Asymptomatic neonatal herpes simplex virus infection in mice leads to long-term cognitive impairment.

Neonatal herpes simplex virus (nHSV) is a devastating infection impacting approximately 14,000 newborns globally each year. Infection is associated with high neurologic morbidity and mortality, making early intervention and treatment critical. Clinical outcomes of symptomatic nHSV infections are well-studied, but little is known about the frequency of, or outcomes following, sub-clinical or asymptomatic nHSV. Given the ubiquitous nature of HSV infection and frequency of asymptomatic shedding in adults, subclinical infections are underreported, yet could contribute to long-term neurological damage. To assess potential neurological morbidity associated with subclinical nHSV infection, we developed a low-dose (100 PFU) HSV infection protocol in neonatal C57BL/6 mice. At this dose, HSV DNA was detected in the brain by PCR but was not associated with acute clinical symptoms. However, months after initial inoculation with 100 PFU of HSV, we observed impaired mouse performance on a range of cognitive and memory performance tasks. Memory impairment was induced by infection with either HSV-1 or HSV-2 wild-type viruses, but not by a viral mutant lacking the autophagy-modulating Beclin-binding domain of the neurovirulence gene γ34.5. Retroviral expression of wild type γ34.5 gene led to behavioral pathology in mice, suggesting that γ34.5 expression may be sufficient to cause cognitive impairment. Maternal immunization and HSV-specific antibody treatment prevented offspring from developing neurological sequelae following nHSV-1 infection. Altogether, these results support the idea that subclinical neonatal infections may lead to cognitive decline in adulthood, with possible profound implications for research on human neurodegenerative disorders such as Alzheimer's Disease.

Counteraction of interferon-induced antiviral responses by herpes simplex viruses.

The outcome of a viral infection of a host involves the complex interplay of viral determinants of virulence and host resistance factors. Among the first lines of defense for the host in attempts to control viral infection are the interferons (IFNs). A large body of work has now shown that the IFNs are a family of soluble proteins that serve to mediate antiviral effects, to regulate cell growth, and to modulate the activation of immune responses. The innate antiviral activities of IFNs are exceedingly potent and rapid. It is, therefore, not surprising that so many viruses have evolved ways to either preclude the synthesis of IFNs or evade downstream antiviral events. Such evasion allows for the virus to spread before the development of a specific adaptive immune response and likely represents a pivotal determinant of virulence for the invading virus. This review describes some of the research on herpes simplex virus (HSV) that has elucidated genes involved in evasion of the IFN response. In particular, the roles of specific viral genes in resistance to the antiviral effects of PKR and RNaseL are described, along with other HSV genes and loci associated with resistance to IFN for which mechanisms have yet to be described.

Temporal regulation of herpes simplex virus type 2 VP22 expression and phosphorylation.

The VP22 protein of herpes simplex virus type 2 (HSV-2) is a major component of the virion tegument. Previous work with HSV-1 indicated that VP22 is phosphorylated during infection, and phosphorylation may play a role in modulating VP22 localization in infected cells. It is not clear, however, when phosphorylation occurs in infected cells or how it is regulated. Less is known about the synthesis and phosphorylation of HSV-2 VP22. To study the complete biosynthetic history of HSV-2 VP22, we generated a monoclonal antibody to the carboxy terminus of VP22. Using immunoprecipitation and Western blot analyses, we show that HSV-2 VP22 can be found in three distinct isoforms in infected cells, two of which are phosphorylated. Like HSV-1 VP22, HSV-2 VP22 is synthesized ca. 4 h after infection, and the isoform later incorporated into virions is hypophosphorylated. In addition, we demonstrate for the first time (i) that newly synthesized VP22 is phosphorylated rapidly after synthesis, (ii) that this phosphorylation occurs in a virus-dependent manner, (iii) that the HSV-2 kinase UL13 is capable of inducing phosphorylation of VP22 in the absence of other viral proteins, (iv) that phosphorylated VP22 is very stable in infected cells, (v) that phosphorylated isoforms of VP22 are gradually dephosphorylated late in infection to produce the virion tegument form, and (vi) that this dephosphorylation occurs independently of viral DNA replication or virion assembly. These results indicate that HSV-2 VP22 is a stable protein that undergoes highly regulated, virus-dependent phosphorylation events in infected cells.

Herpes simplex virus type 1 corneal infection results in periocular disease by zosteriform spread.

In humans and animal models of herpes simplex virus infection, zosteriform skin lesions have been described which result from anterograde spread of the virus following invasion of the nervous system. Such routes of viral spread have not been fully examined following corneal infection, and the possible pathologic consequences of such spread are unknown. To investigate this, recombinant viruses expressing reporter genes were generated to quantify and correlate gene expression with replication in eyes, trigeminal ganglia, and periocular tissue. Reporter activity peaked in eyes 24 h postinfection and rapidly fell to background levels by 48 h despite the continued presence of viral titers. Reporter activity rose in the trigeminal ganglia at 60 h and peaked at 72 h, concomitant with the appearance and persistence of infectious virus. Virus was present in the periocular skin from 24 h despite the lack of significant reporter activity until 84 h postinfection. This detection of reporter activity was followed by the onset of periocular disease on day 4. Corneal infection with a thymidine kinase-deleted reporter virus displayed a similar profile of reporter activity and viral titer in the eyes, but little or no detectable activity was observed in trigeminal ganglia or periocular tissue. In addition, no periocular disease symptoms were observed. These findings demonstrate that viral infection of periocular tissue and subsequent disease development occurs by zosteriform spread from the cornea to the periocular tissue via the trigeminal ganglion rather than by direct spread from cornea to the periocular skin. Furthermore, clinical evidence is discussed suggesting that a similar mode of spreading and disease occurs in humans following primary ocular infection.

Disruption of virion host shutoff activity improves the immunogenicity and protective capacity of a replication-incompetent herpes simplex virus type 1 vaccine strain.

The virion host shutoff (vhs) protein encoded by herpes simplex virus type 1 (HSV-1) destabilizes both viral and host mRNAs. An HSV-1 strain with a mutation in vhs is attenuated in virulence and induces immune responses in mice that are protective against corneal infection with virulent HSV-1, but it has the capacity to establish latency. Similarly, a replication-incompetent HSV-1 strain with a mutation in ICP8 elicits an immune response protective against corneal challenge, but it may be limited in viral antigen production. We hypothesized therefore that inactivation of vhs in an ICP8(-) virus would yield a replication-incompetent mutant with enhanced immunogenicity and protective capacity. In this study, a vhs(-)/ICP8(-) HSV-1 mutant was engineered. BALB/c mice were immunized with incremental doses of the vhs(-)/ICP8(-) double mutant or vhs(-) or ICP8(-) single mutants, or the mice were mock immunized, and protective immunity against corneal challenge with virulent HSV-1 was assessed. Mice immunized with the vhs(-)/ICP8(-) mutant showed prechallenge serum immunoglobulin G titers comparable to those immunized with replication-competent vhs(-) virus and exceed those of mice immunized with the ICP8(-) single mutant. Following corneal challenge, the degrees of protection against ocular disease, weight loss, encephalitis, and establishment of latency were similar for vhs(-)/ICP8(-) and vhs(-) virus-vaccinated mice. Moreover, the double deleted vhs(-)/ICP8(-) virus protected mice better in all respects than the single deleted ICP8(-) mutant virus. The data indicate that inactivation of vhs in a replication-incompetent virus significantly enhances its protective efficacy while retaining its safety for potential human vaccination. Possible mechanisms of enhanced immunogenicity are discussed.

Specific phenotypic restoration of an attenuated virus by knockout of a host resistance gene.

To produce disease, viruses must enter the host, multiply locally in host tissues, spread from the site of entry, and overcome or evade host immune responses. At each stage in this infectious process, specific microbial and host genes determine the ultimate virulence of the virus. Genetic approaches have identified many viral genes that play critical roles in virulence and are presumed to target specific components of the host innate and acquired immune response. However, formal proof that a virulence gene targets a specific protein in a host pathway in vivo has not been obtained. Based on cell culture studies, it has been proposed that the herpes simplex virus type 1 gene ICP34.5 (ICP, infected cell protein) enhances neurovirulence by negating antiviral functions of the IFN-inducible double-stranded RNA-dependent protein kinase R or PKR [Chou, J., Chen, J.J., Gross, M. & Roizman, B. (1995) Proc. Natl. Acad. Sci. USA 92, 10516-10520]. Herein, we show that a virus that has been attenuated by deletion of ICP34.5 exhibits wild-type replication and virulence in a host from which the PKR gene has been deleted. We show that restoration of virulence is specific to ICP34.5 and PKR by using additional host and viral mutants. The use of recombinant viruses to infect animals with null mutations in host defense genes provides a formal genetic test for identifying in vivo mechanisms and targets of microbial virulence genes.

Herpes simplex virus virion host shutoff (vhs) activity alters periocular disease in mice.

During lytic infection, the virion host shutoff (vhs) protein of herpes simplex virus (HSV) mediates the rapid degradation of RNA and shutoff of host protein synthesis. In mice, HSV type 1 (HSV-1) mutants lacking vhs activity are profoundly attenuated. HSV-2 has significantly higher vhs activity than HSV-1, eliciting a faster and more complete shutoff. To examine further the role of vhs activity in pathogenesis, we generated an intertypic recombinant virus (KOSV2) in which the vhs open reading frame of HSV-1 strain KOS was replaced with that of HSV-2 strain 333. KOSV2 and a marker-rescued virus, KOSV2R, were characterized in cell culture and tested in an in vivo mouse eye model of latency and pathogenesis. The RNA degradation kinetics of KOSV2 was identical to that of HSV-2 333, and both showed vhs activity significantly higher than that of KOS. This demonstrated that the fast vhs-mediated degradation phenotype of 333 had been conferred upon KOS. The growth of KOSV2 was comparable to that of KOS, 333, and KOSV2R in cell culture, murine corneas, and trigeminal ganglia and had a reactivation frequency similar to those of KOS and KOSV2R from explanted latently infected trigeminal ganglia. There was, however, significantly reduced blepharitis and viral replication within the periocular skin of KOSV2-infected mice compared to mice infected with either KOS or KOSV2R. Taken together, these data demonstrate that heightened vhs activity, in the context of HSV-1 infection, leads to increased viral clearance from the skin of mice and that the replication of virus in the skin is a determining factor for blepharitis. These data also suggest a role for vhs in modulating host responses to HSV infection.

Interferons regulate the phenotype of wild-type and mutant herpes simplex viruses in vivo.

Mechanisms responsible for neuroattenuation of herpes simplex virus (HSV) have been defined previously by studies of mutant viruses in cultured cells. The hypothesis that null mutations in host genes can override the attenuated phenotype of null mutations in certain viral genes was tested. Mutants such as those in infected cell protein (ICP) 0, thymidine kinase, ribonucleotide reductase, virion host shutoff, and ICP34.5 are reduced in their capacity to replicate in nondividing cells in culture and in vivo. The replication of these viruses was examined in eyes and trigeminal ganglia for 1-7 d after corneal inoculation in mice with null mutations (-/-) in interferon receptors (IFNR) for type I IFNs (IFN-alpha/betaR), type II IFN (IFN-gammaR), and both type I and type II IFNs (IFN-alpha/beta/gammaR). Viral titers in eyes and ganglia of IFN-gammaR-/- mice were not significantly different from congenic controls. However, in IFN-alpha/betaR-/- or IFN-alpha/beta/gammaR-/- mice, growth of all mutants, including those with significantly impaired growth in cell culture, was enhanced by up to 1,000-fold in eyes and trigeminal ganglia. Blepharitis and clinical signs of infection were evident in IFN-alpha/betaR-/- and IFN-alpha/beta/gammaR-/- but not control mice for all viruses. Also, IFNs were shown to significantly reduce productive infection of, and spread from intact, but not scarified, corneas. Particularly striking was restoration of near-normal trigeminal ganglion replication and neurovirulence of an ICP34.5 mutant in IFN-alpha/betaR-/- mice. These data show that IFNs play a major role in limiting mutant and wild-type HSV replication in the cornea and in the nervous system. In addition, the in vivo target of ICP34.5 may be host IFN responses. These experiments demonstrate an unsuspected role for host factors in defining the phenotypes of some HSV mutants in vivo. The phenotypes of mutant viruses therefore cannot be interpreted based solely upon studies in cell culture but must be considered carefully in the context of host factors that may define the in vivo phenotype.

Analysis of the basal and inducible activities of the ICPO promoter of herpes simplex virus type 1.

Sequences from -420 to -70 from the ICPO transcriptional start site of herpes simplex virus type 1 are dispensable for reactivation from latency. A putative cAMP-response element (CRE) outside of this region was non-functional in both murine neuroblastoma (NB41A3) and rat pheochromocytoma (PC12) cells. Also, poor binding of cAMP-response element binding protein (CREB) was observed. Sequences from -95 to -37 are important for constitutive activity in NB41A3, PC12 and baby hamster kidney (BHK) cells. The TATA box and Sp1 site were also shown to be major contributors to constitutive activity. Finally, high constitutive activity of a deleted construct (-420 to -1) in NB41A3 and BHK cells suggests transcription initiates upstream of -420 in the absence of VP16. The implications of these observations regarding ICPO expression during the virus life-cycle are discussed.

The UL4 gene of herpes simplex virus type 1 is dispensable for latency, reactivation and pathogenesis in mice.

The UL4 gene of herpes simplex virus type 1 is predicted to encode a 21.5 kDa protein of 199 amino acids. Although UL4 is dispensable for growth in cell culture, its function is not known. In the present study, the promoter of UL4 was examined and found to contain a cAMP-response element which bound the transcription factor CREB, and was strongly activated by cAMP. A recombinant virus, termed UL4HS, was constructed with a nonsense linker inserted into the UL4 open reading frame, to make a truncated UL4 protein of 60 amino acids. In addition, a marker-rescued virus, UL4HSMR, was constructed. Western immunoblot analysis revealed a 23 kDa band in extracts of wild-type and marker-rescued virus infected cells which was missing for UL4HS. Only modest differences were observed in the abilities of wild-type and UL4-mutant viruses to grow in Vero cells or in contact-inhibited mouse C3H/10T1/2 cells. In addition, there were only modest differences between the ability of UL4HS to replicate in murine corneas and trigeminal ganglia relative to wild-type viruses, and reactivation of UL4HS from latency was unaffected. Taken together, these data demonstrate that UL4 is dispensable for latency and pathogenesis in mice.

Protection from primary infection and establishment of latency by vaccination with a herpes simplex virus type 1 recombinant deficient in the virion host shutoff (vhs) function.

A herpes simplex virus type 1 (HSV-1) recombinant virus deficient in the virion host shutoff (vhs) function was assessed for its ability to function as a live-attenuated vaccine. Protection of mice from wild-type challenge infection and the establishment and reactivation of HSV-1 latency was measured in a mouse ocular model. Challenge virus replication in corneas and trigeminal ganglia was significantly reduced for vaccinated mice. Consistent with these findings, the vaccinated groups showed no clinical signs during acute infection and high levels of virus-specific IgG and neutralizing antibodies were induced. The establishment of and reactivation from latency in trigeminal ganglia from the vaccinated group were also significantly reduced relative to controls. These data suggest that vhs deletion mutants may have significant utility as live-attenuated HSV-1 vaccines.

Postexposure vaccination with a virion host shutoff defective mutant reduces UV-B radiation-induced ocular herpes simplex virus shedding in mice.

A herpes simplex virus type 1 (HSV-1) recombinant (UL41NHB) deficient in the virion host shutoff (vhs) function was tested as a therapeutic vaccine in an ultraviolet (UV) light-induced mouse ocular reactivation model. Mice were infected with HSV-1 via the cornea. Following the establishment of latency by HSV-1 the mice were subsequently vaccinated intraperitoneally with one dose of UL41NHB or with uninfected cell extract. Mice were subsequently UV-irradiated to induce viral reactivation and during the 7 days post-UV irradiation, numbers of mice shedding virus were reduced from 13/23 (57%) to 3/25 (12%), and numbers of virus-positive eye swabs were reduced from 40/161 (25%) to 6/175 (3%) by the vaccine (P < 0.001). These data suggest that deletion of vhs may be a useful strategy in the development of attenuated therapeutic HSV vaccines.

Identification of the UL4 protein of herpes simplex virus type 1.

Herpes simplex virus type 1 gene UL4 is predicted to encode a 199-amino-acid protein with a molecular mass of 21 5 kDa. We report here identification of this protein and its localization in the nuclei of infected cells. Antisera raised against oligopeptides corresponding to the C terminus of the predicted UL4 protein were used for identification of a 25 kDa protein as the product of the UL4 gene. This protein was not detected in cells infected with a UL4 defective mutant virus, but was synthesized by coupled in vitro transcription-translation of the UL4 gene. Synthesis of the 25 kDa protein was blocked by phosphonoacetic acid, an inhibitor of DNA synthesis, indicating that the UL4 gene is expressed with gamma kinetics. Subcellular fractionation showed the protein to be localized in the nucleus. It was not detected in virions or light particles.

Analysis of conserved domains of UL41 of herpes simplex virus type 1 in virion host shutoff and pathogenesis.

The herpes simplex virus type 1 virion host shutoff protein has four domains whose sequences are conserved only among neurotropic herpesviruses. Mutant viruses with 29- and 31-amino-acid deletions in domains III and IV but outside of the domain required for interaction with VP16 were generated. The mutants failed to induce cellular RNA degradation and showed impaired virulence in mice. Domains III and IV are therefore required for both shutoff and virulence.

Role of cis-acting sequences of the ICPO promoter of herpes simplex virus type 1 in viral pathogenesis, latency and reactivation.

A mutant herpes simplex virus type 1, termed delta Tfi, with a 350 bp deletion of the Sp1, NF-kappaB, TAATGARATs, C/EBP and F2 DNA-binding elements from -420 to -70 relative to the transcriptional start site of ICPO (Vmw 110), was generated and characterized. The efficiency of plating of delta Tfi was reduced on Vero cells and it expressed correctly initiated ICPO RNA in the presence of cycloheximide, although RNA levels were 2.5-fold lower than with wild-type (KOS) and marker-rescued (delta TfiR) viruses. This was consistent with a demonstrated reduction in ICPO protein expression for delta Tfi at early times post-infection and a 3-fold reduction in ICPO-dependent transactivation of the ICP6 promoter. KOS, delta Tfi and delta TfiR replication in murine corneas and trigeminal ganglia were comparable when measured on a complementing cell line, but delta Tfi titres appeared 15- to 50-fold lower when measured on Vero cells. delta Tfi was correspondingly less virulent than wild-type or marker-rescued viruses in both immunocompetent and SCID mice. delta Tfi, however, established and reactivated from latency with efficiencies comparable to wild-type and marker-rescued viruses. These results demonstrate that although this deletion of the ICPO promoter results in lower levels of ICPO in vitro and decreased virulence in vivo, the establishment of and reactivation from latency are unaffected. This indicates that elements which regulate ICPO expression and virulence during acute infection may be distinct from those involved in reactivation.

Role of the virion host shutoff (vhs) of herpes simplex virus type 1 in latency and pathogenesis.

The herpes simplex virus type 1 (HSV-1) UL41 gene product, virion host shutoff (vhs), has homologs among five alphaherpesviruses (HSV-1, HSV-2, pseudorabies virus, varicella-zoster virus, and equine herpesvirus 1), suggesting a role for this protein in neurotropism. A mutant virus, termed UL41NHB, which carries a nonsense linker in the UL41 open reading frame at amino acid position 238 was generated. UL41NHB and a marker-rescued virus, UL41NHB-R, were characterized in vitro and tested for their ability to replicate in vitro and in vivo and to establish and reactivate from latency in a mouse eye model. As demonstrated by Western blotting (immunoblotting) and Northern (RNA) blotting procedures, UL41NHB encodes an appropriately truncated vhs protein and, as expected for a vhs null mutant, fails to induce the degradation of cellular glyceraldehyde-3-phosphate dehydrogenase mRNA. The growth of UL41NHB was not significantly altered in one-step growth curves in Vero or mouse C3H/10T1/2 cells but was impaired in corneas, in trigeminal ganglia, and in brains of mice compared with the growth of KOS and UL41NHB-R. As a measure of establishment of latency, quantitative DNA PCR showed that the amount of viral DNA within trigeminal ganglia latently infected with UL41NHB was reduced by approximately 30-fold compared with that in KOS-infected ganglia and by 50-fold compared with that in UL41NHB-R-infected ganglia. Explant cocultivation studies revealed a low reactivation frequency for UL41NHB (1 of 28 ganglia, or 4%) compared with that for KOS (56 of 76, or 74%) or UL41NHB-R (13 of 20 or 65%). Taken together, these results demonstrate that vhs represents a determinant of viral pathogenesis.

The roles of the cAMP-response element and TATA box in expression of the herpes simplex virus type 1 latency-associated transcripts.

A quantitative ribonuclease protection assay (RPA) was developed in order to rapidly and accurately measure the levels and timing of latency-associated transcript (LAT) expression in ganglia latently infected with wild-type and mutant herpes simplex virus (HSV). Use of this assay in parallel with measurement of viral titers in murine trigeminal ganglia demonstrated that the peak of viral replication precedes the peak and subsequent plateau of LAT expression. This plateau of LAT expression was unaltered from Day 7 through the end of the experimental period on Day 28, suggesting that LAT does not further accumulate during latency of wild-type virus. RPA analyses of trigeminal ganglia latently infected with HSV-1 mutants containing specific alterations in the LAT TATA box, cyclic AMP-response element (CRE), and both TATA and CRE were performed. Mutation of the upstream TATA box reduced LAT expression to 25% of wild-type or marker-rescued virus levels, whereas mutation of the CRE did not significantly affect LAT expression in vivo whether in the presence or absence of the TATA box. These experiments demonstrate a specific requirement for the upstream promoter TATA box for wild-type LAT expression. Further examination of the role of the CRE and the TATA box by transient expression assays suggests that the CRE is important for inducible activity and that its interaction with the TATA box requires stereospecific alignment.

A structural and functional comparison of the latency-associated transcript promoters of herpes simplex virus type 1 strains KOS and McKrae.

The promoters of the latency-associated transcripts (LATs) of herpes simplex virus type 1 (HSV-1) strains KOS and McKrae were compared to examine their influence upon the reactivation phenotypes of these two strains. Unlike strain KOS, McKrae is readily reactivable using in vivo reactivation models. We found greater than 96% sequence conservation between KOS and McKrae in the LATs promoter region, and both promoters showed equivalent basal and inducible activities. An inter-strain recombinant (termed MK13) was constructed in which the LATs promoter of HSV-1 McKrae was recombined into the background of HSV-1 strain KOS. In a murine u.v. light-induced reactivation model, virus shedding was detected by eye swabbing in two of 44 (5%) mice infected with KOS, 20 of 42 (48%) mice infected with McKrae and none of 45 (0%) mice infected with MK13. These data show that the LATs promoters of these viruses are structurally and functionally similar and that transfer of the LATs promoter from McKrae into KOS is insufficient to confer a reactivatable phenotype.