Glenn A. Gentry

The Equine Herpesviruses

The equine herpesviruses comprise a diverse group of three antigenically distinct biological agents of protean manifestation in the horse, causing a variety of natural infections that vary from the subclinical to fatal generalized disease. Equine herpesvirus type 1 (EHV-1), also known as equine abortion virus (EAV), or equine rhinopneumonitis virus, is a major cause of respiratory disease and abortion in the horse. This agent, in particular, appears to be a model system for in vitro and in vivo study of disease, persistent infection, biochemistry of viral infection, and biochemical and oncogenic transformation. This review will therefore emphasize certain biological features of this equine herpesvirus, the best studied of the group. Equine herpesvirus type 2 (EHV-2), or equine cytomegalovirus (ECMV), is a ubiquitous, loosely defined, antigenically heterogeneous, usually slowly growing group of viruses, causing no known disease. Equine herpesvirus type 3 (EHV-3), equine coital exanthema (ECE) virus, is the causative agent of a relatively mild progenital exanthema of both mare and stallion.

HERPESVIRAL DEOXYTHYMIDINE KINASES CONTAIN A SITE ANALOGOUS TO THE PHOSPHORYL-BINDING ARGININE-RICH REGION OF PORCINE ADENYLATE KINASE; COMPARISON OF SECONDARY STRUCTURE PREDICTIONS AND CONSERVATION

Twelve herpesviral deoxythymidine kinases were examined for regions of sequence similarity by multiple alignment. Six highly conserved sites were observed. Site 1 corresponded to a glycine-rich loop that forms part of the ATP-binding pocket in porcine adenylate kinase (PAK), and site 5 corresponded to a region in PAK, located on one lobe of the cleft, that contains arginine residues that bind substrate phosphoryl groups. Site 3, consisting of the motif -DRH-, is thought to be involved in thymine/deoxythymidine recognition; site 4, which is nearby, probably participates in this function as well. The functions of sites 2 and 6 have not been identified. Secondary structure predictions were made by the Garnier method and averaged for each position in the multiple alignment. The structure predicted for all six sites was typically a short flexible region (turn or coil) at or adjacent to the site, flanked by rigid structures (helix or sheet) on either side.

Viral thymidine kinases and their relatives

Thymidine kinases were described for cellular life long before it was shown that they could also be encoded by viruses, but the viral thymidine kinase genes were the first to be sequenced. These enzymes have been extraordinarily useful to the researcher, serving first to help label DNA, then to get thymidine analogs incorporated into DNA for therapeutic and other purposes and more recently to move genes from one genome to another. Knowledge of the nucleotide and amino acid sequences of these enzymes has allowed some deductions about their possible three-dimensional structure, as well as the location on the polypeptide of various functions; it has also allowed their classification into two main groups: the herpesviral thymidine/eukaryotic deoxycytidine kinases and the poxviral and cellular thymidine kinases; the relationships of the mitochondrial enzyme are still not clear.

The Medicinal Value of Tropical Rainforest Plants from Paluma, North Queensland, Australia

A pharmacological survey of flora of the Paluma rainforest including 90 species representing 19 families has been carried out. Crude plant extracts have been screened for cytotoxic, antibacterial, antifungal, and antiviral activity, as well as brine shrimp lethality. Of these, 27 extracts exhibited remarkable cytotoxic activity, 23 showed antimicrobial activity, and 7 showed promising antiviral activity. Thus, 53 of the plant species examined showed marked bioactivity in one or more bioassays; a “hit rate” of 59%. These results underscore the phytomedicinal potential of Australian tropical rainforests.

Equine herpesvirus in vivo: cyclic production of a DNA density variant with repetitive sequences.

Abstract The DNA of a strain of equine herpesvirus type 1 passed more than 500 times in Syrian hamsters (EHV-1ha) has been analyzed by CsCl equilibrium density gradient ultracentrifugation, analytical sedimentation, and DNA-DNA reassociation kinetics. The viral DNA consisted of light and heavy species having densities in CsCl of 1.716 and 1.724 g/cm 3 , which correspond to guanine plus cytosine contents of 56 and 64%, respectively. These values were confirmed by T m measurements. Similar molecular weight values were obtained by analytical sedimentation for the light (87.9 × 10 6 ) and heavy (81.8 × 10 6 ) DNA species. The heavier species was produced in a cyclic manner. Hamsters infected with virus containing a high proportion of the heavy species gave reduced virus yields and survived longer. The genetic relatedness of the two viral DNA species of EHV-1ha was compared by examining the ability of each to reanneal with 32 P-labeled viral DNA of the tissue culture strain (L-M cell) of EHV-1 (EHV-1tc). The lighter (1.716 g/cm 3 ) species of EHV-1ha was composed of unique sequences completely homologous to the entire EHV-1tc genome, while the heavier species (1.724 g/cm 3 ) consisted of sequences homologous to approximately 50% of the EHV-1tc genome. Of these homologous sequences, 40–60% (20–30% of the entire EHV-1tc genome) were reiterated. Further, analyses of the EHV-1tc genome (fragmented and unfragmented) by thermal chromatography on hydroxylapatite and in neutral preparative CsCl equilibrium density gradients revealed considerable intramolecular heterogeneity in nucleotide distribution. Finally, analysis of the structural polypeptides of virions of EHV-1ha which contained the heavy and light DNA species revealed that the following two major viral proteins were missing from virions containing the heavier DNA species: VP8, an envelope protein with a molecular weight of 173,000, and VP23, a nucleocapsid protein with a molecular weight of 38,000.

Thymine Salvage, Mitochondria, and the Evolution of the Herpesviruses

The salvage of thymine is an apparently ubiquitous feature of free-living lifeforms as well as of mitochondria, chloroplasts and most of the large DNA viruses. Assumptions and data are described which explain the evolution of thymine salvage in prokaryotes, animal cells, and large DNA viruses, in terms of deoxythymidine kinase and its relationship to mitochondria. Specifically, it is suggested that regulation of deoxythymidine kinase (by end-product inhibition) has evolved as a means of assuring a constant supply of thymine compounds for the mitochondria and that the degree to which this regulation is present in the deoxythymidine kinases of the various herpesviruses correlates with the degree of dependence of their replicative cycle on the continued health of the mitochondria of their host cells.

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

Abstract 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 Epstein-Barr virus genome encodes deoxythymidine kinase activity in a nested internal open reading frame.

The Epstein-Barr virus (EBV) BXLF1 fragment open reading frame (LORF), though to encode deoxythymidine kinase (dTK) activity, and a shorter frame (SORF), starting at an internal in-frame AUG, were isolated by polymerase chain reaction from a plasmid containing the EcoR1 fragment of EBV strain FF-41. These were transfected into dTK-Escherichia coli, producing multiple SORF- or LORF-containing colonies, which expressed dTK. The 243 NH2-terminal residues of the LORF-encoded polypeptide thus are not essential for dTK activity. SORF, with 1,092 bp, is predicted to encode a 36- to 37-kD polypeptide, in the size range of other herpesviral dTKs.

HSV1-Specific Thymidylate Kinase Activity in Infected Cells

Several 5-methoxymethyldeoxyuridine (MMdU)-resistant mutants of herpes simplex virus type 1 (HSV1) were classified by measuring their sensitivities to the deoxythymidine kinase (dTK)-dependent antiviral drugs 9-(2-hydroxyethoxymethyl)-guanine (acyclovir, ACV), 1-beta-D-arabinofuranosylthymine (araT), and E-(2)-5-bromovinyldeoxyuridine (BVdU) and to the dTK-independent antiviral drug phosphonoacetate (PAA). Compared to wild-type (WT) virus, all five of the dTK- mutants were highly resistant (greater than or equal to 500-fold) to BVdU and MMdU, moderately resistant to ACV (50- to 100-fold) and araT (10- to 20-fold), but not resistant to PAA. The dTK of the mutant MMdUr-20 (dTK+) appeared to phosphorylate dTMP less well than that of the WT virus, while its affinity for deoxythymidine was not altered. Two other drug-resistant HSV mutants-S1 (isolated against ACV) and B3 (isolated against BVdU)--also showed reduced phosphorylation of dTMP. This suggests that alterations in both dTK and thymidylate kinase activities may determine sensitivity to antiviral drugs.

Purification and characterization of deoxythymidine kinase (dTK) induced in dTK− 3T3 mouse cells by equine herpesvirus type 1 (EHV-1)

Abstract Infection of mouse 3T3 cells deficient in cytosol deoxythymidine kinase (dTK − ) with the Kentucky A strain of EHV-1 resulted in a 20- to 30-fold increase in cytosol dTK activity. The EHV-1-induced dTK was partially purified from such cells by affinity chromatography on deoxythymidine (dT)-Sepharose and characterized by electrophoretic, enzymatic, and immunological criteria. The purified EHV-1 dTK migrated in polyacrylamide gels with an R f of 0.25 and sedimented in glycerol gradients with an S value of 5.2, corresponding to a molecular weight of 85,000. Deoxycytidine could not serve as an alternative substrate for the viral-induced enzyme. A rabbit antiserum prepared against EHV-1-infected horse cells neutralized the viral-induced dTK activity purified from infected mouse (dTK − 3T3) cells, but not the kinase activities from uninfected 3T3 cells. EHV-1 dTK differed from 3T3 cytosol dTK also in the greater resistance of the viral enzyme to feedback inhibition by dTTP and dCTP, its inhibition by arabinosylthymine, and its ability to utilize a variety of nucleoside triphosphates as phosphate donors. The purified EHV-1 kinase could be distinguished from 3T3 mitochondrial dTK by electrophoretic mobility and inhibition by anti-(EHV-1) serum. These results lend further support to the hypothesis that the dTK induced by EHV-1 is coded by the virus genome.