Characterization and susceptibility to antiviral agents of herpes simplex virus type 1 containing a unique thymidine kinase gene with an amber codon between the first and the second initiation codons

Summary A herpes simplex virus type 1 (HSV-1) containing a thymidine (TK) gene with an amber mutation at the 8th position counted from the first AUG codon was isolated from a child with acute gingivostomatitis. The virus was predicted to express a mutant viral translated from the 2nd AUG codon at the 46th amino acid position and consisting of 331 amino acids. The virus was as sensitive to acyclovir (ACV), 5-bromovinyl-2′-deoxyuridine (BVdU), 1-β-D-arabinofuranosyl-(E)-5-(2-bromovinyl)uracil (BVaraU), and 1-β-D-arabinofuranosylthymine (araT) as a wild-type HSV-1. The mutant TK showed the same level of TK activity as the wild-type TK at reaction temperatures of 34 °C, 37 °C and 39 °C. ACV, BVdU, BVaraU, and araT inhibited the replication of the TK-deficient and drug-resistant HSV-1 and HSV-2 in 293T cells in which the mutant TK was expressed to the same extent as in cells in which intact HSV-1-TK was expressed, whereas BVdU and BVaraU inhibited the replication of these viruses less strongly in cells in which HSV-2-TK was expressed. It can be concluded that the mutant HSV-1 exists in nature as a variant and possesses the necessary phosphorylation activities to form ACV-monophosphate from ACV, to form BVdU-diphosphate through BVdU-monophosphate from BVdU, and to form BVaraU-diphosphate through BVaraU-monophosphate from BVaraU. These results indicate that the mutant HSV-1-TK with a deletion of the first 45 amino acid residues is phenotypically the same as that of wild-type HSV-1-TK in terms of the phosphorylation activity of TK-associated anti-herpes virus drugs. Correspondence: Masayuki Saijo, Special Pathogens Laboratory, Department of Virology 1, National Institute of Infectious Diseases, 4-7-1 Gakuen, Musashimurayama, Tokyo 208-0011, Japan.     

Problems associated with the use of (E)-5-(2-bromovinyl)-2''-deoxyuridine for typing herpes simplex virus.

When 0.5 microgram of (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVdU) per ml was incorporated directly into cell culture medium inoculated with eight known positive specimens, one herpes simplex virus type 1 (HSV-1) isolate grew in the presence of BVdU and was misidentified. By plaque assay, the titers of 15 HSV-1 strains were reduced by more than 3 log10 by BVdU, and the titers of 16 HSV-2 strains were reduced by less than 2 log10. Titers of HSV-1 acyclovir-resistant strains were reduced by less than 1.5 log10, which was characteristic of HSV-2 strains. Thus, typing of HSV isolates in the presence of BVdU by plaque assay is reliable only if information regarding previous antiviral therapy is obtained.     

5-Methoxymethyldeoxyuridine-resistant mutants of herpes simplex virus type 1

5-Methoxymethyldeoxyuridine (MMdU)-resistant (MMdU^r) mutants of herpes simplex virus type 1 (HSV-1) were isolated by a single passage of the virus in MMdU at 100 μg/ml (3.7 × 10^?4M). The isolates were cloned and passed in the presence of MMdU at 5 μg/ml (0.18 × 10^?4M). All MMdU^r mutants showed considerable cross-resistance to the nucleoside analogs acycloguanosine (ACG), (E)-5-bromovinyldeoxyuridine (BVdU), and arabinosylthymine (araT), but were as sensitive as the parental strain to phosphonoacetate (PAA). One mutant, MMdU^r-20, induced significant deoxythymidine kinase (dTK) activity. Because it was only 30 times more resistant to BVdU than the parental wild-type virus, while mutants MMdU^r-2 and -12-3 were about 10,000 times more resistant, it was suspected that the mutation was in the dTK locus. All three mutants, however, showed a similar pattern of resistance to the other nucleoside analogs ACG, MMdU, and araT. These results suggest differences in the active sites for PAA and nucleoside analogs (with respect to the viral DNA polymerase), and also among the nucleoside analogs (with respect to the viral dTK).     

The equine herpesvirus type 1 (EHV-1) homolog of herpes simplex virus type 1 US9 and the nature of a major deletion within the unique short segment of the EHV-1 KyA strain genome.

The DNA sequence of the short (S) genomic component of the equine herpesvirus type 1 (EHV-1)KyA strain has been determined recently in our laboratory. Analysis of a 1353-bp BamHI/PvuII clone mapping at the unique short/terminal inverted repeat (Us/TR) junction revealed 507 bp of Us and 846 bp of TR sequences as well as an open reading frame (ORF) that is contained entirely within the Us. This ORF encodes a potential polypeptide of 219 amino acids that shows significant homology to the US9 proteins of herpes simplex virus type 1 (HSV-1), EHV-4, pseudorabies virus (PRV), and varicella zoster virus (VZV). The US9 polypeptides of the two equine herpesviruses exhibit 50% identity but are twice as large as their counterparts in HSV-1, PRV, and VZV. All five US9 proteins are enriched for serine and threonine residues and share a conserved domain of highly basic residues followed by a region of nonpolar amino acids. DNA sequence and Southern blot hybridization analyses revealed that the Us of EHV-1 KyA differs from the Us of EHV-1 KyD and AB1 in that the ORFs encoding glycoproteins I and E and a unique 10-kDa polypeptide are deleted from the KyA genome. These data demonstrate that the predicted 10-kDa protein unique to EHV-1 is nonessential for replication in vitro and that EHV-1 glycoproteins I and E, like their equivalents in HSV-1 and PRV, are also nonessential. These findings and those reported previously by this laboratory and others reveal that the Us segment of EHV-1 comprises nine ORFs, two of which, US4 and 10-kDa ORF, are unique to EHV-1. The gene order of the Us is US2, protein kinase, gG, US4, gD, gI, gE, 10 kDa, and US9.     

Antiherpes activity of some novel analogues of (E)-5-(2-bromovinyl)-2'-deoxyuridine (E-BrVUdR) in two different cell lines

In a series of 5-vinyl-2'-deoxyuridine (VUdR) analogues (5-(2-X-vinyl)-UdRs) the (E)-5-(2-bromovinyl)-UdR (E-BrVUdR) proved the most potent inhibitor of plaque formation of two herpes simplex virus type 1 (HSV-1) strains in human embryonic lung fibroblast (HELF) and African green monkey kidney (Vero) cell cultures. The (Z)-5-(2-bromovinyl)-UdR (Z-BrVUdR) isomer and the 5-(2,2-dibromovinyl)-UdR (Br2VUdR) analogue were 10-20 times less efficient, whereas the (E)-5-(2-cyanovinyl)-UdR (CNVUdR) and the (E)-5-(2-carboxyvinyl)-UdR (COOHVUdR) derivative were only marginally active (10(3)-10(4) times less than E-BrVUdR). The antiherpes potential of the 5-(2-X-vinyl)-UdRs was compared with that of 5-iodo-, 5-fluoro-, 5-formyl- and 5-ethyl-UdR (IUdR, FUdR, fUdR, EUdR) as well as of 9-(2-hydroxyethoxymethyl)guanine (acyclovir, ACV), 2'-fluoro-5-iodo-1-beta-D-arabinofuranosyl(ara)-cytosine (FIAC), 2'-fluoro-5-methylarauracil (FMAU), arabinosylthymine (araT) and (E)-5-(2-bromovinyl)- and 5-vinyl-araU (BrVaraU, VaraU). In HELF cells the following order of decreasing activity against HSV-1-77 was found: E-BrVUdR greater than greater than BrVaraU greater than VaraU greater than FIAC greater than FMAU = VUdR = = Z-BrVUdR = ACV = araT = FUdR greater than Br2VUdR greater than greater than IUdR greater than fUdR greater than EUdR greater than CNVUdR greater than COOHVUdR. The inhibition of HSV-1 replication by most of the investigated compounds was somewhat weaker in the plaque inhibition assay on Vero than on HELF cells, but, in the case of the 5-X-araU reference compounds the activity was strongly reduced in Vero cells. In HELF cells the order of decreasing potential against HSV-2 strain 42/78 (HSV-2-42/78) was: FIAC = FMAU greater than greater than araT greater than IUdR = VUdR greater than ACV = FUdR = greater than fUdR = EUdR = VaraU greater than E-BrVUdR greater than Z-BrVUdR greater than Br2VUdR greater than greater than BrVaraU; CNVUdR and COOHVUdR were nearly inactive.     

Use of chimeric nectin-1(HveC)-related receptors to demonstrate that ability to bind alphaherpesvirus gD is not necessarily sufficient for viral entry.

Human nectin-1 (HveC, Prr1), a member of the immunoglobulin superfamily and a receptor for the entry of herpes simplex viruses 1 and 2 (HSV-1, HSV-2), pseudorabies virus (PRV), and bovine herpesvirus 1 (BHV-1), binds to viral gD. For HSV-1, HSV-2, and PRV, the gD-binding region of nectin-1 has been localized to the N-terminal V-like domain. To determine whether the two C-like domains of nectin-1 influenced gD binding and entry activity, genes encoding chimeric proteins were constructed. Portions of nectin-1 were replaced with homologous regions from nectin-2 (HveB, Prr2), a related protein with ability to mediate the entry of PRV, HSV-2, and Rid mutants of HSV-1, but not HSV-1 or BHV-1. Also, one or more domains of nectin-1 were fused to the two membrane-proximal Ig domains of CD4, a protein with no herpesvirus entry or gD-binding activity. The chimeric proteins were expressed in Chinese hamster ovary cells, which normally lack alphaherpesvirus entry receptors, and detected on the cell surface by one or more anti-nectin-1 monoclonal antibodies. One chimeric protein (nectin-1 amino acids 1-124 fused to CD4) failed to bind to soluble forms of HSV-1, HSV-2, PRV, and BHV-1 gD and, as expected, also failed to mediate entry of the viruses from which these gDs were derived. The other chimeric receptors bound all forms of gD. Some mediated the entry of all the viruses tested but others mediated entry of some but not all the viruses. We conclude that binding of gD to the nectin-1 V domain is not sufficient for entry activity, that there are structural requirements for entry activity independent of gD binding, and that these requirements are different for the several alphaherpesviruses that can use nectin-1 as a receptor.     

Completely assembled virus particles detected by transmission electron microscopy in proximal and mid-axons of neurons infected with herpes simplex virus type 1, herpes simplex virus type 2 and pseudorabies virus

The morphology of alphaherpesviruses during anterograde axonal transport from the neuron cell body towards the axon terminus is controversial. Reports suggest that transport of herpes simplex virus type 1 (HSV-1) nucleocapsids and envelope proteins occurs in separate compartments and that complete virions form at varicosities or axon termini (subassembly transport model), while transport of a related alphaherpesvirus, pseudorabies virus (PRV) occurs as enveloped capsids in vesicles (assembled transport model). Transmission electron microscopy of proximal and mid-axons of primary superior cervical ganglion (SCG) neurons was used to compare anterograde axonal transport of HSV-1, HSV-2 and PRV. SCG cell bodies were infected with HSV-1 NS and 17, HSV-2 2.12 and PRV Becker. Fully assembled virus particles were detected intracellularly within vesicles in proximal and mid-axons adjacent to microtubules after infection with each virus, indicating that assembled virions are transported anterograde within axons for all three alphaherpesviruses.     

Induction of deoxythymidine kinase activity in several mammalian cell lines after infection with six different strains of equine herpesvirus type 1 (EHV-1)

Infection of cells derived from three different mammalian species with any one of six strains of EHV-1 resulted in a 5- to 10-fold increase in the deoxythymidine kinase (dTK) activity when assayed in the presence of 100 μM dTTP. Increased dTK activity was also demonstrated in liver homogenates prepared from EHV-1-infected Syrian hamsters. Antibody elicited against the EHV-1 dTK induced in horse cells neutralized the enzyme activity induced by EHV-1 infection of horse, mouse, hamster, and monkey cells but not the dTKs of uninfected, nonequine cells. EHV-1 induction of dTK was confirmed by demonstration of the inability of the virus strains to replicate in the presence of arabinosylthymine (ara-T) in cells which themselves were totally resistant to the drug. Mutants of EHV-1 resistant to 5-bromodeoxyuridine and ara-T and lacking the ability to induce dTK activity (dTK⁻) were also isolated. Electrophoretic analysis of cell extracts in polyacrylamide gels revealed a new peak of dTK activity (R_f = 0.25) after EHV-1 infection of some cell types (e.g., dTK⁻ 3T3 cells, owl monkey kidney cells, etc.). Other cell types, whose cytosol dTK migrated with an R_f (0.25) identical with that of the EHV-1 enzyme, contained no new electrophoretic peak of dTK activity after infection. In these latter infected cells, however, the dTK activity with an R_f value of 0.25 was partially inhibited by anti-(EHV-1) serum, partially resistant to 100 μM dTTP, and displayed activity even with CTP as a phosphate donor, suggesting that, in these particular cell types, the EHV-1-induced dTK and the host cell cytosol dTK co-electrophorese. These results indicate that infection of cells with all strains of EHV-1 results in the induction of a new, virus-coded dTK activity.     

The nucleotide sequence of the equine herpesvirus 4 gC gene homologue

The genomic position of an equine herpesvirus 4 (EHV-4) gene homologue of the herpes simplex virus 1 (HSV-1) gC gene was determined by Southern analysis and DNA sequencing. The gene lies within a 2-kbp Bg/II-EcoRI fragment mapping between 0.15 and 0.17 within the long unique component of the EHV-4 genome and is transcribed from right to left. Putative promoter elements were identified upstream of the 1455-bp open reading frame which encodes a 485-amino-acid protein of unglycosylated molecular weight 52,513. Computer-assisted analysis of the primary sequence predicts the protein possesses a domain structure characteristic of a type 1 integral membrane glycoprotein. Four domains were distinguished--(i) an N-terminal signal sequence, (ii) a large extracellular domain containing 11 putative N-linked glycosylation sites, (iii) a hydrophobic transmembrane domain, and (iv) a C-terminal charged domain. Comparison of the predicted amino acid sequence to that of other herpesvirus glycoproteins indicated identities of between 22 and 29% with HSV-1 gC, HSV-2 gC, VZV gpV, PRV gIII, BHV-1 gIII, and MDV A antigen and of 79% with EHV-1 gp13. A gene with no apparent homologue in HSV-1 or VZV maps immediately downstream of the EHV-4 gC gene homologue.     

Induction of receptors for complement and immunoglobulins by herpesviruses of various species

Cells infected with herpes simplex virus type 1 (HSV-1) express a viral glycoprotein on the cell surface, which can function as a receptor for a cleavage product of complement factor 3 (C3b), and it has been suggested that this has biological relevance in the infected host (Smiley et al., 1985, J. Virol. 19, 217). As herpesviruses of different species share common determinants on their glycoproteins, a possible conservation of biological function was investigated for bovine herpesviruses type 1 and 2 (BHV-1 and -2), equine herpesvirus type 1 (EHV-1) and HSV-1 and -2, respectively. Only HSV-1 and EHV-1 induced C3b-receptors on infected cells. Nevertheless, BHV-1 infected cells could be killed by complement-dependent neutrophil mediated cytotoxicity (CDNC) as could EHV-1-infected cells. HSV-1-infected cells were not killed by this mechanism, but were highly susceptible to direct C-lysis. Four different scenarios for interaction between herpesvirus-infected cells and the nonspecific host defense system are presented.     

Enhanced resistance to herpes simplex virus type 1 infection in transgenic mice expressing a soluble form of herpesvirus entry mediator

Herpesvirus entry mediator (HVEM) is a member of the tumor necrosis factor (TNF) receptor family used as a cellular receptor by virion glycoprotein D (gD) of herpes simplex virus (HSV). Both human and mouse forms of HVEM can mediate entry of HSV-1 but have no entry activity for pseudorabies virus (PRV). To assess the antiviral potential of HVEM in vivo, three transgenic mouse lines expressing a soluble form of HVEM (HVEMIg) consisting of an extracellular domain of murine HVEM and the Fc portion of human IgG1 were generated. All of the transgenic mouse lines showed marked resistance to HSV-1 infection when the mice were challenged intraperitoneally with HSV-1, but not to PRV infection. The present results demonstrate that HVEMIg is able to exert a significant antiviral effect against HSV-1 infection in vivo.     

Acyclovir resistance in herpes simplex virus type 1: biochemical and functional studies on the thymidine kinase of the highly resistant R100 strain

The biochemical and functional properties of the thymidine kinase (TK) of the herpes simplex virus type 1 mutant R100, that is highly resistant to 9-(2-hydroxyethoxymethyl)guanine (acyclovir), are reported in comparison with the properties of its parental strain, wt. The mutant induced the production of a TK activity that accounted for only 10% of the wt one. This feature was not apparently related to a defective expression of the TK gene but it was rather connected to some functional characteristics of R100 enzyme. Although affinities of this enzyme for ATP and thymidine were unchanged, apparent Vmax values for thymidine were much reduced. In addition, affinities for antiviral analogues acyclovir, 9-(1,3-dihydroxymethyl)guanine (DHPG), 5-(2-bromovinyl)2'-deoxyuridine (BVdU), and 5-iodo-2'deoxycytidine (IdCyd) were drastically diminished (between 50-fold and more than 100-fold). This mutation therefore seems to affect the active site of the enzyme which is involved in the catalytic conversion of thymidine and in the binding of the analogues. The above features of HSV-1 R100 seem quite distinct from those of previously described HSV-1 resistant mutants.     

Herpes simplex virus type 2 and pseudorabies virus associated growth factors and their role in the latency in vitro.

A putative herpes simplex virus type 2 (HSV-2) growth factor (HSGF-2) was detected in a crude extract from virus infected mouse embryo cells. This factor, similar to previously described pseudorabies virus (PRV) associated growth factor (PRGF) was shown to have ability to morphologically transform non-transformed cells and to repress the transformed phenotype of transformed cells. Both activities could be neutralized with two, out of seven monoclonal antibodies directed against glycoprotein B of HSV-2. Both PRGF and HSGF-2 were detected in human embryo lung cells latently infected with PRV or HSV-2 either at 41 degrees C, or in the presence of phosphonoacetic acid. Human alpha-2 interferon, when present in medium of latently infected cells enhanced the production of both HSGF and PRGF. On the contrary, when latently infected cells were treated with 5-azacytidine the synthesis of both PRGF and HSGF-2 was completely blocked and the virus reactivated from latency replicated to higher titers than in non-treated cells. The role of PRGF and HSGF-2 in the establishment, maintenance and reactivation of latency, as well as in cellular transformation is discussed.     

Antigenic relatedness of equine herpes virus types 1 and 3

Summary Antiserums prepared in specific pathogen free (SPF) ponies were used in direct and indirect immunofluorescence, immunodiffusion, complement fixation and serum neutralization procedures to study the interrelationships of the three types of equine herpes viruses (EHV-1, EHV-2, and EHV-3). Equine cell cultures infected with each type virus fluoresced when stained with homologous conjugated anti-serum. In reciprocal tests EHV-1 and EHV-3 cross-fluoresced, but EHV-2 did not cross-fluoresce. Non-infected cell cultures did not fluoresce when stained with the 3 conjugates. EHV-1 and EHV-3 cross-fluoresced in reciprocal indirect fluorescent antibody tests, but no cross-fluorescence was shown with EHV-2. Antigens representing each type of equine herpes virus reacted with their homologous antiserum in the immunodiffusion test. In reciprocal tests, a common line(s) of identity formed with EHV-1 and EHV-3; however, the precipitin line(s) was not common with EHV-2. Antigen prepared from noninfected embryonic mule skin (EMS) cell cultures did not react with any of the antiserums. Specific complement-fixing antibodies were present in antiserums when tested against their homologous antigens. In reciprocal complement fixation tests EHV-1 and EHV-3 cross-reacted, but no cross-reactivity was shown with EHV-2. Significant levels of neutralizing antibody were in an antiserum when tested against homologous virus, whereas cross-neutralization was not detectable in reciprocal tests. These studies indicate that each type of equine herpes virus contains specific antigenic components, and EHV-1 and EHV-3 share a common antigen(s) that is not shared with EHV-2.With 8 Figures     

Characterisation of IE and UL5 gene products of equine herpesvirus 1 using DNA inoculation of mice

Summary.  The equine herpesvirus 1 (EHV-1) strain HVS25A regulatory genes IE and UL5, encoding homologues of herpes simplex virus 1 (HSV-1) ICP4 and ICP27 respectively, were cloned into a eukaryotic expression vector and the DNA injected intramuscularly into mice. Antibodies produced in this way detected the IE or UL5 gene products as diffuse material in nuclei of RK13 cells transfected with the individual genes but as discrete punctate or large aggregates in RK13 cells infected with EHV-1. Western blotting on EHV-1 infected RK13 cells showed multiple IE products of 120–200 kDa and a UL5 product of 52 kDa. Inoculation with plasmids expressing EHV-1 IE or UL5 provided limited protection against EHV-1 challenge in mice as determined by increased virus clearance from lungs on day 2 post-challenge and a reduction in severity of lung histopathology. However, this protection was relatively weak compared with that provided by inoculation of DNA encoding EHV-1 glycoprotein D (gD), possibly reflecting the importance of neutralising antibody in this model. Accepted May 19, 2000 Received March 20, 2000