Charles C. Randall

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.

Biochemical studies of the maturation of herpesvirus nucleocapsid species

Three distinct species of nucleocapsids of equine herpesvirus type-1, designated as either light (L), intermediate (I), or heavy (H) on the basis of their densities in Renografin-76 density gradients (Perdue et al. 1975), were characterized with respect to their amino acid content, DNA content, and role in the maturation process. Preparations of L and I nucleocapsids, shown previously to lack a densely staining core within the capsid, exhibited virtually identical amino acid compositions. Preparations of H nucleocapsids, which possess densely staining cores, contained significantly more lysine, glutamic acid, and serine than did L and I capsids and, in general, more closely resembled the enveloped virion in amino acid composition. The increased content of lysine and glutamic acid in H nucleocapsids indicates that polypeptide IVa (30,000 mol wt), which is present only in H nucleocapsids, is rich in these amino acids. These amino acids may be present as polylysine and polyglutamic acid which have been shown to collapse DNA and function in the DNA packaging event of bacterial viruses (Laemmli, 1975). DNA isolated from preparations of each of the three nucleocapsid species was characterized and shown to be identical to the DNA of enveloped virus in density (1.716 g/cm(3)) and size (50-55 S). The intranuclear production of the three nucleocapsid species was studied with respect to both the time and rate of synthesis of each and the relative amount of each species present during infection. All three species appeared at 6-8 hr postinfection and were produced in a logarithmic fashion until 15 hr postinfection. The ratio of L:I:H particles, based on the percentage that each species comprised of the total purified nucleocapsid population, remained constant and was approximately 45:45:10 at all times postinfection. Analysis of the fate of each of the three intranuclear nucleocapsid classes by pulse-chase experiments indicated that I and H species are removed from the nucleus by participation in the maturation process, but L capsids remain in the nucleus throughout infection and are defective by-products of virus assembly. Based on the evidence accumulated in this study and on previous data (Perdue et al. 1974, 1975), a model depicting equine herpesvirus maturation is proposed. This model proposes that the viral DNA is inserted into I nucleocapsids and that this process is accompanied by condensation of the internal protein resulting in the formation of a dense core and hence an H nucleocapsid.

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.

Virus-specific RNA and DNA in nuclei of cells infected with fowlpox virus.

Abstract Poxviruses appear to be assembled exclusively in the cytoplasm of infected cells, and it has been generally held that all replicative events are restricted to the cytoplasm. Previous work in this laboratory has shown that fowlpox virus (FPV) stimulates DNA synthesis both in vivo and in vitro , which, together with the ability of FPV to cause hyperplasia in vivo , suggests that the host cell nucleus is affected during FPV infection. To investigate this possibility, FPV-infected cells were pulse labeled with either [ 14 C]thymidine or [ 14 C]uridine at various times after infection, harvested, and separated into nuclear and cytoplasmic fractions. By hybridization techniques, both viral RNA and DNA were detected in the nucleus of infected cells from 24 to 72 hr after infection. Less than 10% of the total labeled nuclear DNA is viral specific at 24 hr after infection, but by 48–72 hr, more than 50% of the newly synthesized nuclear DNA is viral specific. The newly synthesized nuclear viral-specific DNA represents more than half of the total labeled viral-specific DNA found in the infected cell at all times studied. In contrast, the viral-specific RNA located in the nucleus always comprises the minor fraction of the total FPV-specific RNA. At 24 hr after infection, approximately 10% of the newly synthesized viral-specific RNA was located in the nucleus, while at 72 hr, about one-third of the total labeled viral-specific RNA could be found in the nuclear fraction. FPV infection does not result in the rapid cessation of host cell nucleic acid synthesis. The amount of ribosomal RNA synthesized in FPV-infected cells from 30 to 48 hr p.i. was found to be only slightly less than in mock-infected cells. Significant host cell DNA synthesis could also be demonstrated until approximately 48 hr after infection.

Purification and characterization of equine herpesvirus-induced DNA polymerase

Abstract Infection of cells with equine herpesvirus type 1 (EHV-1) or type 3 (EHV-3) resulted in the induction of a DNA polymerase activity distinguishable from host cell DNA polymerases by its high salt requirement for maximal activity. By column chromatography on DEAE-cellulose, DNA-cellulose, phosphocellulose, and hydroxyapatite, the EHV-1-induced polymerase was purified 500-fold with 1–2% recovery of total activity from the nuclei of infected hamster livers. The final enzyme preparation was homogeneous as judged by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. Calculations based on Stokes radius, sedimentation coefficient, and electrophoretic mobility indicated that the native enzyme is composed of a single subunit having a molecular weight of 160,000. The purified enzyme exhibited anomalous gel filtration behavior indicating molecular asymmetry. It required Mg 2+ , dithiothreitol, alkaline pH (8–9), all four deoxyribonucleoside triphosphates, and 150 m M salt [K 2 SO 4 , (NH 4 ) 2 SO 4 , or K 2 HPO 4 ] for maximal activity, and utilized templates in the following order of preference: activated DNA (100%), poly(dA)·oligo(dT) (40%), poly(dC)·oligo(dG) (21%), native DNA (7%), denatured DNA (4%), and poly(rA)·oligo(dT) (3%). N-Ethylmaleimide, Zn 2+ , and phosphonoacetate acted as inhibitors of the EHV-1-induced DNA polymerase. Antiserum elicited against the EHV-1 DNA polymerase induced in hamsters inactivated the viral enzyme from infected mouse and equine cell cultures. However, the DNA polymerase induced by EHV-3 and DNA polymerases present in uninfected cells were not inhibited by the antiserum. These results support the hypothesis that a new, virus-coded DNA polymerase is induced after equine herpesvirus infection.

Transcription of equine herpesvirus type 1: evidence for classes of transcripts differing in abundance.

Abstract Scatchard analysis of RNA-DNA hybridization saturation kinetics of viral RNA from equine herpesvirus type 1 (EHV-1)-infected cells revealed the synthesis of two classes of RNAs distinguishable by relative molar concentration. A relationship between the time of infection and the concentration of each viral RNA class was detected such that at 1.5–3.5 hr postinfection, a period before the initiation of viral DNA synthesis, the ratio of the major to minor class was observed to be 82.7 to 1. However at 4.5–6.5 hr. a period after the initiation of viral DNA synthesis, this ratio had decreased to 24.0 to 1. indicating that regulation of the synthesis of the major and minor classes was occurring. Summation hybridization analyses using a mixture of RNAs from 3.5 (early RNA) and 6.5 (late RNA) hr postinfection demonstrated that the transcripts present in each class remained relatively constant throughout the infection. These studies show that the transcriptional pattern of the genome of EHV-1 is similar to that of herpes simplex virus type 1.

Replication of equine herpesvirus type I: resistance to hydroxyurea.

Abstract Hydroxyurea (HU) at concentrations that rapidly and completely inhibit mammalian cellular DNA synthesis does not prevent either equine herpesvirus type 1 (EHV-1) DNA synthesis or virus replication. Analysis by CsCl isopycnic centrifugation of DNA synthesized in HU treated, EHV-1 infected L-M cell cultures demonstrated the synthesis of only viral DNA, whereas both cellular and viral DNA were synthesized in uninhibited, infected cultures. With regard to the mechanism of HU-resistance of EHV-1 DNA synthesis, these studies showed the following: (1) Selective degradation of cellular DNA and increased nuclease activity to provide deoxyribonucleotides for viral DNA synthesis were not induced by infection and/or the inhibitor, (2) HU did not selectively inhibit cellular DNA polymerase activity as both cellular and viral DNA polymerase activities were present in HU-treated, infected cells and were not inhibited by HU added to the in vitro enzyme assay, (3) Cycloheximide inhibition of protein synthesis revealed the requirement of a protein(s) other than viral polymerase at 4 hr postinfection for viral DNA synthesis in the presence of HU. These results do not rule out the possibility that a HU-resistant, viral-induced ribonucleotide reductase activity may be responsible for the HU-resistance of this herpesvirus.

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.

Replication of Equine Herpesvirus Type 1 and Type 3: Resistance to Hydroxyurea and Thymidine

The replication of equine herpesvirus type 1 (EHV-1) and type 3 (EHV-3) was unimpeded in three different cell types-equine epithelial cells, equine fibroblasts, and mouse fibroblasts-which had been blocked in their capacity to synthesize host DNA by 2.5 mM hydroxyurea (HU) or 2 mM thymidine (TdR). The rate of DNA synthesis in uninfected or equine herpesvirus-infected cells in the presence of HU or TdR was measured by pulse-labeling cell samples with a labeled DNA precursor at different times after infection. DNA synthesis in uninfected cultures was completely inhibited by both compounds. However, in cells infected with EHV-1 or EHV-3 and incubated in the presence of HU or TdR, a burst of DNA synthesis occurred which coincided in time with that of virus-specific DNA replication in infected cells without inhibitors. Analysis by cesium chloride isopycnic centrifugation confirmed that the DNA made in drug-treated, infected cells was viral. A possible mechanism of the HU and TdR resistance of the equine herpesviruses is the induction of a ribonucleotide reductase which is insensitive to inhibition by these compounds.

Cellular fatty acids during fowlpox virus infection of three different host systems

Abstract Fatty acid compositions of host cells during fowlpox virus infection have been studied by complementary thin-layer and gas-liquid chromatographic techniques. Three different host systems have been studied: the in vivo infection of the chick scalp, the in ovo infection of the chick embryo chorioallantoic membrane, and the in vitro infection of chick embryo fibroblast cell cultures. Extensive alterations in fatty acid composition of the host cell accompany the fowlpox infection of the chick scalp. The noticeable trends are toward greater unsaturation and a depletion of odd numbered fatty acids. In contrast, the fatty acid compositions of the chorioallantoic membrane and chick embryo cell cultures are not altered significantly following fowlpox virus infection. A slight increase in pentaenoic fatty acids was the only consistent effect of infection in these systems. To the extent that the fatty acid composition of a cell reflects its fatty acid metabolism, it can be concluded that the alterations in fatty acid metabolism observed in the chick scalp are not a constant feature of fowlpox virus infection, and therefore probably represent a host-determined response to infection.