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

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 (? = 1.724 g/cm³) in addition to standard EHV-1 DNA (? = 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.     

Properties of the genome of equine herpesvirus type 3

Methods developed for the isolation and purification of equine herpesvirus type 3 (EHV-3) DNA were shown to yield quantities of intact infectious DNA molecules suitable for characterization by physicochemical, biochemical, and electron microscopic methods. Preparations of EHV-3 DNA were shown by CsCl analytical ultracentrifugation to be comprised of a single species of DNA with a buoyant density of 1.727 g/cm³ which corresponds to a G + C content of 67.9%. Rate velocity centrifugation studies revealed that EHV-3 DNA has a sedimentation coefficient of approximately 55.4 S which corresponds to a molecular weight value of 90 to 100 megadaltons (Md). Intact viral DNA molecules were found to be 96 to 100 Md by electron microscopy, and restriction enzyme analyses with BamHI, EcoRI, and HindIII yielded an average molecular weight of 96.2 Md. Restriction enzyme and blot hybridization methods revealed that: (i) four 0.5 M EcoRI fragments were present, (ii) these four 0.5 M fragments shared significant homology, (iii) three EcoRI terminal fragments were identified and two of these were 0.5 M, and (iv) the two 0.5 M EcoRI end fragments hybridized only to one of the two 1.0 M terminal fragments identified for BamHI or HindIII. These findings indicated that a set of DNA sequences located at one terminus is repeated within the genome and that two populations of molecules exist with regard to the orientation of these repeat sequences. Electron microscopic examination of reannealed EHV-3 DNA molecules revealed structures that contained a single-strand loop equivalent to a duplex molecular weight of 5.3 Md at one end of the molecule contiguous to a double-strand region which terminated in a long single-strand segment. These structures were identical in morphology to those observed for EHV-1 DNA which has an overall genomic structure of an L (long) region covalently linked to an S (short) region; the S region consists of a unique segment (Us) bracketed by inverted repeat sequences (Henry et al., 1981; O'Callaghan et al., 1981). The above findings indicate that the 96.2-Md EHV-3 genome is comprised of an L region of approximately 73–76 Md covalently linked to a 20- to 23-Md S region; the S region contains a 5.3-Md Us segment bounded by 8-Md inverted repeat sequences that allow the S region to invert relative to the fixed L region and the genome to exist in two isomeric arrangements.     

Equine cytomegalovirus: Cultural characteristics and properties of viral DNA

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 × 10⁷ 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/cm³ 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 × 10⁶ which was confirmed by restriction endonuclease analyses which indicated a value of 126 × 10⁶ 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.     

Alterations in virus protein synthesis and capsid production in infection with DI particles of herpesvirus

High multiplicity, undiluted passage of equine herpesvirus type 1 (EHV-1) in L-M cells resulted in the rapid production of virus particles whose genome was genetically less complex, contained more reiterated DNA sequences and exhibited a greater buoyant density (rho = 1.724 g/ml) than the DNA (rho = 1.716 g/ml) of standard virus. These data and the finding that these particles inhibited the replication of standard virus in interference assays confirmed that these were defective interfering (DI) particles (Henry et al. 1979). Additional evidence for this has been obtained from the pattern of cyclic fluctuation in infectious virus titre through 17 serial passages as well as from the pronounced variation in the particle to plaque ratio for each passage. Total particle production was markedly reduced in cells infected with virus preparations containing DI particles and quantification of major cell-associated EHV-1 capsid species by electron microscopy and analysis in Renografin density gradients indicated that this reduction occurred at the level of capsid assembly. Although total capsid production was reduced in cells infected with DI particle preparations, the synthesis of I (immature) capsids increased relative to that of L (empty) capsids and these alterations in the assembly of capsid species could be related to changes in the synthesis of capsid proteins. In cells infected with EHV-1 preparations rich in DI particles, the synthesis of major capsid protein 150000 was greatly reduced, whereas core protein 46000, a major component of I capsids, was overproduced as compared to standard virus infection. Capsids produced in cells infected with virus preparations rich in DI particles were identical in polypeptide composition to those made in standard virus infection.     

Equine herpesvirus-induced alterations in nuclear RNA and DNA polymerase activities.

Hamster hepatic cells infected in vivo with equine herpesvirus, type 1, were used for the preparation of isolated nuclei, which were assayed for RNA and DNA polymerases. An endogenous RNA polymerase II activity (sensitive to α-amanitin) was inhibited early and progressively during infection. The radiolabeled RNA products, synthesized in vitro were characterized by analytical centrifugation in sucrose-formamide gradients and by hybridization analysis. The bulk of the RNA synthesized by uninfected and 9-hr-postinfection (p.i.) nuclei sedimented at 10–18 S and 10–14 S, respectively. Purified RNA from uninfected nuclei hybridized exclusively to hamster DNA, whereas RNA from infected nuclei hybridized predominantly to viral DNA (12% of the input radioactivity measured as counts per minutes) and to a lesser degree to hamster DNA (1% of the input counts). Endogenous DNA polymerase activity, assayed in the presence of 150 mM K₂SO₄, was completely inhibited in uninfected nuclei; however, an induced DNA polymerase, active under the same conditions, was detected as early as 2 hours p.i. Overall, a 35-fold increase in activity of the “high salt DNA polymerase” was noted between 2 and 9 hr p.i. Radiolabeled DNA synthesized in vitro by 9-hr-p.i. nuclei was purified, treated with a single-strand-specific endonuclease, and separated by isopycnic CsCl centrifugation. Two species with densities equivalent to viral (1.716 g/cm³) and host cell (1.702 g/cm³) DNA, respectively, were resolved. Hybridization analysis demonstrated that purified DNA, synthesized in vitro by 9-hr-p.i. nuclei, was complementary to viral DNA (38% of the input counts).     

Ribonucleotides in newly synthesized DNA of herpes simplex virus

Newly synthesized DNA of herpes simplex virus type 1 (HSV-1), obtained from primary rabbit kidney cells pulse-labeled with [³H]thymidine or [³H]uridine at 6 hr postinfection, was purified by two cycles of centrifugation in CsCl density gradients. These intracellular viral DNA preparations hybridized specifically with homologous HSV-1 DNA but not with host cell DNA or E. coli DNA. Upon denaturation by alkali, the [³H]thymidine-labeled HSV-1 DNA cleaved to smaller pieces. The alkali-labile material in the viral DNA was identified as ribonucleotides on the basis of the following observations: (1) When labeled with [³H]uridine for short periods, the labeled viral “DNA” was susceptible to RNase and NaOH, and all the radioactivity was confined to the nucleoside [³H]uridine; however, upon longer labeling periods (up to 20 hr), the [³H]uridine-labeled viral “DNA” became more susceptible to DNase, as most but not all of the [³H]uridine was converted to deoxyribonucleosides. (2) Denaturation of [³H]uridine-labeled double-stranded HSV-1 “DNA” (?_Cs2SO4 = 1.45 g/cm³) by heat shifted the buoyant density to single-stranded DNA region (?_Cs2SO4 = 1.48?1.50 g/cm³) but not to single-stranded RNA region; however, treatment with hot NaOH considerably reduced the radioactivity of this “DNA.” Treatment with DNase, but not with pronase, shifted the buoyant density to the heavier RNA region of the gradient. Heat-denatured DNA but not the native DNA was susceptible to single-strand specific nuclease S1.     

Properties of the DNA product of reverse transcription of turnip yellow mosaic virus RNA preparations.

Four populations of adenovirus type 16 incomplete particles with different buoyant densities in CsCl have been identified. They contain less DNA than complete particles. The standard viral genome has a molecular weight of 23 × 10⁶. The molecular weights of the incomplete DNA molecules range from 1 × 10⁶ to 16 × 10⁶, increasing with increasing density of the particles in CsCl. Characterization of isolated DNA by equilibrium density centrifugation in CsCl, DNA-DNA hybridization, and analysis of DNA labeled with [³H]thymidine before infection shows that both viral and host cell-specific DNAS are incorporated into incomplete virus particles.     

Adeno-associated virus autointerference.

We have analyzed an autointerference phenomenon exhibited by adeno-associated virus type 2 (AAV) when grown in KB cells coinfected with adenovirus type 2 as the helper. Infectious AAV particles that banded at 1.41 g/cm³ in CsCl were purified by three cycles of centrifuging in CsCl equilibrium gradients. When cells were infected with an increasing multiplicity of these AAV particles there was a corresponding decrease in production of infectious progeny AAV. There was also an AAV multiplicity-dependent inhibition of production of infectious adenovirus and inhibition of Ad DNA replication. The viral DNA in the Hirt supernatant fraction extracted from cells infected with different multiplicities of AAV was analyzed in neutral sucrose gradients. At low multiplicities of infection with AAV, the main AAV DNA species synthesized was the mature 14.5 S (standard) viral genome. In higher multiplicity infections with AAV increasing amounts of aberrant 10 S AAV DNA molecules accumulated and the proportion of 14.5 S AAV DNA decreased. Restriction endonuclease cleavage showed that the 10 S DNA was enriched for the left- or right-hand terminal regions of the AAV genome. These molecules may be analogous to the previously characterized aberrant DNA molecules found in light-density AAV particles. Thus, the AAV autointerference is correlated with production of the aberrant deleted AAV genomes.     

Replication of equine herpesvirus type I: resistance to hydroxyurea.

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.     

9-beta-D-Arabinofuranosyladenine as a tool to study herpes simplex virus DNA replication in vitro.

9-β-d-Arabinofuranosyladenine (araAdo) strongly suppresses herpes simplex virus (HSV) DNA synthesis in intact cell systems. After incubation with araAdo, two HSV-DNA fractions can be isolated by neutral isopycnic CsCl density gradient centrifugation, a light fraction with a buoyant density of 1.726 g/cm³, and a heavy fraction with a density of 1.738 g/cm³. After recentrifugation in neutral CsCl gradients, the light and heavy fractions are detected at a density of 1.729 and 1.741, respectively. Analysis of the two HSV-DNA fractions by hydroxylapatite chromatography and by digestion with nuclease S₁ revealed that the light fraction consists predominantly of native DNA and the heavy fraction of denatured DNA. AraAdo is incorporated into HSV-DNA and has been shown to be present in terminal positions at the 3′-hydroxyl position. The HSV-DNA pieces, containing incorporated araAdo, are of low molecular weight (less than 2.6 × 10⁶) and are not assembled to higher molecular weight aggregates.     

Characterization of DNA from polyhedral inclusion bodies of the nucleopolyhedrosis single-rod virus pathogenic for Orgyia pseudotsugata.

A nucleotide sequence complexity of 88.5 × 10⁶ was determined for the DNA of the nucleopolyhedrosis single-rod (unicapsid) virus of Orgyia pseudotsugata using optical renaturation. In addition, the genome size was determined to be 85 × 10⁶ by comparison of EcoR1 restriction endonuclease fragments with markers of known size using agarose gel electrophoresis. A G + C concentration of 44% for the viral DNA was estimated from its melting properties and buoyant density in CsCl. Evidence from buoyant density in CsCl indicates that DNA which is occluded in the polyhedral matrix but not associated with virions is of viral origin.     

Subfractionation of CsCl-purified H-1 parvovirus on metrizamide gradients.

The different density classes of H-1 parvovirus, collected within 30 hr of parection of parasynchronous cultures, following the standard CsCl purification step, have been shown to be heterogeneous. Rebanding of the denser form (HF, ? = 1.46 g/cm³) and the less dense form (LF, ? = 1.42 g/cm³) of infectious virus in the nonionic density generating solute, metrizamide, showed that both HF and LF virus bands were heterogeneous in density. The infectivity banded with isotopically labeled virus protein and DNA at 1.32 g/cm³ for both HF and LF virus. Amounts of protein and DNA which varied from preparation to preparation, but which were greater from the HF virus band, were distributed throughout the rest of the gradient, but predominated in a peak at a density of 1.2 g/cm³. The protein in this peak was without hemagglutinating activity but had the molecular weights and proportions of the H-1 virion proteins (VP1, VP2′, and VP2). The DNA was of the same size as H-1 DNA monomers and its proportion to the protein was similar to that of the infectious peak. The DNA was susceptible to micrococcal nuclease digestion. The nature of this noninfectious viral material thus seemed to be incompletely assembled virus. Radio-labeled H-1 virus collected after 72 hr of infection formed a discrete single peak in both CsCl (? = 1.42 g/cm³), and metrizamide gradients (? = 1.32 g/cm³). There was no significant amount of the 1.20 g/cm³ viral protein-DNA complex in these mature preparations.     

Marker rescue of temperature-sensitive mutants by defective DNA of herpes simplex virus type 1.

High-density, defective DNA (?_CsCl = 1.732 g/cm³) produced in infections of the 15th through 20th undiluted serial passage of herpes simplex virus type 1 (HSV-1), Strain KOS, was purified by preparative CsCl density gradient ultracentrifugation. Purified defective DNA expressed no discernible cytopathic effects when transfected into receptive cell monolayers. Cleavage of defective DNA preparations with EcoRI restriction endonuclease revealed four major and three minor bands upon electrophoresis in agarose gels. Restriction endonuclease HindIII failed to cleave defective DNA.Marker rescue studies revealed that of 13 temperature-sensitive mutants representing 12 complementation groups, only mutants in group B were rescued by defective DNA. These studies demonstrate, therefore, that defective DNA is enriched for at least part of the B cistron and suggest that this cistron is located in the region of the viral genome from which defective DNA is generated.     

Respiratory and neurological disease in rabbits experimentally infected with equid herpesvirus 1

Equid herpesvirus type 1 (EHV-1) is an important pathogen of horses worldwide, associated with respiratory, reproductive and/or neurological disease. A mouse model for EHV-1 infection has been established but fails to reproduce some important aspects of the viral pathogenesis. Then, we investigated the susceptibility of rabbits to EHV-1 aiming at proposing this species as an alternative model for EHV-1 infection. Weanling rabbits inoculated intranasal with EHV-1 Kentucky D (10⁷ TCID₅₀/animal) shed virus in nasal secretions up to day 8–10 post-inoculation (pi), presented viremia up to day 14 pi and seroconverted to EHV-1 (virus neutralizing titers 4 to 64). Most rabbits (75%) developed respiratory disease, characterized by serous to hemorrhagic nasal discharge and mild to severe dyspnea. Some animals (20%) presented neurological signs as circling, bruxism and opisthotonus. Six animals died during acute disease (days 3–6); infectious virus and/or viral DNA were detected in the lungs, trigeminal ganglia (TG), olfactory bulbs (OBs) and cerebral cortex/brain (CC). Histological examination showed necrohemorrhagic, multifocal to coalescent bronchointerstitial pneumonia and diffuse alveolar edema. In two rabbits euthanized at day 50 pi, latent EHV-1 DNA was detected in the OBs. Dexamethasone administration at day 50 pi resulted in virus reactivation, demonstrated by virus shedding, viremia, clinical signs, and increase in VN titers and/or by detection of virus DNA in lungs, OBs, TGs and/or CC. These results demonstrate that rabbits are susceptible to EHV-1 infection and develop respiratory and neurological signs upon experimental inoculation. Thus, rabbits may be used to study selected aspects of EHV-1 biology and pathogenesis, extending and complementing the mouse model.     

Herpes simplex type 1 infection of nonpermissive rat XC cells

Summary Herpes simplex virus type 1 (HSV-1) infection of non permissive XC cells (a rat cell line transformed by Rous sarcoma virus) was studied. Using virus labeled with³H-thymidine it was shown that adsorption is similar to that in a permissive system. By electron microscopy enveloped particles were observed in cytoplasmic vesicles in XC cells but not in the permissive system. However input viral DNA was degraded both in non permissive cells (XC) and permissive cells (HEp-2) and the degradation products were found incorporated into cellular DNA in the first case or into viral DNA in the second case.In the non permissive XC cells, it was possible to detect a small amount of incorporation of radioactive precursors into the viral DNA, identified by its buoyant density in CsCl of 1.726 g/cm³ and by hybridization with viral DNA. This DNA has the size of the native viral genome and its uptake of radioactive precursors was only partially inhibited by phosphonoacetic acid, a specific inhibitor of HSV-DNA polymerase. With permissive HEp-2 cells in the presence of such inhibitor, the obtained data are roughly the same as with XC cells, both in the presence or in the absence of phosphonoacetic acid. These results suggest that the observed viral DNA synthesis in XC cells is not a true replication but, further, a repair synthesis and, also, that the same events might take place in the permissive system before the onset of viral DNA replication.With 4 Figures     

Evidence of precursors of defective measles virus

Cytoplasmic extracts of Vero cells infected with wild strain Edmonston measles virus were found to contain at least two distinct nucleocapsid species. The two most prominent species of nucleocapsids sedimented at 200S and 110S and contained RNA of molecular weight 6.0×10⁶ and 0.6×10⁶ daltons respectively. Both species of nucleocapsids had a density of 1.31 g/cm³ in CsCl. A third species sedimenting at 170S was not present in all experiments and was not characterized in detail. Infection of cells with undiluted-passage virus usually resulted in production of mostly 110S nucleocapsids while both 110S and 200S species were found when diluted-passage virus was used. These results suggest that measles virus may produce distinct classes of defective virus which contain segments of RNA representing as little as 10% of the complete viral genome.     

Intermediates in type 5 adenovirus DNA replication

Intermediates in the replication of type 5 adenovirus DNA in KB cells have been extracted and partially characterized. Replicating molecules sediment faster than 31 S viral DNA at neutral pH and slower than 35 S single-stranded viral DNA at pH 12.5. This indicates that no strands longer than 1 genome length and no covalently closed circular forms are present. The behaviour of the replicating DNA on BND-cellulose columns and the increased buoyant density in CsCl (approximately 0.008 g/cm³ higher than that of viral DNA) indicate that the intermediates contain single-stranded DNA. Electron microscopy of purified replicating DNA has shown the presence of two classes of molecules: linear branched molecules containing single-stranded DNA at the branching point and often on one of the arms, and unbranched molecules which can be either entirely double-stranded, entirely single-stranded, or partially single-stranded. It is concluded that the intermediates essentially consist of linear Y-shaped structures, and that at least one of the strands is discontinuously synthesized.     

Viral nucleic acid sequences in transformed cells: IV. A study of the sequences of adenovirus 5 DNA and RNA in four lines of Adenovirus 5-transformed rodent cells using specific fragments of the viral genome

Specific fragments of Adenovirus 5 DNA were produced by digestion of intact, ³²P-labeled viral DNA with restriction endonucleases Eco R1 and and Hpa 1. The kinetics of renaturation of each fragment and of complete Adenovirus 5 DNA were measured in the presence of DNA extracted from four lines of Adenovirus 5-transformed rodent cells and from nontransformed control cells. All four transformed cell lines contained sequences homologous to the Hpa 1 fragment comprising the left 4% of the viral genome, but varied in the other Adenovirus 5 DNA sequences which were present: three lines of transformed cells contain segments of DNA extending from the left hand end to points 35, 40, and 12% along the viral genome and carry no other Adenovirus 5 DNA sequences. The fourth line also contains sequences homologous to the left half of the viral genome, but these could not be precisely defined. Therefore, the gene(s) encoded by the left end of Adenovirus 5 DNA must specify any viral gene functions expressed in transformed cells.Separated strands of the three Eco R1 fragments and certain Hpa 1 fragments of ³²P-labeled Adenovirus 5 DNA were hybridized with unlabeled, cytoplasmic RNA extracted from each of the four transformed cell lines. In each case, about 10% only of the r strand sequences of the largest Eco R1 fragment were complementary to transformed cell RNA. These sequences have been mapped to the left end of the viral genome using Hpa 1 fragment strands. The same sequences are shown to be expressed as mRNA during the early phase of an Adenovirus 5 lytic infection.