Dennis J. O'Callaghan

Structure of the genome of equine herpesvirus type 3

Abstract The molecular structure of the genome of equine herpesvirus type 1 (EHV-1) was determined by restriction endonuclease mapping studies. Primary restriction enzyme digestion of purified EHV-1 DNA, either unlabeled, 32P04 labeled, or [3H]TdR labeled, gave the following cleavage patterns: EcoRI yielded 17 fragments of 23.4 to 1.3 megadaltons (Mdalton); BglII, 16 fragments of 24.5 to 1.0 Mdalton; XbaI, 15 major fragments of 18.6 to 1.7 Mdalton; and BamHI,17 fragments of 13.7 to 2.8 Mdalton. Several fragments were present in 0.5 M amounts while all others were 1.0 M no 0.25 M fragments were detected. Secondary restriction enzyme digestion of these isolated fragments with various enzymes, analysis of terminal fragments using both the methods of λ 5′ exonuclease digestion and end labeling with polynucleotide kinase, and blot hybridization experiments with 32P-labeled BamHI fragments indicated that this herpesvirus genome is a 92-Mdalton linear, double-stranded DNA molecule and is comprised of two segments designated as L (Long) and S (Short) which are 71.6 and 20.4 Mdalton, respectively. The 0.5 M fragments are located at the ends of the S region, an arrangement which allows the S region to invert relative to the L region; thus, two structural arrangements (isomers) of the genome exist. In addition, areas of heterogeneity were detected at the L terminus, within the S segment, and at a split variable locus in the L region.

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.

Expression of altered ribonucleotide reductase activity associated with the the replication of the epstein-barr virus

Abstract The expression of the Epstein-Barr virus (EBV) genome can be regulated in the epithelial/Burkitt hybrid (D98/HR-1) cell line and Raji lymphoblastoid nonproducer cell line by induction with 5-iododeoxyuridine (IUdR) or by superinfection with EBV. Extracts of control and induced D98/HR-1 and Raji cells were assayed for ribonucleotide reductase activity in the presence and absence of hydroxyurea (HU). Enzyme activity of control D98/HR-1 and Raji cells was inhibited by greater than 70%, by HU at both low (2 × 10−4 M) bot high (5 × 10−4 M) concentrations; however, the reductase activities of IUdR-induced D98/HR-1 cells, superinfected Raji cells, and IUdR-induced Raji cells were resistant to both levels of HU, and enzyme activities of 85 to +100% of control values were obtained in all cases. Under conditions that allowed only partial expression of the EBV genome (before removal of IUdR; early time after superinfection), very significant levels (70–85°k) of HU-resistant enzyme activity were obtained in the presence of 2 × 10−4 M HU, whereas only 30 to 45% of control reductase activity was observed at the high HU concentration. Mixing experiments employing combinations of various D98/HR-1 control and induced cell extracts indicated that the HU-resistant reductase activity present in induced 1)98/HR-1 cells was due to the presence of an altered enzyme activity and not due to some nonenzymatic factor(s). Additional experiments, in which HU was preincubated with control and induced cell extracts, showed that the HU-resistant ribonucleotide reductase in extracts of induced D98/HR-1 cells was not due to inactivation of the inhibitor by an enzyme or factor present in these cells. These findings of an altered ribonucleotide reductase activity associated with EBV replication, considered in light of similar findings for equine herpesvirus type 1 and herpes simplex virus types 1 and 2, suggest that alteration of this enzyme activity may be a feature of herpesvirus replication.

Biological and biochemical properties of defective interfering particles of equine herpesvirus type 1.

Abstract Defective interfering (DI) particles of equine herpesvirus type 1(EHV-1) were purposely generated in an in vitro system of L-M cells by repeated high-multiplicity, serial, undiluted passage. Quantitation of infectious virus revealed a definite cyclic pattern which increased in magnitude with continued passage; additional experiments indicated that these fluctuations in virus titer were due to the presence of a population of DI particles as judged by interference assays. Attempts to separate standard and defective EHV-1 were unsuccessful. Analysis of DNA isolated from mixed populations of these virions revealed the presence of a high-density (H) variant DNA (ϱ = 1.724 g/cm3) in addition to standard EHV-1 DNA (ϱ = 1.716 g/cm3). Furthermore, it was found that the relative amount of this H-DNA in each passage corresponded very closely to the fluctuations in infectious virus titer. Sedimentation velocity studies of DNA isolated from populations of virions rich in H-DNA (>99%) indicated that the variant genomes were the same size as the standard EHV-1 genome (50–55 S). Comparisons of purified virion populations from 17 high-multiplicity passages with regard to particle counts, relative amount of H-DNA, and infectious virus titer indicated that the relative interference capacity of EHV-1 DI particles increased significantly with continued passage. Although the factor(s) responsible for the increased interference activity is unknown, DNA-DNA hybridization analyses of selected passages rich in H-DNA indicated that the genomes of EHV-1 DI particles became genetically less complex with passage and contained significant amounts of reiterated sequences. The possible mechanisms of the evolution of EHV-1 DI particles and their role in the interference process are discussed.

Equine cytomegalovirus: Cultural characteristics and properties of viral DNA

Abstract Equine cytomegalovirus (equine herpesvirus type 2; ECMV) exhibited cultural characteristics typical of the cytomegalovirus group. Ninety-six to one hundred twenty hours were required to reach a maximum titer of 1 × 10 7 PFU/ml in infected cells, from which no more than 50% of infectious virus produced was released into the supernatant fluid. Only cells of equine or rabbit origin were permissive for virus replication. Ultrastructural investigation of ECMV-infected cells revealed the presence of three types of intranuclear nucleocapsids (empty capsidc, capsidc with a cross-shaped, electron-lucent core, and mature capsidc with an electron-dense core). The mature capsids appeared to acquire their envelope at the nuclear membrane. Infected cells were characterized by nuclei containing marginated chromatin in a large, electron-dense inclusion substance. Viral DNA extracted and purified from virions, nucleocapsids, or infected cells (Hirt fractionation) demonstrated an average density of 1.716 g/cm 3 which corresponds to a G+C content of 57.7%. Sedimentation analyses of ECMV DNA in neutral sucrose gradients using phage T4 and equine herpesvirus type 1 (EHV-1) DNA as markers indicated a sedimentation coefficient of approximately 61 S. Sedimentation in alkaline sucrose suggested that the ECMV genome is a non-cross-linked, double-stranded DNA, possibly possessing nonligated areas either within the sugar phosphate backbone of the molecule or within specific alkali-labile regions. Sedimentation analyses of ECMV DNA yielded a molecular weight of approximately 121 × 10 6 which was confirmed by restriction endonuclease analyses which indicated a value of 126 × 10 6 Determination of the number, size, and molarity of ECMVDNA fragments generated by digestion with restriction enzymes revealed that ECMV DNA differs markedly in molecular structure from the genome of EHV-1.

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.

A specific viral DNA sequence is stably integrated in herpesvirus oncogenically transformed cells

Abstract The integration patterns of viral DNA sequences in three hamster embryo cell lines independently derived by transformation with equine herpesvirus type 1 (EHV-1) have been investigated by DNA blot hybridization analyses for the restriction enzymes Eco RI, Bgl II, Xba I and Bam HI with 32 P-labeled selected DNAs from a collection of cloned EHV-1 restriction enzyme fragments as probes. These EHV-1-transformed cell lines contained subgenomic portions of the viral genome in an integrated state at multiple sites in the host genome. At least one copy of a viral DNA sequence mapping colinearly from 0.32 to 0.38 map units within the EHV-1 genome was common among these three EHV-1 transformed cell lines. The 0.32–0.38 viral DNA sequence was maintained stably even after 125 cell passages, whereas sequences from other positions in the EHV-1 genome were lost progressively during continued cell passage. The significance of the findings that these oncogenically transformed cell lines harbor a specific region of the EHV-1 genome is discussed with regard to stable maintenance of the oncogenically transformed state.

Oncogenic transformation by equine herpesviruses (EHV). I. Properties of hamster embryo cells transformed by ultraviolet-irradiated EHV-1.

Abstract Permissive primary hamster embryo cells (HEF) were morphologically transformed in vitro by uv-irradiated standard equine herpesivrus type 1 (EHV-1). Cell lines (LSEH) were established and were shown to exhibit growth and biological properties typical of transformed cells. These properties included an altered, network-like cellular morphology, loss of contact inhibition, increased saturation density, decreased generation time, immortality in culture, formation of colonies in soft agar, growth at low concentrations of serum, and resistance to EHV-1 superinfection. These LSEH transformed cell lines were oncogenic in both newborn and immunocompetent inbred LSH syngeneic hamsters, and LSEH tumor cell lines were established from the tumors which were identified as malignant fibrous sarcomas. Both the LSEH transformed and tumor cell lines were determined to be virus nonproducers, since released virus could be neither detected by electron microscopy, infectious center assays, or cocultivation assays nor induced by iododeoxyuridine (IUdR) or growth at higher temperature. Analysis of the hybridization kinetics of 125I- or 32P-labeled EHV-1 DNA with DNA from low-passaged LSEH transformed cell lines, tumor tissues, or LSEH tumor cell lines revealed that viral DNA sequences were present in all cells and that the amount of these viral DNA sequences varied from one cell line to another. In the transformed cell lines, these values ranged from 3.3 to 20.9% of the EHV-1 genome in amounts of 0.3 to 2.2 copies/cell, whereas the DNA of tumor tissues and low-passaged LSEH tumor cell lines contained from 2.2 to 12.2% of the EHV-1 genome in amounts of 1.2 to 4.0 copies/cell. These viral DNA sequences were expressed since viral antigens were detected in LSEH transformed cells, tumor tissues, and tumor cell lines. Neutralizing antibody, ND50 titers ranging from 1 8 to 1 100 , was detected in the sera of hamsters that had been inoculated with either LSEH transformed or LSEH tumor cells. In addition, EHV-1-specific speypeptides were demonstrated in the cell membrane, cytoplasm, and perinuclear region of both LSEH transformed and tumor cell lines by indirect immunofluorescence using antisera to purified EHV-1 virions or antisera to EHV-1 infected HEF cell extracts.

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.