Vaccinia Virus Replication I. Requirement for the Host-Cell Nucleus

Using cytochalasin B-induced enucleation techniques, we examined the ability of vaccinia virus to replicate in the absence of the host-cell nucleus in several mammalian cell lines. It was found that virus-infected enucleated cells (cytoplasts) prepared from BSC-40, CVC, and L cells were incapable of producing infectious progeny virus. The nature of this apparent nuclear involvement was studied in detail in BSC-40 cells. Modulations designed to maximize cytoplast integrity and longevity, such as reduction of the growth temperature and initial multiplicity of infection, did not improve virus growth in cytoplasts. Sodium dodecyl sulfate-polyacrylamide gel analysis of the [(35)S]methionine pulse-labeled proteins synthesized in vaccinia virus-infected cytoplasts demonstrated that both early and late viral gene products were being expressed at high levels and with the proper temporal sequence. Vaccinia virus cytoplasmic DNA synthesis, as measured by [(3)H]thymidine incorporation, peaked at 3 h postinfection and was 70 to 90% of control levels in cytoplasts. However, in the cytoplasts this DNA was not converted to a DNase-resistant form late in infection, which was consistent with the failure to isolate physical particles from infected cytoplasts. Treatment of vaccinia virus-infected cells with 100 mug of rifampin/ml from 0 to 8 h to increase the pools of viral precursors, followed by subsequent removal of the drug, resulted in a threefold increase virus yield. This treatment had no effect on virus-infected cytoplasts. Finally, vaccinia virus morphogenesis was studied under an electron microscope in thin sections of virus-infected cells and cytoplasts which had been prepared at various times during a single-step growth cycle. It was apparent that, although early virus morphogenetic forms appeared, there was no subsequent DNA condensation or particle maturation in the cytoplasts. These results suggest that vaccinia virus requires some factor or function from the host-cell nucleus in order to mature properly and produce infectious progeny virus.     

Genetic evidence for vaccinia virus-encoded DNA polymerase: isolation of phosphonoacetate-resistant enzyme from the cytoplasm of cells infected with mutant virus.

Phosphonoacetate (PAA), at concentrations of 200 micrograms/ml or more, prevented growth of vaccinia virus in HeLa and BSC-1 cells. Spontaneous vaccinia virus mutants, selected at high PAA levels, were resistant to the antiviral effects of the drug. The action of PAA was directed toward an early viral function, since the drug was inhibitory only during the first 4 h of the approximately 15-h growth cycle. Conversely, significant reversal of the antiviral effects was obtained only when the drug was removed at or before the fourth hour of infection. Incorporation of [3H]thymidine into cytoplasmic viral DNA was severely inhibited in cells infected with wild-type virus but not in cells infected with mutant virus. Virus-induced DNA polymerase isolated from the cytoplasm of cells infected with wild-type or mutant virus had indistinguishable chromatographic properties on DEAE-cellulose and phosphocellulose columns. However, the wild-type enzyme was inhibited by relatively low concentrations of PAA, whereas 10-fold higher concentrations were needed for equivalent inhibition of the mutant enzyme. Kinetic analysis indicated that PAA inhibition was noncompetitive with deoxyribonucleoside triphosphates; Ki values for wild-type and mutant DNA polymerases were approximately 25 and 300 microM, respectively. Inhibition of wild-type DNA polymerase was immediate and complete even when PAA was added after initiation of DNA synthesis in vitro, suggesting that chain elongation was affected. These results established that the DNA polymerase is a target of the antiviral action of PAA and provided genetic evidence that this enzyme is virus encoded.     

Effect of aphidicolin on vaccinia virus: isolation of an aphidicolin-resistant mutant.

Vaccinia virus growth in BSC-1 and HeLa cells was inhibited by aphidicolin concentrations of 20 microM or more. Virus yield, which decreased only when the drug was added early in infection, was reduced several 100-fold by 80 microM aphidicolin. Viral inhibition was reversed by the suspension of the infected cells in drug-free medium. DNA synthesis in uninfected cells was reduced about 10-fold by 1 microM aphidicolin. In infected cells, aphidicolin concentrations over 10 microM were needed to reduce DNA synthesis to the same extent as in uninfected cells. Fractionation of infected cells which were incubated with 1 microM drug showed that cytoplasmic viral DNA synthesis was resistant to this aphidicolin concentration. The radioactivity associated with crude nuclei from these cells was estimated to be from vaccinia DNA synthesis. Spontaneous virus mutants which were resistant to 80 microM aphidicolin did not appear. However, after mutagenesis, mutants were generated which formed large plaques in medium with 80 microM drug. In cells with replicating aphidicolin-resistant virus, DNA synthesis was about four times more resistant to 80 microM aphidicolin than in cells with replicating wild-type virus. Chromatographic patterns of viral DNA polymerase isolated from cells with wild-type or resistant virus were similar. However, in an in vitro assay, 50% inhibition of enzyme activity was obtained with ca. 75 and 188 microM aphidicolin for the wild-type and resistant DNA polymerases, respectively. Viral enzymes were much more resistant to the drug than were the cell polymerases.     

Establishment and Maintenance of the Interferon-Induced Antiviral State: Studies in Enucleated Cells

Requirements for the physical presence of the cell's nucleus for the establishment and maintenance of the interferon-induced antiviral state were investigated. Enucleated chicken embryo fibroblasts were obtained by cytochalasin B treatment during centrifugation. The inhibition of vaccinia virus cytoplasmic DNA synthesis, monitored by autoradiography, was used to measure the antiviral activity resulting from interferon treatment. The antiviral state is not established in cells treated with interferon after removal of their nuclei. On the other hand, cells first treated with interferon for 6 or 12 h and then enucleated express the antiviral state. Furthermore, the antiviral state is maintained in enucleated cells for 16 h after enucleation. The antiviral state appears to be more stable in enucleates than in the residual nucleated cells found in the same cultures. Single cells of antiviral populations are found to be either fully permissive or fully restrictive to vaccinia DNA synthesis. The effect of an increasing intracellular multiplicity of infectious virus is to overcome the antiviral cell's block against viral DNA synthesis.     

Simian virus 40 host range/helper function mutations cause multiple defects in viral late gene expression.

Simian virus 40 (SV40) deletion mutants dlA2459 and dlA2475 express T antigens that lack the normal carboxy terminus. These mutants are called host range/helper function (hr/hf) mutants because they form plaques at 37 degrees C on BSC-1 and Vero monkey kidney cell lines but not on CV-1p monkey kidney cells. Wild-type SV40 can provide a helper function to permit growth of human adenoviruses in monkey kidney cells; the hr/hf mutants cannot. Progeny yields of hr/hf mutants are also cold sensitive in all cell lines tested. Patterns of viral macromolecular synthesis in three cell lines (Vero, BSC-1, and CV-1) at three temperatures (40, 37, and 32 degrees C) were examined to determine the nature of the growth defect of hr/hf mutants. Mutant viral DNA replication was similar to that of the wild type in all three cell lines, indicating that the mutations affect late events in the viral lytic cycle. In mutant-infected Vero cells, in which viral yields were highest, late mRNA levels were similar to those observed during wild-type infection. Levels of viral late mRNA from mutant-infected CV-1 and BSC-1 cells at 32 and 37 degrees C were reduced relative to those of wild-type-infected cells. The steady-state level of the major viral capsid protein, VP1, in mutant-infected CV-1 cells was reduced to the same extent as was late mRNA. The synthesis of agnoprotein could not be detected in mutant-infected CV-1 cells but was readily detected in CV-1 cells infected by wild-type SV40. Primer extension analyses indicated that most late mRNAs from mutant-infected CV-1 cells utilize start sites downstream from the major wild-type cap site (nucleotide 325) and the agnoprotein initiation codon (nucleotide 335). These results indicate that deletion of the carboxyl-terminal domain of T antigen affects viral late mRNA production, both quantitatively and qualitatively. The agnoprotein is detected late in the wild-type SV40 lytic cycle and is thought to play a role in the assembly or maturation of virions. Reduced hr/hf progeny yields could result from decreased capsid protein synthesis and, in the absence of detectable levels of agnoprotein, from inefficient use of available capsid proteins.     

Location of DNA-binding proteins and disulfide-linked proteins in vaccinia virus structural elements.

Treatment with sodium dodecyl sulfate (SDS) converted the vaccinia virus strain IHD-J into particles of two types: (i) ghosts which possessed a thin-membrane vesicle derived from basement part of the virus membrane with attached lateral bodies and a membranous structure derived from the core wall and (ii) aggregates of a DNA-nucleoprotein eluted from the core. These particles lacked lipids, and all the viral phospholipids were detected in the SDS-soluble fraction. The viral membrane was composed of an SDS-soluble coat layer and the basement membrane, and the basement membrane was maintained by a mechanism other than the lipid bilayer. By comparisons of protein species in morphologically distinct subviral particles prepared by several solubilizing methods, protein compositions of viral structural elements were suggested as follows: 25,000-molecular-weight viral protein-17,000-molecular-weight viral protein ( VP25K - VP17K ), viral basement membrane; VP13 . 8K , major component of the lateral body; VP70K , VP69K , VP66K , and VP64K , minor components of the lateral body; VP61K , outer layer of core wall; VP57K - VP22K , inner layer of core wall; and VP27K - VP13K , nucleoprotein. These structural elements found in the SDS-insoluble particles dissolved in the same SDS solution under reducing conditions, indicating that the disulfide linkages seem to have a principal role in maintaining their morphological integrity. VP57K , VP27K , VP13 . 8K , and VP13K were revealed to possess affinity for DNA. Denatured calf thymus DNA and viral DNA in double- or single-stranded form associated equally well with these proteins, but RNA did not bind. Therefore, it was strongly suggested that disulfide-linked VP27K - VP13K represented the nucleoproteins of vaccinia virus. A structural model of vaccinia virus is proposed and discussed.     

Vaccinia Virus Replication in Enucleate BSC-1 Cells: Particle Production and Synthesis of Viral DNA and Proteins

The growth of vaccinia virus in monolayers of BSC-1 cells enucleated by centrifugation in the presence of cytochalasin B has been studied. No evidence for the production of infectious virus in these cells was obtained, and the production of virus particles was reduced to 8.3% compared with the yield from cytochalasin-treated, uncentrifuged cells. Virus DNA and early and late polypeptides were synthesized with normal timing in enucleate cells, but in reduced amounts; cleavage of structural polypeptide precursors P4a and Px also occurred in enucleate cells. Factories containing immature virus particles were demonstrated in enucleate cells by electron microscopy; these factories were reduced in number and size compared with those found in cytochalasin-treated, uncentrifuged cells.     

Isolation and characterization of defective simian virus 40 genomes which complement for infectivity.

A new variant of simian virus 40 (EL SV40), containing the complete viral DNA separated into two molecules, was isolated. One DNA species contains nearly all of the early (E) SV40 sequences, and the other DNA contains nearly all of the late (L) viral sequences. Each genome was encircled by reiterated viral origins and termini and migrated in agarose gels as covalently closed supercoiled circles. EL SV40 or its progenitor appears to have been generated in human A172 glioblastoma cells, as defective interfering genomes during acute lytic infections, but was selected during the establishment of persistently infected (PI) green monkey cells (TC-7). PI TC-7/SV40 cells contained EL SV40 as the predominant SV40 species. EL SV40 propagated efficiently and rapidly in BSC-1, another line of green monkey cells, where it also formed plaques. EL SV40 stocks generated in BSC-1 cells were shown to be free of wild-type SV40 by a number of criteria. E and L SV40 genomes were also cloned in the bacterial plasmid pBR322. When transfected into BSC-1 cell monolayers, only the combination of E and L genomes produced a lytic infection, followed by the synthesis of EL SV40. However, transfection with E SV40 DNA alone did produce T-antigen, although at reduced frequency.     

Deoxyribonucleic Acid Replication in Simian Virus 40-Infected Cells: III. Comparison of Simian Virus 40 Lytic Infection in Three Different Monkey Kidney Cell Lines

A comparative study of simian virus 40 (SV40) lytic infection in three different monkey cell lines is described. The results demonstrate that viral deoxyribonucleic acid (DNA) synthesis and infectious virus production begin some 10 to 20 hr earlier in CV-1 cells and primary African green monkey kidney (AGMK) cells than in BSC-1 cells. Induction of cellular DNA synthesis by SV40 was observed in CV-1 and AGMK cells but not with BSC-1 cells. Excision of large molecular weight cellular DNA to smaller fragments was easily detectable late in infection of AGMK cells. Little or no excision was observed at comparable times after infection of CV-1 and BSC-1 cells. The different kinds of responses of these three monkey cell lines during SV40 lytic infection suggest the involvement of cellular functions in the virus-directed induction of cellular DNA synthesis and the excision of this DNA from the genome.     

Pattern of Protein Synthesis in Monkey Cells Infected by Simian Virus 40

After infection of several permanent monkey cell lines by simian virus 40 (SV40), four additional protein bands can be detected by simple sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole-cell extracts. These bands appear only after the onset of viral deoxyribonucleic acid (DNA) synthesis, and inhibitors of DNA synthesis prevent their appearance. Three of them correspond to three previously identified capsid components, VP1, VP2, and VP3. The fourth protein band, which does not correspond to a previously identified virion component, is induced by SV40 infection of CV-1 and BSC-1 cultures but not by infection of MA-134 cultures.     

Vaccinia virus RNA polymerase associated with nuclei of infected HeLa cells.

Though vaccinia virus DNA and RNA replication take place predominantly in the cytoplasm of an infected cell, virus formation requires the presence of a functional nucleus in a yet undefined manner. When the nuclei from cells infected for 3 h are isolated and purified, they are found to synthesize five times more RNA in vitro than do corresponding nuclei from noninfected cells. Fifty percent of the RNA synthesized in vitro by nuclei from infected cells is vaccinia specific, and this vaccinia RNA synthesis is resistant to alpha-amanitin concentrations up to 100 micrograms/ml. Furthermore, when the RNA polymerase activities of these nuclei are separated on DEAE-Sephadex columns, 56% of the total nuclear enzyme activity is found to be the vaccinia-specific RNA polymerase known to be alpha-amanitin resistant. The nucleus associated vaccinia RNA polymerase represents 18% of the total cellular vaccinia RNA polymerase. This synthesis of vaccinia RNA in the nucleus may explain the nuclear requirement for vaccinia virus maturation.     

Mapping the genomic location of the gene encoding alpha-amanitin resistance in vaccinia virus mutants.

To facilitate the determination of the genomic location of the vaccinia virus gene(s) encoding alpha-amanitin resistance (alpha r) (Villarreal et al., J. Virol. 51:359-366, 1984), a collection of alpha r, temperature-sensitive (ts) mutants were isolated. The premise of these experiments was that mutants might be found whose dual phenotypes were the result of a single or two closely linked mutations. Genetic analyses of the alpha rts mutant library revealed two mutants, alpha rts7 and alpha rts12, that apparently fit this criterion; in alpha rts7 the two lesions were indistinguishable, whereas in alpha rts12 the two mutations were closely linked but separable. Cloned vaccinia virus HindIII DNA fragments were used to marker rescue the temperature-sensitive phenotype of these two dual mutants. The temperature-sensitive lesion of alpha rts7 was rescued by the HindIII N fragment (1.5 kilobases), whereas alpha rts12 was rescued by the neighboring HindIII M fragment (2.0 kilobases). The progeny virions of the alpha rts7 HindIII-N rescue reverted to an alpha-amanitin-sensitive phenotype, whereas the alpha rts12 HindIII-M progeny were still resistant to the drug. Taken together, these data indicate that the gene encoding alpha-amanitin resistance maps to the HindIII N fragment and provides evidence for the existence of essential vaccinia virus genes in a region of the genome previously believed to be nonessential for replication in tissue culture. Biochemical analyses revealed that both mutants were capable of synthesizing DNA as well as early and late viral proteins at the permissive and nonpermissive temperatures. At the nonpermissive temperature alpha rts12 and alpha rts7 were unable to process the major core precursors P94 and P65 into VP62 and VP60.     

Recurring defective variants of simian virus 40 containing monkey DNA segments.

Four independently and newly isolated defective variants of simian virus 40 have been characterized. All four are very similar, if not identical, to two previously and independently isolated variants (Wakamiya et al., J. Biol. Chem. 254:3584-3591, 1979; J. Papamatheakis, E. Kuff, E. Winocour, and M. F. Singer, J. Biol. Chem. 255:8919-8927, 1980). The documented similarities include restriction endonuclease maps and the presence of the same monkey DNA segments covalently linked to simian virus 40 DNA sequences. Each of the newly described variants was first detected upon serial passaging of wild-type simian virus 40 at a high multiplicity of infection at 33 degrees C as recently described (M. F. Singer and R. E. Thayer, J. Virol. 35:141-149, 1980). A variety of experiments support the idea that the various isolates were independent and do not reflect inadvertent cross-contamination. Two of the new isolates arose during passage of wild-type strain 777 virus in BSC-1 cells, one during passage of strain 776 in BSC-1 cells, and one during passage of strain 776 in primary African green monkey kidney cells. The two variants obtained after passage of strain 776 were shown to contain a particular recognition site for restriction endonuclease MboII within their simian virus 40 DNA segments, as do the two previous isolates. This site is not present in wild-type strain 776 DNA but is shown here to be present in wild-type strain 777 DNA. The surprising recurrence of closely related variants and particularly the unexpected presence of the endo R.MboII site in variants derived from passaging strain 776 suggest that these variants may arise by mechanisms other than recombination between the initial infecting viral genome and the host DNA.     

Viral and Host Deoxyribonucleic Acid Synthesis in Shope Fibroma Virus-infected Cells as Studied by Means of High-Resolution Autoradiography

Incorporation of (3)H-thymidine by BSC-1 cells infected with Shope fibroma virus was studied by means of high-resolution electron microscopic radioautography. One-hour pulses with the radioactive precursor were given at various times after infection, during a one-step growth cycle of the virus. In the cytoplasm of infected cells, reacted grains occurred over foci of viroplasm; these foci are believed to represent the true sites of viral deoxyribonucleic acid (DNA) replication. Shope fibroma virus DNA synthesis began before 3 hr postinfection, reached a maximum at 8 to 9 hr, and then declined rapidly. It was demonstrated that the decline in (3)H-thymidine uptake is correlated with the onset of viral morphogenesis. In comparison with the noninfected culture, the nuclear labeling, which reflects host DNA metabolism, was slightly reduced by 4 hr postinfection. Inhibition became more marked as infection progressed, and host DNA synthesis was almost completely suppressed in late stages of viral development.     

Simian virus 40 agnoprotein facilitates normal nuclear location of the major capsid polypeptide and cell-to-cell spread of virus.

The simian virus 40 agnoprotein is a 61-amino-acid, highly basic polypeptide that is coded within the 5' leader of late 16S mRNAs. To better understand agnoprotein function and to more effectively differentiate cis-from trans-acting effects of an agnogene mutation, we constructed a mutant virus that carries a single-base-pair substitution and fails to produce agnoprotein. pm 1493 contains a T/A to A/T transversion at sequence position 335. This mutation converts the agnoprotein initiation codon from ATG to TTG, preventing synthesis of the protein. The mutant displays only a modest growth defect in CV-1P and AGMK cells and no defect in BSC-1 cells. Early-gene expression, DNA replication, synthesis of late viral products, and the kinetics of virion assembly all appear normal in pm 1493-infected CV-1P cells. Immunofluorescent studies, however, indicate that localization of the major capsid polypeptide VP1 is different in mutant- than wild-type virus-infected cells. Furthermore, the lack of agnoprotein led to inefficient release of mature virus from the infected cell. Agnogene mutants could be severely compromised in their ability to propagate in monkeys given their reduced capacity for cell-to-cell spread.     

Preferential replication of a class of host-substituted defective simian virus 40 variants at low temperature

The host-substituted variant termed CVP8/1/P2 (EcoRI res) was first isolated several years ago after serial passage of simian virus 40 strain 777 on BSC-1 cells at 37 degrees C. When BSC-1 are coinfected with wild-type simian virus 40 strain 777 and variant CVP8/1/P2 (EcoRI res), the variant rapidly becomes the dominant species produced, often representing as much as 80% of the total DNA I synthesized after infection. We present evidence that the replicative advantage of the variant was increased when the infection was carried out at 33 rather than 37 degrees C. Also described are nine new and independent serial passage experiments carried out at 33 degrees C with several purified wild-type virus stocks, including strain 776, and both BSC-1 and primary African green monkey kidney cells. In each series variants related to CVPs/1/P2 (EcoRI res) were detected in the progeny viral genomes after four serial passages. Hybridization data suggest that at least some of these variant DNA I molecules contain simian virus 40 DNA sequences, monkey alpha-component DNA sequences (highly repetitive), and the infrequently reiterated monkey DNA sequences found in CVP8/1/P2 (EcoRI res), all covalently linked as in CPV8/1/P2 (EcoRI res). It appears that this type of variant emerges with some frequency during infection and is then preferentially replicated at 33 degrees C, thereby becoming readily detectable in passaged stocks. A variety of control experiments indicated that the repeated emergence of similar, if not identical, variants is unlikely to be the result of inadvertent cross-contamination or the presence of detectable amounts of the variant in the plaque-purified viral stocks.     

Hydroxyurea-resistant vaccinia virus: overproduction of ribonucleotide reductase.

Repeated passages of vaccinia virus in increasing concentrations of hydroxyurea followed by plaque purification resulted in the isolation of variants capable of growth in 5 mM hydroxyurea, a drug concentration which inhibited the reproduction of wild-type vaccinia virus 1,000-fold. Analyses of viral protein synthesis by using [35S]methionine pulse-labeling at intervals throughout the infection cycle revealed that all isolates overproduced a 34,000-molecular-weight (MW) early polypeptide. Measurement of ribonucleoside-diphosphate reductase (EC 1.17.4.1) activity after infection indicated that 4- to 10-fold more activity was induced by hydroxyurea-resistant viruses than by the wild-type virus. A two-step partial purification which yielded greater than 90% of the induced ribonucleotide reductase activity in the fraction obtained by 35% saturation with ammonium sulfate resulted in a substantial enrichment for the 34,000-MW protein from extracts of wild-type and hydroxyurea-resistant-virus-infected, but not mock-infected, cells. In the presence of the drug, the isolates incorporated [3H]thymidine into DNA earlier and at a rate substantially greater than that of the wild type, although the onset of DNA synthesis was delayed in both cases. In the absence of the drug, the attainment of a maximum viral DNA synthesis rate was accelerated after infection by drug-resistant isolates. The drug resistance trait was markedly unstable in all isolates. In the absence of selective pressure, plaque-purified isolates readily segregated progeny that displayed a wide range of resistance phenotypes. The results of this study indicate that vaccinia virus encodes a subunit of ribonucleotide reductase which is a 34,000-MW early protein whose overproduction confers hydroxyurea resistance on reproducing viruses.     

Expression of polyomavirus virion proteins by a vaccinia virus vector: association of VP1 and VP2 with the nuclear framework.

The polyomavirus proteins VP1, VP2, and VP3 move from their cytoplasmic site of synthesis into the nucleus, where virus assembly occurs. To identify cellular or viral components which might control this process, we determined the distribution of VP1, VP2, and VP3 in a soluble fraction, a cytoplasmic cytoskeleton fraction, and a nuclear framework fraction of infected cells. All three proteins were detected in a detergent-extractable form immediately after their synthesis in polyomavirus-infected cells. Approximately 50, 25, and 40% of pulse-labeled VP1, VP2, and VP3, respectively, associated with the skeletal framework of the nucleus within 10 min after their synthesis. The remaining portion of each labeled protein failed to accumulate on the nuclear framework during a 40-min chase and was degraded. When expressed separately by recombinant vaccinia viruses, VP1 and VP2, but not VP3, accumulated on the nuclear framework. This association was not dependent on other polyomavirus proteins or viral DNA. The amount of total VP1 and VP2 which was bound to the nuclear framework approximated 45 and 20%, respectively. Indirect immunofluorescence demonstrated an exclusive nuclear localization of VP1 in situ. In coinfection experiments, a greater percentage of total VP2 and VP3 was bound to the nuclear framework of cells which cosynthesized VP1. These results indicate that although VP1 and VP2 can bind independently to the insoluble nuclear framework, the association of VP3 with this nuclear structure is promoted by the presence of VP1.