderatelydelayed in IFN- ${alpha}$ /ßR^-/- mice (Fig. 3B), and titers of333d41^R were lower than those of SB5 in the brains of 129 mice(P = 0.005) (Fig. 4A). 333d41^R possesses wild-type vhs activity,as determined by degradation of cellular glyceraldehyde-3-phosphatedehydrogenase mRNA (data not shown). 333-vhsB, 333d41, and 333d41^Rviruses were constructed from wild-type HSV-2 strain 333, whereasSB5 is a clonal isolate of 333. It is possible that a subtledifference in the virulence of 333 and SB5 exists, perhaps dueto clonal selection or different passage histories. Alternatively,a secondary mutation could have arisen in the construction ofthe 333d41^R virus. In preliminary studies, 333-vhsB^R, a differentrescue virus which replicates like SB5 in the cornea (47), wasequivalent to 333d41^R in the genital model (data not shown).This result suggests that 333d41^R does not contain an additionalattenuating mutation relative to 333-vhsB and, instead, it impliesthat a difference in the pathogenicity of SB5 and 333 is discriminatedin the vaginal model of HSV infection. We have taken the conservativeapproach of comparing 333d41 to the 333d41^R rescue virus ratherthan to SB5 to evaluate statistical significance of differencesresulting from the vhs mutation.

To rigorously demonstrate vhs-specific phenotypic restorationof HSV-2 vhs mutants in IFN- ${alpha}$ /ßR^-/- mice, groups of129 and IFN- ${alpha}$ /ßR^-/- mice were infected with a 333 straincontaining a deletion in the tk gene, HSV-2 ${Delta}$ TK^- (27). tk isdispensable for HSV replication in dividing cells but not inneurons (20), and tk-deficient viruses are less virulent thanwild-type virus (7, 13, 15). As expected, replication of ${Delta}$ TK^-in the genital mucosa of 129 mice and IFN- ${alpha}$ /ßR^-/- micewas equivalent to that of wild-type and rescue viruses (Fig.2), and genital inflammation in 129 mice was mild (Fig. 3A).In distinct contrast to 333d41, the virulence of ${Delta}$ TK^- in IFN- ${alpha}$ /ßR^-/-mice was not restored to wild-type levels (Fig. 3B), and ${Delta}$ TK^-could be detected only at low levels in the nervous systemsof IFN- ${alpha}$ /ßR^-/- mice (Fig. 4B). These observations indicatethat the enhanced capacity of 333d41 to replicate and causedisease in IFN- ${alpha}$ /ßR^-/- mice was specific to the vhslesion.

Another criterion of specificity for viral inhibitors of hostproteins involved in key antiviral pathways is the demonstrationthat recovery of virulence occurs only in the absence of theparticular host protein. This criterion may be difficult tomeet for a viral protein such as vhs that likely employs therelatively nonspecific mechanism of mRNA degradation to blockhost antiviral responses. To begin to dissect IFN-stimulatedantiviral pathways inhibited by vhs, as well as to evaluatespecificity, we focused on PKR. PKR, one of the best-characterizedIFN- ${alpha}$ /ß-stimulated genes, directs the shutoff of hostcell protein synthesis. We investigated the role of HSV-2 vhsin inhibition of the PKR pathway by using mice that lack PKR(PKR^-/-). Interestingly, we found that replication in the genitalmucosa (Fig. 5A) and virulence (Fig. 5B) of 333d41 were notrestored in PKR^-/- mice relative to wild-type virus. Titersof vhs-deficient virus were higher in PKR^-/- mice than in wild-type129 mice only in the spinal cord (Fig. 5C; P = 0.0016). ThePKR-deficient phenotype of PKR^-/- mice was confirmed by thecapacity to restore replication of the ${gamma}$ 34.5-deficient HSV-1strain, 17termA (1) (data not shown). These results indicatethat vhs does not counteract the antiviral activities of PKRand that restoration of replication and virulence of vhs-deficientvirus is not a universal phenomenon in mice with genetic defects(even defects in a specific IFN- ${alpha}$ /ß-induced antiviralpathway). vhs instead must interfere with a separate antiviralmechanism stimulated by IFN- ${alpha}$ /ßR engagement.

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FIG. 5. Pathogenesis of 333d41 is not restored in PKR^-/- mice. Groups of seven to nine wild-type 129 and PKR^-/- mice were infected i.vag. with 2 x 10⁶ PFU of SB5 or 333d41. n = 4 for 129/SB5. (A) The titer of virus in vaginal swab samples was determined by standard plaque assay. Data represent the geometric mean ± standard error of the mean (SEM). (B) Mice were scored for signs of disease as described in the legend for Fig. 3. Values represent the arithmetic mean ± SEM. (C) Viral titers in brain, brainstem, and spinal cord tissues were determined as described in the legend for Fig. 4. Dashed lines indicate limits of detection.

vhs-deficient HSV-1 does not regain the capacity to replicateand cause disease in the corneas of IFN- ${alpha}$ /ßR^-/- mice(25), indicating that HSV-1 vhs is not an inhibitor of the IFN- ${alpha}$ /ßresponse in the eye. We therefore investigated whether a uniqueproperty of genital epithelial cells or of HSV-2 vhs resultedin HSV-2 interference with the IFN- ${alpha}$ /ß response inthe genital tract. As shown in Fig. 6A, the HSV-1 vhs null mutantUL41NHB (49) did not replicate efficiently in the genital mucosaof 129 mice after i.vag. inoculation. Titers of UL41NHB wereonly slightly elevated in IFN- ${alpha}$ /ßR^-/- mice and werenot increased relative to those of wild-type HSV-1 (Fig. 6A).Virulence of wild-type virus was enhanced by infection of IFN- ${alpha}$ /ßR^-/-mice, but vhs-deficient HSV-1 remained attenuated (Fig. 6B).Neither virus was detectable in the nervous system (data notshown). Thus, recovery of a vhs mutant's capacity to replicateand cause disease similar to wild-type virus in the absenceof an IFN- ${alpha}$ /ß response is unique to HSV-2 vhs and isnot a general property of the i.vag. route of infection.

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FIG. 6. Pathogenesis of vhs-deficient HSV-1 is not restored in IFN- ${alpha}$ /ßR^-/- mice. Groups of four to five 129 and IFN- ${alpha}$ /ßR^-/- mice were infected i.vag. with 2 x 10⁶ PFU of HSV-1 KOS or vhs-deficient UL41NHB. (A) Replication in the genital mucosa. The titer of virus in vaginal swab samples was determined by standard plaque assay. Values represent the geometric mean ± the standard error of the mean (SEM). The dashed line indicates the limit of detection. (B) Severity of genital and neurological disease. Mice were scored for signs of disease as described in Materials and Methods. Values represent the arithmetic mean ± SEM.

DISCUSSION

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Discussion

The vhs protein clearly contributes to HSV virulence in thehost, because vhs-deficient mutants of HSV are highly attenuatedin various mouse models of HSV infection (47, 49). One hundred-to 1,000-fold less replication occurs in the genital mucosaafter infection with vhs-deficient HSV-2, and little virus reachesthe nervous system. We have made several observations regardingthe role of HSV-2 vhs in pathogenesis that largely explain theattenuation of vhs-deficient viruses. First, we determined thatadaptive immunity is not responsible for attenuation of vhs-deficientHSV-2 or HSV-1 during acute infection by demonstrating thatthe mutant viruses remain attenuated during primary infectionof SCID mice. Second, we have shown that vhs interferes withan aspect(s) of the IFN- ${alpha}$ /ß response, because replicationand especially virulence of vhs-deficient HSV-2 are significantlyincreased relative to wild-type and rescue viruses in IFN- ${alpha}$ /ßR^-/-mice. Third, inhibition of the IFN- ${alpha}$ /ß response isa unique property of the HSV-2 vhs protein, because vhs-deficientHSV-1 replication and virulence do not improve relative to wild-typeHSV-1 after i.vag. (Fig. 2 to 4) or corneal (25) infection ofIFN- ${alpha}$ /ßR^-/- mice. Fourth, vhs does not assist HSV-2evasion of the IFN- ${alpha}$ /ß response by interfering withthe PKR pathway. The IFN- ${alpha}$ /ß response rapidly constructsa formidable barrier to virus infection; the dramatic effectof IFN- ${alpha}$ /ß on the pathogenesis of a virus without vhsas a countermeasure attests to the significance of HSV-2 vhsas a modulator of this innate host defense mechanism.

In order to prove specificity in the restoration of the wild-typephenotype to vhs-deficient viruses in IFN- ${alpha}$ /ßR^-/- mice,it was important to demonstrate that (i) an attenuated viralstrain with a mutation in a gene irrelevant to IFN- ${alpha}$ /ßremains attenuated in IFN- ${alpha}$ /ßR^-/- mice, and (ii) thevhs mutant virus is not restored in a host with a differentgenetic lesion (26). We have satisfied the first of these specificitycriteria with the tk^- mutant virus, ${Delta}$ TK^-. We observed that ${Delta}$ TK^-replicated poorly in the nervous systems of 129 mice and, unlike333d41, did not replicate well in the nervous systems of IFN- ${alpha}$ /ßR^-/-mice, even with an inoculum of 2 x 10⁶ PFU. Therefore, restorationwas specific to the vhs mutation. Leib et al. (25) observedthat a tk^- HSV-1 mutant inoculated at the same dose replicatedmore efficiently in the corneal epithelium of IFN- ${alpha}$ /ß/ ${gamma}$ R^-/-mice than wild-type mice but was barely detectable in the nervoussystem through 5 days postinfection. Taken together, these datasuggest that loss of ${alpha}$ /ß or ${alpha}$ /ß/ ${gamma}$ IFN receptorsdoes not globally restore the capacity of HSV mutants to replicatein the nervous system.

Infection of PKR^-/- mice satisfied the second criterion forspecificity in that 333d41 was not restored for replicationor virulence. While the PKR mutation is not independent of theIFN- ${alpha}$ /ß receptor signaling pathway, our data indicatethat this separate host genetic mutation does not restore vhs-deficientviruses to virulence and, therefore, restoration is specificto the IFN- ${alpha}$ /ßR mutation. The fact that attenuationof 333d41 was not reversed by deletion of the host PKR geneis intriguing. This result suggests that although PKR mediatesan important antiviral pathway that is induced by IFN- ${alpha}$ /ßRengagement, PKR is not the primary target of vhs-mediated hostinhibition. HSV already encodes two proteins, US11 and ICP34.5,demonstrated to block PKR-mediated protein synthesis inhibitionat different stages (3, 4, 17, 29). With two specific inhibitorsof the PKR pathway, it may not be necessary for HSV to encodeyet another inhibitor. Lack of PKR pathway disruption also suggeststhat vhs may discriminately inhibit host antiviral functions.However, if vhs utilizes its RNase activity to inhibit induciblemediators of the IFN- ${alpha}$ /ß response, it may have a limitedcapacity to block the PKR pathway due to preformed PKR in cellsthat can be directly activated by virus infection (14). Determiningwhich IFN-related antiviral pathways are opposed by vhs andthe mechanisms of vhs action will be the focus of future experiments.

If the mechanism of IFN- ${alpha}$ /ß pathway inhibition by vhsdoes involve RNA degradation, as seems likely, vhs could beunique as a general, relatively nonspecific inhibitor of theinducible IFN- ${alpha}$ /ß response. In this manner, vhs couldbe broadly useful to the virus across cell types and tissuesthat may vary in their dependence on individual IFN-inducedantiviral pathways. Nevertheless, some evidence for tissue specificityin HSV-2 vhs interference with the IFN- ${alpha}$ /ß responsemay have arisen in our present experiments. Restoration of vhs-deficientHSV-2 replication in the genital mucosa of IFN- ${alpha}$ /ßR^-/-mice was not comprehensive because titers were lower than thosefor wild-type virus at 3 and 4 days postinfection. Despite reducedmucosal replication, vhs-deficient virus replicated as vigorouslyas wild-type virus in the spinal cord and brainstem. Thus, differencesmay exist in the particular antiviral pathways stimulated byIFN- ${alpha}$ /ßR engagement in specific tissues, and some ofthese pathways, for example, PKR, may be less inhibited by vhsthan others. Such cell-type-specific differences in IFN inductionhave been noted in reovirus infections of cardiac myocytes versusskeletal muscle cells (45). In the genital mucosa, NK cells,neutrophils, or even early antiviral T-cell responses may alsohelp reduce vhs-deficient virus replication after 48 h. We arecurrently investigating the reason for the later decline invhs-deficient virus titer in the mucosa of IFN- ${alpha}$ /ßR^-/-mice relative to that with wild-type virus. Production of IFN- ${alpha}$ /ßand IFN-stimulated gene induction in the genital mucosa willbe interesting areas of future investigation.

Our observation that HSV-2 vhs counteracts the IFN- ${alpha}$ /ßresponse fits logically into a picture of HSV-2 pathogenesisfor three reasons. First, vhs enters the cytoplasm with theinfecting virion and, thus, can immediately counter rapid IFNsynthesis or receptor-mediated induction of the antiviral state.Indeed, IFN- ${alpha}$ can suppress immediate-early gene expression (28,37, 38, 42), so the virus must have an immediate counterresponse.Second, because vhs degrades mRNA, it could counteract a widevariety of host responses essential to induction of the antiviralstate, including IFN-ß and - ${alpha}$ 4 produced by the initiallyinfected cells. Finally, HSV-2 is even more resistant to IFN- ${alpha}$ /ßthan HSV-1 (24), and HSV-2 vhs is faster and approximately 40-foldmore active than HSV-1 vhs (10, 11, 19). An effect of HSV-2vhs on the host IFN- ${alpha}$ /ß response therefore should bemore readily discernible than an effect of HSV-1 vhs. Our resultshighlight an important difference in the pathogenic mechanismsof HSV-2 and HSV-1.

We observed that the presence or absence of functional lymphocytesemphatically does not alter the pathogenesis of either HSV-1(data not shown) or HSV-2 vhs-deficient viruses in a naïvehost. This observation indicates that the adaptive immune responseis not responsible for attenuating the pathogenesis of virusesthat lack vhs activity, at least during a rapidly progressingprimary infection in BALB/c mice. Because the IFN- ${alpha}$ /ßR^-/-mice used had a 129 genetic background, it is formally possiblethat vhs could interfere with primary adaptive immune responsesin 129 mice. However, the generally greater resistance of 129mice to HSV infection should make recovery of virulence by vhs-deficientvirus more challenging.

Despite observations that vhs interferes with the expressionof MHC class I and class II molecules (18, 54, 55), our resultwas not entirely surprising given that replication of vhs-deficientviruses in the genital mucosa of wild-type mice is curtailedwithin 48 h. Nevertheless, vhs could play a role in adaptiveimmune responses during reactivation in an immune host, whenevasion of memory cytotoxic T-lymphocyte responses would confera significant advantage to the virus. Our results also do notpreclude the possibility that vhs retards the induction of adaptiveimmune responses through its interference with MHC expressionand potentially through its inhibition of the IFN- ${alpha}$ /ßresponse. IFNs induce the activation and maturation of dendriticcells (6, 30), the most potent antigen-presenting cell typefor naïve T cells. IFN- ${alpha}$ /ß thus establishes animportant link between early innate responses and developingantiviral adaptive immunity. If less IFN- ${alpha}$ /ß is producedby wild-type virus-infected cells than by cells infected withvhs-deficient virus, the adaptive immune response to wild-typevirus may be delayed or reduced in magnitude. We are currentlyinvestigating this possibility. In any event, our observationthat vhs counteracts the innate antiviral IFN- ${alpha}$ /ß responseduring acute infection, coupled with its capacity to down-regulateMHC molecules crucial in adaptive immune responses, mark vhsas a multifunctional immune evasion mechanism of HSV-2.

ACKNOWLEDGMENTS

We are indebted to Jim Smiley for providing the 333-vhsB and ${Delta}$ TK^- viruses, Rick Thompson for the 17termA virus, and MichelAguet and Bob Silverman for extending to us use of IFN- ${alpha}$ /ßR^-/-and 129 PKR^-/- mice, respectively. Construction of the 333d41and 333d41^R viruses was performed in the laboratory of DavidLeib. We thank Rob Reass for expert technical assistance, DavidLeib for helpful comments on the manuscript, and members ofthe Leib and Virgin laboratories for advice and discussion.This work was supported by Public Health Service award CA75052.

Jenny A. Murphy and Rebecca J. Duerst contributed equally tothis work.

FOOTNOTES

* Corresponding author. Mailing address: Dept. of Molecular Microbiology and Immunology, Saint Louis University School of Medicine, 1402 S. Grand Blvd., St. Louis, MO 63104. Phone: (314) 577-8321. Fax: (314) 773-3403. E-mail: morrisla{at}slu.edu.

REFERENCES

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