Multigenic control of virus virulence in intertypic recombinants of herpes simplex virus

The virulence of herpes simplex virus (HSV) type 1 x type 2 intertypic recombinants was determined following infection of corneas of outbred New Zealand White rabbits. None of the four recombinants was as virulent for rabbits as type 1 parent. All the four recombinants having an insert of type 2 virus genome between 0.35 and 0.576 map units (m.u.) and/or 0.82 and 1.00 m.u. exhibited intermediate virulence between their type 1 and type 2 parents. The results indicate that intertypic recombinants are moderated in their virulence independent of their parental virulence and therefore there exists a multigenic control of HSV virulence.     

Localization of a herpes simplex virus neurovirulence gene dissociated from high-titer virus replication in the brain.

Previous studies with the herpes simplex virus type 1 X type 2 intertypic recombinant RS6 suggested that the genomic region from 0.11 to 0.14 map units is involved in neurovirulence (R. T. Javier, R. L. Thompson, and J. G. Stevens, J. Virol. 61:1978-1984, 1987). To study this further, we isolated an RS6-derived herpes simplex virus intertypic recombinant (R13-1) which has a genetic defect within this area. After inoculation into mouse brains, R13-1 was found to be approximately 10,000-fold less neurovirulent than either the wild-type type 1 or type 2 parental virus. However, R13-1 replicated in the mouse brain to titers resembling those of the wild-type parents. Further comparisons with wild-type counterparts indicated that R13-1 expressed equivalent levels of the enzyme thymidine kinase and replicated to intermediate levels in primary mouse embryo fibroblasts maintained at the normal body temperature for mice. Using marker rescue techniques combined with in vivo selection, we found that recombination between unit-length R13-1 DNA and a cloned type 1 DNA fragment spanning the region from 0.11 to 0.14 map units (EcoRI-d, 0.079 to 0.192 map units) generated viruses with a wild-type neurovirulence phenotype. To further refine the genomic region of interest, we performed marker rescue experiments using two EcoRI-d subclones, EcoRI/BamHI dc (0.079 to 0.143 map units) and BamHI/EcoRI and (0.143 to 0.192 map units), representing the left and right halves of the EcoRI d fragment, respectively. In these experiments the EcoRI/BamHI dc clone, but not the BamHI/EcoRI ad clone, yielded recombinant viruses exhibiting wild-type neurovirulence. These results show that at least one herpes simplex virus gene function associated with neurovirulence is located within a 9.1-kilobase region at 0.079 to 0.143 map units of the viral genome. Perhaps more significantly, the results indicate that this neurovirulence property functions independently of high-titer virus replication in the brain.     

Rescue of a herpes simplex virus type 1 neurovirulence function with a cloned DNA fragment.

A herpes simplex virus type 1 (HSV-1) genetic function that is required for viral replication in the murine central nervous system was unambiguously localized. Thus, cosmid clones of either HSV-1 HindIII fragment C (0.64 to 0.87 map units) or fragment B (0.64 to 0.83 plus 0.91 to 1.0 map units) were employed to restore neurovirulence to an intertypic recombinant (RE6) that is specifically deficient in this property. The neurovirulent recombinants were generated in cell culture by cotransfecting the clone fragments and unit-length RE6 DNA and then selected in mouse brains. Either fragment efficiently conferred neurovirulence to RE6, demonstrating that no short region unique sequences are required. Analyses of the genomic structures of the neurovirulent recombinants showed that, in every case, HSV-1 information from 0.71 to 0.83 map units was incorporated into the RE6 genome. Cleavage of HindIII fragment C with EcoRI eliminated its capacity to rescue RE6. Virulence could be restored by the addition of HSV-1 BamHI fragment L (0.71 to 0.74 map units) that spans an EcoRI site at 0.72 map units. The precise location of this HSV-1 neurovirulence function is discussed.     

Attenuation of herpes simplex virus neurovirulence with picornavirus cis-acting genetic elements

Viral pathogenesis depends on a suitable milieu in target host cells permitting viral gene expression, propagation, and spread. In many instances, viral genomes can be manipulated to select for propagation in certain tissues or cell types. This has been achieved for the neurotropic poliovirus (PV) by exchange of the internal ribosomal entry site (IRES), which is responsible for translation of the uncapped plus-strand RNA genome. The IRES of human rhinovirus type 2 (HRV2) confers neuron-specific replication deficits to PV but has no effect on viral propagation in malignant glioma cells. We report here that placing the critical gamma(1)34.5 virulence genes of herpes simplex virus type 1 (HSV) under translation control of the HRV2 IRES results in neuroattenuation in mice. In contrast, IRES insertion permits HSV propagation in malignant glioma cell lines that do not support replication of HSV recombinants carrying gamma(1)34.5 deletions. Our observations indicate that the conditions for alternative translation initiation at the HRV2 IRES in malignant glioma cells differ from those in normal central nervous system (CNS) cells. Picornavirus regulatory sequences mediating cell type-specific gene expression in the CNS can be utilized to target cancerous cells at the level of translation regulation outside their natural context.     

Tetrahydrouridine specifically facilitates deoxycytidine incorporation into herpes simplex virus DNA.

As reported by Jamieson and Subak-Sharpe (J. Gen. Virol. 31:303-313, 1976), exogenous deoxycytidine is very poorly incorporated into herpes simplex virus DNA. Here it is shown that this incorporation was dramatically increased in the presence of tetrahydrouridine (THU), a specific inhibitor of cytidine-deoxycytidine deaminase. Thus, the exclusion of deoxycytidine from herpes simplex virus DNA probably results from massive degradation by the deaminase, which is consistent with the observation that in the absence of THU, most of the nucleotides formed from exogenous deoxycytidine are dUMP. The effect of tHU upon deoxycytidine incorporation was specific for herpes simplex virus-infected cells; THU did not increase deoxycytidine incorporation into DNA of uninfected cells. Therefore, one might expect THU to enhance the antiviral activity of 1-beta-D-arabinofuranasylcytosine since this analog is also readily deaminated. However, THU increased both the antiviral activity and the cell toxicity only slightly and to about the same extent. Therefore, the metabolism of 1-beta-D-arabnofuranosylcytosine is different from that of deoxycytidine in herpes simplex virus-infected cells.     

Bioreactor production of recombinant herpes simplex virus vectors

Serotypical application of herpes simplex virus (HSV) vectors to gene therapy (type 1) and prophylactic vaccines (types 1 and 2) has garnered substantial clinical interest recently. HSV vectors and amplicons have also been employed as helper virus constructs for manufacture of the dependovirus adeno-associated virus (AAV). Large quantities of infectious HSV stocks are requisite for these therapeutic applications, requiring a scalable vector manufacturing and processing platform comprised of unit operations which accommodate the fragility of HSV. In this study, production of a replication deficient rHSV-1 vector bearing the rep and cap genes of AAV-2 (denoted rHSV-rep2/cap2) was investigated. Adaptation of rHSV production from T225 flasks to a packed bed, fed-batch bioreactor permitted an 1100-fold increment in total vector production without a decrease in specific vector yield (pfu/cell). The fed-batch bioreactor system afforded a rHSV-rep2/cap2 vector recovery of 2.8 x 10(12) pfu. The recovered vector was concentrated by tangential flow filtration (TFF), permitting vector stocks to be formulated at greater than 1.5 x 10(9) pfu/mL.     

Control of expression of the herpes simplex virus-induced deoxypyrimidine triphosphatase in cells infected with mutants of herpes simplex virus types 1 and 2 and intertypic recombinants.

Infection of cells with herpes simplex virus type 1 (HSV-1) induces high levels of deoxypyrimidine triphosphatase. The majority of the enzyme activity is found in infected cell nuclei. A similar activity is induced by HSV type 2 (HSV-2) which, in contrast to the HSV-1 enzyme, fractionates to more than 99% in the soluble cytoplasmic extract. Of a series of temperature-sensitive mutants of HSV-1 studied, only the immediate-early mutants in complementation group 1-2 (strain 17 mutants tsD and tsK and strain KOS mutant tsB2) induced reduced levels of triphosphatase at nonpermissive temperature. Of a series of temperature-sensitive mutants of HSV-2 strain HG52, ts9 and ts13 failed to induce wild-type levels of the enzyme at nonpermissive temperature; ts9 was the most defective mutant with regard to triphosphatase expression of both herpes simplex virus serotypes. After shift-up from permissive to nonpermissive temperature, triphosphatase activity in cells infected with ts9 decreased rapidly, whereas all other mutants continued to exhibit enzyme levels comparable with controls kept at the permissive temperature. The type 1-specific nuclear expression of the triphosphatase was mapped physically by the use of HSV-1 x HSV-2 intertypic recombinants, based on enzyme levels different by more than two orders of magnitude found in nuclei of HSV-1- and HSV-2-infected cells. The locus for the type-specific expression maps between 0.67 and 0.68 fractional length on the HSV genome.     

Incorporation of CD4 into virions by a recombinant herpes simplex virus.

Two herpes simplex virus type 1 (HSV-1) recombinants were constructed by inserting the human CD4 gene into the HSV-1 genome between the gC promoter and the gC structural gene. These viruses, designated K delta T/CD4 and K082/CD4, synthesized a significant quantity of CD4. CD4 was expressed on the surface of infected cells at levels substantially higher than on the surface of HUT78 cells, a CD4+ cell line. Most significantly, a small but detectable quantity of CD4 was incorporated into virions produced by the recombinant viruses. This was demonstrated both by immunoprecipitation of CD4 from purified virions and by neutralization of the recombinant virions by OKT4 and complement. These results suggest that specific virion incorporation signals are not strictly required for inclusion of glycoproteins into HSV-1 virions. It may be possible to utilize this ability to alter the host range or tissue specificity of HSV-1.     

Physical mapping of paar mutations of herpes simplex virus type 1 and type 2 by intertypic marker rescue.

Mutations (paar) in herpes simplex virus (HSV) which confer resistance to phosphonoacetic acid involve genes associated with virus-induced DNA polymerase activity. Two mutants of HSV (HSV-1 tsH and HSV-2 ts6) produce a thermolabile DNA polymerase activity. In this study, the ts lesions present in these mutants and those present in two independent phosphonoacetic acid-resistant mutants of HSV-1 and HSV-2 (paar-1 and paar-2) have been physically mapped by restriction endonuclease analysis of recombinants produced between HSV-1 and HSV-2 by intertypic marker rescue. All four mutations mapped within a 3.3-kilobase pair region around map unit 40. The accuracy of the method is reflected by the mapping results for tsH and paar-2, which were found to lie in the same 1.3-kilobase pair region. paar-1 was found to lie to the right of ts6. Virus-induced DNA polymerase is thought to have a molecular weight of 150,000, necessitating a gene with a coding capacity of 4.6 kilobase pairs. The four mutations mapped in this study all lie within a region smaller than this, but the results do not yet prove that all four lesions reside in this or any single gene.     

Genetic variability of herpes simplex virus: development of a pathogenic variant during passaging of a nonpathogenic herpes simplex virus type 1 virus strain in mouse brain.

Herpes simplex virus type 1 ANG (HSV-1 ANG) is originally nonpathogenic for inbred mice upon intraperitoneal intravenous, or intravaginal inoculation. In contrast, mice died of encephalitis within 4 to 5 days after intracerebral inoculation with this strain. HSV-1 ANG was serially passaged in mouse brains. In two independent series, peripherally pathogenic virus variants had developed and accumulated in the virus progeny after 12 to 15 intracerebral passages. In mixed infections both nonpathogenic and pathogenic viruses replicated at the primary site of infection and spread to various organs. However, only the pathogenic phenotype could be recovered from the spinal cord and the brain. Comparison of the restriction enzyme cleavage patterns of pathogenic ANG and nonpathogenic ANG virus DNAs revealed distinct alterations in the S-segment (US) sequences bounded by coordinates 0.953 and 0.958 in the prototype orientation and by coordinates 0.862 to 0.867 in the IS orientation of the viral genome. However, it is not known whether these alterations are physiologically relevant to the observed changes in pathogenicity. When coinjected intraperitoneally at 50 to 100-fold excess, the nonpathogenic HSV-1 ANG protected mice against its own pathogenic variant as well as against other pathogenic HSV-1 strains. Pathogenic HSV-1 ANG proved to be genetically and phenotypically stable for at least 25 serial passages in tissue culture at either high or low multiplicity of infection.     

Neuron-specific restriction of a herpes simplex virus recombinant maps to the UL5 gene.

We have previously shown that, when compared with either parent, a herpes simplex virus type 1/herpes simplex virus type 2 intertypic recombinant (R13-1) is attenuated by 10,000-fold with respect to neurovirulence in mice. Despite this, after intracranial inoculation, R13-1 replicated to titers of 10(5) PFU per brain. We present evidence that the restriction is specific for replication in neurons and have taken a three-step approach in determining the basis of the attenuation by (i) characterizing cellular tropism of the virus in both central and peripheral nervous systems, (ii) defining where in the viral replication cycle the restriction is manifest, and (iii) identifying the genetic basis of the restriction through marker rescue analysis. Following inoculation into the animal, R13-1 viral antigens predominate in nonneuronal cells, and the block to replication in neurons was found to be beyond the level of adsorption and penetration. Despite the restricted replication within neurons, the virus established a latent infection in spinal ganglia and could be reactivated by in vitro cocultivation of the ganglia. In studies carried out in cell culture, R13-1 was found to replicate normally in mouse embryo fibroblasts and primary mouse glial cells but was restricted by 1,000-fold in primary mouse neurons and PC12 cells. R13-1 appeared to produce normal levels of early RNA in these cells, but production of DNA and late RNA was less than that of the wild type. Marker rescue analysis localized the fragment responsible for restoring neurovirulence to UL5, a component of the origin-binding complex implicated in replication of the viral genome. Our results with this virus, with a cell-specific restriction, suggest that a neuron-specific component is involved in viral replication.     

Characterization of a recombinant herpes simplex virus which expresses a glycoprotein D lacking asparagine-linked oligosaccharides.

Glycoprotein D (gD) is an envelope component of herpes simplex virus essential for virus penetration. gD contains three sites for addition of asparagine-linked carbohydrates (N-CHO), all of which are utilized. Previously, we characterized mutant forms of herpes simplex virus type 1 gD (gD-1) lacking one or all three N-CHO addition sites. All of the mutants complemented the infectivity of a gD-minus virus, F-gD beta, to the same extent as wild-type gD. Here, we show that recombinant viruses containing mutations in the gD-1 gene which eliminate the three N-CHO signals are viable. Two such viruses, called F-gD(QAA)-1 and F-gD(QAA)-2, were independently isolated, and the three mutations in the gD gene in one of these viruses were verified by DNA sequencing. We also verified that the gD produced in cells infected by these viruses is devoid of N-CHO. Plaques formed by both mutants developed more slowly than those of the wild-type control virus, F-gD(WT), and were approximately one-half the size of the wild-type. One mutant, F-gD(QAA)-2, was selected for further study. The QAA mutant and wild-type gD proteins extracted from infected cells differed in structure, as determined by the binding of monoclonal antibodies to discontinuous epitopes. However, flow cytometry analysis showed that the amount and structure of gD found on infected cell surfaces was unaffected by the presence or absence of N-CHO. Other properties of F-gD(QAA)-2 were quite similar to those of F-gD(WT). These included (i) the kinetics of virus production as well as the intracellular and extracellular virus titers; (ii) the rate of virus entry into uninfected cells; (iii) the levels of gB, gC, gE, gH, and gI expressed by infected cells; and (iv) the turnover time of gD. Thus, the absence of N-CHO from gD-1 has some effect on its structure but very little effect on its function in virus infection in cell culture.     

Genetic determinants of dengue type 4 virus neurovirulence for mice.

Mouse-adapted dengue type 4 virus (DEN4) strain H241 is highly neurovirulent for mice, whereas its non-mouse-adapted parent is rarely neurovirulent. The genetic basis for the neurovirulence of the mouse-adapted mutant was studied by comparing intratypic chimeric viruses that contained the three structural protein genes from the parental virus or the neurovirulent mutant in the background sequence of nonneurovirulent DEN4 strain 814669. The chimera that contained the three structural protein genes from mouse neurovirulent DEN4 strain H241 proved to be highly neurovirulent in mice, whereas the chimera that contained the corresponding genes from its non-mouse-adapted parent was not neurovirulent. This finding indicates that most of the genetic loci for the neurovirulence of the DEN4 mutant lie within the structural protein genes. A comparison of the amino acid sequences of the parent and its mouse neurovirulent mutant proteins revealed that there were only five amino acid differences in the structural protein region, and three of these were located in the envelope (E) glycoprotein. Analysis of chimeras which contained one or two of the variant amino acids of the mutant E sequence substituting for the corresponding sequence of the parental virus identified two of these amino acid changes as important determinants of mouse neurovirulence. First, the single substitution of Ile for Thr-155 which ablated one of the two conserved glycosylation sites in parental E yielded a virus that was almost as neurovirulent as the mouse-adapted mutant. Thus, the loss of an E glycosylation site appears to play a role in DEN4 neurovirulence. Second, the substitution of Leu for Phe-401 also yielded a neurovirulent virus, but it was less neurovirulent than the glycosylation mutant. These findings indicate that at least two of the genetic loci responsible for DEN4 mouse neurovirulence map within the structural protein genes.     

Vaccine protection against simian immunodeficiency virus by recombinant strains of herpes simplex virus

An effective vaccine for AIDS may require development of novel vectors capable of eliciting long-lasting immune responses. Here we report the development and use of replication-competent and replication-defective strains of recombinant herpes simplex virus (HSV) that express envelope and Nef antigens of simian immunodeficiency virus (SIV). The HSV recombinants induced antienvelope antibody responses that persisted at relatively stable levels for months after the last administration. Two of seven rhesus monkeys vaccinated with recombinant HSV were solidly protected, and another showed a sustained reduction in viral load following rectal challenge with pathogenic SIVmac239 at 22 weeks following the last vaccine administration. HSV vectors thus show great promise for being able to elicit persistent immune responses and to provide durable protection against AIDS.     

Identification of a common antigen of herpes simplex virus bovine herpes mammillitis virus, and B virus.

In immunoelectrophoretic analyses one common antigen was demonstrated in antigen preparations from herpes simplex virus types 1- and 2- (HSV-1 and HSV-2), bovine herpes mammillitis (BHM) virus-, and B virus-infected cells solubilized by Triton X-100. The antigen was also demonstrated in solubilized purified HSV-1 and BHM virus. The common antigen was identified as antigen 11 of HSV-1 or HSV-2. Differences were found in the polypeptide composition of the related antigens when isolated from the four different herpesviruses, but a glycopolypeptide with a molecular weight of 125,000 was present in each of the four different antigen preparations, indicating that this polypeptide carried the common antigenic determinants.     

Genetic analysis of type-specific antigenic determinants of herpes simplex virus glycoprotein C.

Herpes simplex virus type 1 (HSV-1) glycoprotein C (gC-1) elicits a largely serotype-specific immune response directed against previously described determinants designated antigenic sites I and II. To more precisely define these two immunodominant antigenic regions of gC-1 and to determine whether the homologous HSV-2 glycoprotein (gC-2) has similarly situated antigenic determinants, viral recombinants containing gC chimeric genes which join site I and site II of the two serotypes were constructed. The antigenic structure of the hybrid proteins encoded by these chimeric genes was studied by using gC-1- and gC-2-specific monoclonal antibodies (MAbs) in radioimmunoprecipitation, neutralization, and flow cytometry assays. The results of these analyses showed that the reactivity patterns of the MAbs were consistent among the three assays, and on this basis, they could be categorized as recognizing type-specific epitopes within the C-terminal or N-terminal half of gC-1 or gC-2. All MAbs were able to bind to only one or the other of the two hybrid proteins, demonstrating that gC-2, like gC-1, contains at least two antigenic sites located in the two halves of the molecule and that the structures of the antigenic sites in both molecules are independent and rely on limited type-specific regions of the molecule to maintain epitope structure. To fine map amino acid residues which are recognized by site I type-specific MAbs, point mutations were introduced into site I of the gC-1 or gC-2 gene, which resulted in recombinant mutant glycoproteins containing one or several residues from the heterotypic serotype in an otherwise homotypic site I background. The recognition patterns of the MAbs for these mutant molecules demonstrated that (i) single amino acids are responsible for the type-specific nature of individual epitopes and (ii) epitopes are localized to regions of the molecule which contain both shared and unshared amino acids. Taken together, the data described herein established the existence of at least two distinct and structurally independent antigenic sites in gC-1 and gC-2 and identified subtle amino acid sequence differences which contribute to type specificity in antigenic site I of gC.