HSV molecular biology: general aspects of herpes simplex virus molecular biology.

Comparison of the herpes simplex virus type 1 (HSV-1) DNA sequence with that of other alpha, beta and gamma-herpesviruses, allied with molecular genetic studies have greatly increased understanding of the HSV genome and the functions encoded by individual virus genes and has facilitated the development of rational antiviral strategies. Here we review the coding content of the HSV-1 genome and identify: genes encoding structural components of the capsid, tegument or envelope; genes whose products are essential for growth in tissue culture; and genes that are conserved between members of the alpha, beta and gamma-herpesvirinae. The HSV lifecycle and the main regulation cascade is discussed and genes that present targets for antiviral intervention identified. The protein content of the infectious virion particle is reviewed and compared with that of two additional non-infectious HSV-related particles species (L-particles and pre-DNA replication particles (PREPs)). The potential of HSV-1 L particles and PREP particles as DNA-free HSV-1 vaccine candidates and the desirability of deleting specific gene products from live HSV vaccines is discussed.

The effect of herpes simplex virus type 1 L-particles on virus entry, replication, and the infectivity of naked herpesvirus DNA.

Herpes simplex virus type 1(HSV-1) L-particles are known to be composed mainly of envelope and tegument proteins, to lack the nucleocapsid, and to be noninfectious. Thus L-particles represent interesting vaccine candidates. L-particles at > 1000/cell interfered with HSV-1 virion adsorption and penetration While L-particles did not affect HSV-1 growth kinetics in resting or nonresting BHK cultures infected with purified virions, treatment with L-particles before, or after, transfection with HSV-1 DNA resulted in a progressive increase in plaque numbers (five- to sixfold at 1000 L-particles/cell). Transfection assays using HSV-1 ts mutant DNA (ts 1201) revealed that enhancement was due to induction of otherwise nonreplicating genomes. The enhancement obtained with L-particles produced by WT HSV-1 or by mutants that are either deleted, or defective, in certain gene products was compared. Most important were the Vmw110 (ICP0) and Vmw65 (alpha-TIF) proteins, but VP11/12, VP13/14, and vhs also have a role. The L-particle-associated Vmw175 (ICP 4) protein did not appear be involved. The effect of homologous and heterologous combinations of pseudorabies virus, equineherpesvirus-1, and HSV-1 DNA's and L-particles was investigated in transfection assays. The L-particles of each virus, to varying extent, enhanced the plaquing efficiency of their own DNA but were also effective in heterologous combinations.

Suppression of amber nonsense mutations of herpes simplex virus type 1 in a tissue culture system.

We have investigated the ability of monkey kidney cell lines (SupD3 and SupD12) inducibly expressing an amber suppressor tRNA(ser) to suppress amber nonsense mutations in three genes of herpes simplex virus type 1 (HSV-1). HSV-1 mutant TK4, which contains a nonsense mutation in the non-essential viral thymidine kinase (TK) gene, synthesized a full-length TK polypeptide at about 30% of the wild-type (wt) level in induced SupD3 cells but not in the parental non-suppressor (Sup0) cells. Using complementing cells, we constructed HSV-1 mutants carrying nonsense mutations in an essential gene, UL8, encoding a protein essential for viral DNA replication (ambUL8) or in a partially dispensable gene, UL12, encoding alkaline nuclease (ambUL12). The growth of the mutants in Vero or Sup0 cells was either totally (ambUL8) or severely (ambUL12) impaired, whereas in cells expressing suppressor tRNA the mutants produced infectious virus. However, the yields were much lower than obtained with wt HSV-1. In Vero or Sup0 cells the mutants ambUL8 and ambUL12 failed to synthesize full-length UL8 and UL12 protein products, respectively. Western immunoblotting showed that the virus ambUL12 produced full-length UL12 protein in SupD12 cells which yielded a level of 25.9% of the alkaline nuclease activity of the wt HSV-1 control. Our results show that the levels of suppression of the nonsense mutations in ambUL8 and ambUL12 are insufficient to allow their continuing propagation in the available Sup+ cells. Possible reasons are discussed.

PREPs: herpes simplex virus type 1-specific particles produced by infected cells when viral DNA replication is blocked.

Herpes simplex virus (HSV)-infected cells produce not only infectious nucleocapsid-containing virions but also virion-related noninfectious light particles (L-particles) composed of the envelope and tegument components of the virus particle (J. F. Szilágyi and C. Cunningham, J. Gen. Virol. 62:661-668, 1991). We show that BHK and MeWO cells infected either with wild-type (WT) HSV type 1 (HSV-1) in the presence of viral DNA replication inhibitors (cytosine-beta-D-arabinofuranoside, phosphonoacetic acid, and acycloguanosine) or with a viral DNA replication-defective mutant of HSV-1 (ambUL8) synthesize a new type of virus-related particle that is morphologically similar to an L-particle but differs in its relative protein composition. These novel particles we term pre-viral DNA replication enveloped particles (PREPs). The numbers of PREPs released into the culture medium were of the same order as those of L-particles from control cultures. The particle/PFU ratios of different PREP stocks ranged from 6 x 10(5) to 3.8 x 10(8), compared with ratios of 3 x 10(3) to 1 x 10(4) for WT L-particle stocks. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western immunoblot analyses revealed that true late proteins, such as 273K (VP1-2), 82/81K (VP13/14), and gC (VP8), were greatly reduced or absent in PREPs and that gD (VP17) and 40K proteins were also underrepresented. In contrast, the amounts of proteins 175K (VP4; IE3), 92/91K (VP11/12), 38K (VP22), and gE (with BHK cells) were increased. The actual protein composition of PREPs showed some cell line-dependent differences, particularly in the amount of gE. PREPs were biologically competent and delivered functional Vmw65 (VP16; alpha TIF) to target cells, but the efficiency of complementation of the HSV-1 (strain 17) mutant in1814 was 10 to 30% of that of WT L-particles.

The N-terminal 22 amino acids encoded by the gene specifying the major secreted protein of vaccinia virus, strain Lister, can function as a signal sequence to direct the export of a foreign protein.

Cells infected with vaccinia virus strain Lister secrete a polypeptide of approximate molecular weight 35,000 (35K) into the medium. Previous studies identified a cleavable, hydrophobic region of 17 amino acids in the 35K protein which could potentially function as a signal peptide to target the protein to the secretory pathway. Here we report the use of the expression-secretion signals derived from the 35K gene to direct export and secretion of a foreign protein. Vaccinia virus recombinants carrying the bacterial chloramphenicol acetyl transferase gene (cat) immediately downstream from the promoter and the N-terminal coding sequences of the 35K gene were constructed. Our studies show that the N-terminal 22 or 42 amino acids of the 35K protein direct efficient secretion of the CAT protein. However, due to a cryptic glycosylation site within CAT, glycosylated protein was secreted, which reduced enzymatic activity. Activity was restored in the presence of tunicamycin. Removal of the glycosylation site by site-directed mutagenesis abolished glycosylation with no effect on secretion, although CAT activity was again reduced, possibly due to an effect on the active site. The results presented here demonstrate the feasibility of using the promoter and the signal sequence of the 35K gene to generate recombinant viruses for overexpression and secretion of foreign proteins.

Analysis of intrastrain recombination in herpes simplex virus type 1 strain 17 and herpes simplex virus type 2 strain HG52 using restriction endonuclease sites as unselected markers and temperature-sensitive lesions as selected markers.

The viral and host factors involved in herpes simplex virus (HSV) recombination are little understood. To identify features of the process, recombination in HSV-1 and HSV-2 has been studied by analysing the segregation of unselected markers in the form of restriction endonuclease (RE) sites. By confining parental interactions to only one strain of virus of each serotype, restrictions imposed by non-homology are overcome and differential growth phenotypes can be discounted. The analysis of unselected and selected recombinants using RE sites in conjunction with temperature-sensitive mutations is consistent with (i) HSV being highly recombinogenic, (ii) parental and progeny molecules taking part in the process, (iii) the four genomic isomers participating in recombination, (iv) genome alignment being part of the recombination process and (v) cellular factors in conjunction with genome homology influencing the efficiency of recombination.

The effect of cicloxolone sodium on the replication of vesicular stomatitis virus in BSC-1 cells.

The effect of cicloxolone sodium (CCX) on the replication of vesicular stomatitis virus (VSV) was investigated. The drug was active during all stages of the virus replication cycle, indicating that it does not operate by the specific inhibition of any single essential virus gene product. The drug reduced the number of VSV particles assembled and released by 100- to 1000-fold. Infectious virus yield was reduced 1000- to 10000-fold, giving a 10-fold or greater increase in the particle/p.f.u. ratio. The reduced number of virus particles produced in the presence of CCX results from two superimposed effects: suppression of VSV secondary transcription and viral protein synthesis, and perturbation of virion assembly. The inhibition of VSV assembly is due to impairment of a Golgi apparatus function related to transport of VSV glycoprotein G to the cell surface, and is characterized by accumulation of viral G and M proteins within the cell. Incubation of VSV-infected cells in the presence of two glycosylation inhibitors, tunicamycin and monensin, similarly leads to intracellular accumulation of G and M proteins, suggesting a common mechanism of action affecting VSV virion assembly. The differential effect of CCX concentration on intracellular levels of the L, N and NS proteins was analysed. CCX also possesses a virucidal effect on mature infectious VSV particles in suspension, 300 microM reducing the VSV titre about 10-fold in 24 h at 4 degrees C or 37 degrees C. The mode of antiviral activity against VSV is compared with that against herpes simplex virus.

The effect of cicloxolone sodium on the replication in cultured cells of adenovirus type 5, reovirus type 3, poliovirus type 1, two bunyaviruses and Semliki Forest virus.

The effect of cicloxolone sodium (CCX) on the replication of typical representatives of different virus families [adenovirus type 5 (Ad-5), reovirus type 3 (Reo-3), Bunyamwera and Germiston viruses, poliovirus type 1 (Polio-1) and Semliki Forest virus (SFV)] in tissue culture was investigated. The Golgi apparatus inhibitor monensin (Mon) and CCX were shown to have analogous effects on some aspects of virus replication. Although the Mon-like effect of CCX played no role in the antiviral activity against Ad-5, Reo-3 or Polio-1, it could entirely account for the antiviral activity against the Bunyamwera and Germiston viruses, for which inhibition of glycoprotein processing was responsible for the antiviral activity. In the case of SFV, the Mon-like activity of CCX caused cytoplasmic assembly of fully infectious SFV within vacuoles and thus impaired virus release without altering total infectious virus yield. Fewer Ad-5 and Reo-3 progeny were produced in the presence of the drug. CCX had a dose-dependent biphasic effect on the particle:p.f.u. ratio of the Reo-3 yield. At low CCX concentration (less than 50 microM) the virus yield contained poor quality, non-infectious virus, but at higher CCX concentration (greater than or equal to 100 microM) low quality virus could no longer be successfully assembled. We conclude that the antiviral effect can be manifested in three ways: (i) by a reduction in the virus particle yield produced; (ii) by a loss of quality (relative infectivity); (iii) by a virucidal effect of the drug. We have previously defined three CCX sensitivity classes. Mechanisms (i), (ii) and (iii) operate against viruses belonging to class CCXs-1 [herpes simplex virus (HSV) type 1, HSV-2 and vesicular stomatitis virus], but essentially only (i) and (ii) affect Reo-3 (CCXs-2), whereas (i) and possibly (iii) affect Ad-5 (CCXs-2). In the case of SFV (CCXs-3) none of these mechanisms operate, but relocation of assembled virus is found.

Advantages of branched peptides in serodiagnosis. Detection of HIV-specific antibodies and the use of glycine spacers to increase sensitivity.

The reactivities of antibodies with branched and monomeric peptides were compared in ELISA assays. We found that lower amounts of antibodies could be detected with branched peptides than with monomeric peptides. This was observed with a monoclonal antibody and with antibodies in the sera of various HIV-positive individuals. To investigate the physical aspects of branched peptides important for the observed increase in sensitivity, glycine spacers of different lengths were introduced between the branched lysine core and the epitope reacting with the monoclonal antibody. The effect of the number of glycine residues, both on the sensitivity of antibody detection and on the amount of branched peptide needed to produce a given signal, was studied and the optimum was found at 4-5 residues. We discuss the basis for these findings and conclude that the routine use of branched peptides for serodiagnosis will give both greater sensitivity and appreciable cost savings.

In vivo and in vitro reactivation impairment of a herpes simplex virus type 1 latency-associated transcript variant in a rabbit eye model.

Many recent studies of latent herpes simplex virus type 1 (HSV-1) infections within the nervous system have focused on the diploid genes encoding the latency-associated transcripts (LATs). The impaired explant reactivation of LAT variants from mouse trigeminal ganglia has implicated the LATs in the efficiency or speed of the reactivation process (D. A. Leib, C. L. Bogard, M. Kosz-Vnenchak, K. A. Hicks, D. M. Coen, D. M. Knipe, and P. A. Schaffer, J. Virol. 63:2893-2900, 1989; I. Steiner, J. G. Spivack, R. P. Lirette, S. M. Brown, A. R. MacLean, J. H. Subak-Sharpe, and N. W. Fraser, EMBO J. 8:505-511, 1989). However, it is not known how closely explant reactivation mimics the reactivation process in vivo. In the current study, a LAT variant (1704), parental strain (17+), and rescuant (1704R) were compared in vivo for reactivation of latent infection by iontophoresis in the rabbit eye model and in vitro by explant cocultivation of trigeminal ganglia from rabbits. Following iontophoresis, 17+ and 1704R reactivated in vivo from 76 and 64% of rabbits, respectively, while 1704 reactivated only from 4% (1 of 25) of the animals. In explant reactivation experiments, 17+ and 1704R reactivated from 98 and 67% of rabbit trigeminal ganglia, while 1704 reactivated from only 28% of trigeminal ganglia. The mean time required for the appearance of reactivated 1704 in explant culture, 17 days, was significantly longer than for 17+ and 1704R, 8 to 9 days. Thus, the explant reactivation kinetics in rabbit trigeminal ganglia reflect the behavior of LAT variant 1704 in vivo in the rabbit eye model. These data support the role of the LATs in the reactivation process and support the hypothesis that explant reactivation is a suitable system for analyzing the biological behavior of HSV-1 variants with defined genetic alterations in the LAT gene.

Generation of anti-peptide and anti-protein sera. Effect of peptide presentation on immunogenicity.

The technique of Fmoc chemistry has been applied successfully to the synthesis of branched peptides. The immunogenicity of branched peptides has been compared quantitatively with those of protein-conjugated and resin-linked peptides. Six different peptide sequences were used to immunise rabbits and both antipeptide and anti-protein titres were determined for each serum. The data show that the titres of sera from rabbits immunised with branched peptides were higher than those of rabbits immunised with protein-conjugated peptides which in turn were higher than those immunised with resin-linked peptides. The effect was demonstrated with two strains of rabbits.

The difference in sensitivity to cicloxolone sodium between herpes simplex virus types 1 and 2 maps to the locations of genes UL22 (gH) and UL44 (gC).

Cicloxolone sodium (CCX) is a broad spectrum antiviral agent which has a largely non-specific and complex mode of antiviral action. However, the experimental finding that herpes simplex virus type 2 (HSV-2) (strain HG52) is consistently more sensitive to inhibition by CCX than HSV-1 (117 syn+) additionally implies the specific involvement of HSV genes. HSV-1/HSV-2 intertypic recombinants have been utilized to investigate this genetic difference by comparing their CCX ED50 concentrations. No short stretch of HSV-2 DNA was associated with its greater sensitivity to CCX, implying that two or more non-contiguously located HSV genes are involved. Correlating CCX sensitivity with recombinant virus genome structures allowed separate evaluation of the gene regions encoding glycoproteins gB, gC, gD, gE, gG, gH and gI and this suggests that the gene locations encoding gH and gC determine the CCX sensitivity difference. The selective inhibitor of Golgi apparatus glycosylation, monensin, gave results analogous to those obtained with CCX.

DNA sequence of the gene encoding a major secreted protein of vaccinia virus, strain Lister.

Infection of tissue culture cells with vaccinia virus results in the specific secretion of several polypeptides into the medium. Previous studies identified a protein of approximate Mr 35,000 (35K) which was secreted in large amounts at both early and late times after infection with the Evans strain. We now show that a related protein is secreted by the Lister strain but not by WR, Wyeth nor Tian Tan. The gene encoding the Lister strain 35K protein was mapped within the inverted terminal repeats of the genome. The DNA sequence of this region showed that the ends of this gene are very similar to previously published sequences flanking a gene of WR which encodes a protein of approximate Mr 7,500 (7.5K). Our results suggest that the 7.5K polypeptide of WR may have arisen as a result of a deletion event and is a truncated form of the 35K Lister protein. Site-directed mutagenesis demonstrated that the 35K secreted protein encoded by Lister is not essential for growth in tissue culture.

A herpes simplex virus type 1 variant, deleted in the promoter region of the latency-associated transcripts, does not produce any detectable minor RNA species during latency in the mouse trigeminal ganglion.

In peripheral sensory ganglia latently infected with herpes simplex virus type 1 (HSV-1) transcription is restricted. A set of viral latency-associated transcripts, the LATs, have been characterized by Northern blotting and in situ hybridization. These transcripts have previously been mapped to a 3 kb region of the viral genome within the repeat long region. However, transcription from adjacent regions of the genome can be detected by in situ hybridization, which cannot be detected by Northern blotting. These RNAs are termed minor LATs or m-LAT. In this study we show that in ganglia latently infected with the HSV-1 variant 1704, which is deleted in one complete copy of the LAT gene and in the promoter and 5' portion of the other copy, m-LATs are not detected by in situ hybridization. Furthermore, the levels of DNA in nervous system tissue latently infected with the parental and the 1704 variant virus are similar. Thus we propose that the sequence elements necessary for initiating transcription or stabilizing m-LATs are within the region deleted in variant 1704 that codes for the promoter and the 5' end of the LATs.

Herpes simplex virus type 1 latency-associated transcripts are evidently not essential for latent infection.

The herpes simplex virus type 1 (HSV-1) transcripts that can be detected during latent infection by Northern blot analysis in human and experimental animal sensory ganglia are encoded by diploid genes. To investigate their role in latent infection we studied HSV-1 variant 1704, which has deleted most of the IRL copy of the coding region of these RNAs and has a 1.2-kb deletion that is immediately upstream of the coding region of the TRL copy. During primary infection, 1704 replicated in trigeminal ganglia with kinetics similar to the parent virus (17+) and established latent infection. However, while explant reactivation of latent HSV-1 from trigeminal ganglia was detected in 100% of 17+ infected mice within 7 days, the reactivation of 1704 was significantly delayed, and 31 days elapsed before eight out of nine mice became virus positive. The recognized HSV-1 latency-associated RNAs were not detected during the latent state of 1704 by Northern blot analysis or in situ hybridization, which implies that the 1.2-kb deletion may contain the promoter or other important regulatory elements. The data indicate that detectable levels of these latency-associated transcripts are not required for viral replication, establishment, or maintenance (greater than 6 weeks) of HSV-1 latency in trigeminal ganglia, but suggest a role in reactivation.

Ribonucleotide reductase encoded by herpes simplex virus is a determinant of the pathogenicity of the virus in mice and a valid antiviral target.

The role of the herpes simplex virus (HSV)-encoded ribonucleotide reductase (RR) in the pathogenicity of the virus has been examined by use of mutants with lesions in either the large or small subunit of the enzyme. The virulence of the mutants in mice was reduced by about 10(6)-fold when compared with that of the parental virus (HSV type 1 strain 17), while the virulence of a revertant of one of the mutants was restored to within about 100-fold of that of the parent virus. These experiments demonstrate that activity of the HSV RR is essential for virus pathogenicity in mice and suggests that the enzyme is a valid target for specific antiviral compounds.

Identification and characterization of a varicella-zoster virus DNA-binding protein by using antisera directed against a predicted synthetic oligopeptide.

We have identified, in varicella-zoster virus (VZV)-infected cells, the product of the gene predicted to code for the VZV analog of the herpes simplex virus major DNA-binding protein. The open reading frame of the VZV gene has the potential to code for a protein with a predicted molecular weight of 132,000 (a 132K protein). To detect the protein, a 12-amino-acid oligopeptide corresponding to the carboxyl terminus of the putative open reading frame was synthesized and used to prepare antisera in rabbits. The resulting antibodies reacted specifically in Western immunoblot analysis and immunoprecipitation with a single 130K polypeptide found in VZV-infected cells. The specific reactivity of the antisera with the 130K polypeptide was inhibited by the addition of synthetic peptide. Immunofluorescence studies with the antisera as probe for the 130K polypeptide suggested that this peptide is located predominantly within the nuclei of infected cells. Analysis of proteins that bind to single-stranded DNA immobilized on cellulose matrices indicated that 30 to 50% of the 130K polypeptide is capable of interacting with single-stranded DNA and that this interaction is overcome with 0.5 M NaCl. Thus, we have prepared a specific polyclonal antiserum that identifies a VZV DNA-binding protein whose properties are similar to those of the herpes simplex virus ICP8 (Vmw130) DNA-binding protein.

Herpes simplex virus type 2 establishes latency in the mouse footpad.

BALB/c mice were infected with herpes simplex virus type 2 by inoculation into the footpad. Three months or more after infection, and in the total absence of any evidence of illness, latent virus could be reactivated from both ganglia and footpad tissue but only after explantation and culturing for a week or more. Virus recovery from the footpad could not be prevented by treatment of the infected mice with acycloguanosine for up to 6 months in their drinking water at 40 mg/kg, nor by femoral and sciatic nerve section, nor by combining both treatments.

The effect of triterpenoid compounds on uninfected and herpes simplex virus-infected cells in culture. III. Ultrastructural study of virion maturation.

Electron microscope studies been made of mock-infected or herpes simplex virus (HSV) type 1 (strain 17)- or HSV-2(strain HG52)-infected Flow 2002 cells grown in the absence or presence of various concentrations of cicloxolone sodium (CCX). Fifty non-serial, thin sections of mock-infected or HSV-infected cells, which contained a portion of the nucleus, were examined by transmission electron microscopy. With increasing drug concentration, counts of capsid structures both in the nucleus and cytoplasm showed a decrease in the number of virions per cell section, an increase in the ratio of nuclear to cytoplasmic virus, a relative reduction in the percentage of cytoplasmic enveloped virus particles, but no effect on the ratio of empty to core-containing capsid structures. The high particle to p.f.u. ratio induced by CCX treatment is thus not explained through failure to assemble morphologically mature core-containing capsids. In part it can be explained by non-envelopment, but in addition, specific effects on other virion proteins (tegument and envelope) must be involved. The extracellular virus particle yield was unaffected, indicating that CCX treatment enhances the egress of HSV. In the presence of CCX the encapsidation of HSV DNA into DNase-resistant structures was unaffected.

The antiviral activity against herpes simplex virus of the triterpenoid compounds carbenoxolone sodium and cicloxolone sodium.

Dose-response experiments show that the presence of 300 microM cicloxolone sodium (CCX) or 500 microM carbenoxolone sodium (CBX) during the HSV replication cycle reduced the infectious virus yield by 10,000- to 100,000-fold: CCX is the more potent anti-herpes agent. HSV-2 replication was consistently more severely restricted by either drug than was that of HSV-1. The ED50 values obtained for either drug against HSV-1 or HSV-2 correlate well with data from dose-response curves. CCX, and to a lesser extent CBX, can be cytotoxic but the degree of cytotoxicity varies between cell lines and is also affected by the physiological state of the cells. Triterpenoid drugs exhibit some activity against virus particles in suspension but the effect is small and contributes little to the overall antiviral effect. The drugs appear to be active throughout the replication cycle. In contrast to all other anti-herpesvirus agents in clinical use the triterpenoid compounds do not appear to act directly to block virus DNA synthesis. HSV mutants resistant to ACG and PAA, or lacking a thymidine kinase gene, appear as sensitive as wild-type virus to CCX inhibition. HSV growth in the presence of the drugs resulted in a lower number of assembled virus particles but reduced to a much greater extent the infectious virus yield: thus the progeny virus quality is greatly diminished. Thermolability of progeny virus correlated well with this diminution of quality in increasing CCX concentration. SDS PAGE analysis of the structural proteins of virus particles made in cells treated with 300 microM CCX revealed numerous differences in the relative intensities of protein bands, which is in keeping with the changed quality of the drug-produced virus. SDS PAGE analysis of the polypeptides induced in drug treated infected cells revealed two effects; some polypeptides were synthesised in reduced amounts and the nuclear/cytoplasmic distribution of certain proteins was affected. Post-translational processing by glycosylation and sulphation of both cellular and HSV induced proteins was strongly inhibited by the triterpenoid drugs, while phosphorylation of only a few polypeptides appeared to be affected.