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.     

Vaccinia virus infection of HeLa cells. I. Synthesis of vaccinia DNA in host cell nuclei.

The replication of vaccinia virus is thought to take place exclusively in the cytoplasm of host cells. However, using DNA-DNA hybridization techniques, it can be shown that a significant fraction of the synthesis of vaccinia DNA takes place in the nucleus as well as the cytoplasm. The (3H) thymiding pulse-labeled vaccinia DNA synthesized in the nucleus reaches a maximum at about 3 h after infection, corresponding to the time of maximal DNA synthesis in infected cells. At this time host DNA synthesis drops to about 25% of the rate of the uninfected cells. Even with short labeling times (2 min) the nucleus is found to contain 60% of the incorporated (3H)thymidine, much of which is in vaccinia DNA. Prior inhibition of host nuclear DNA synthesis with mitomycin C, followed by removal of the antibiotic causes a subsequent inhibition of vaccinia DNA synthesis and complete suppression of mature virus. Purified nuclei, isolated from vaccinia-infected cells, also synthesize vaccinia DNA in vitro. Over 90% of the DNA synthesized in vitro by isolated nuclei contain vaccinia-specific sequences.     

Synthesis of viral and host DNA in isolated chromatin from herpes simplex virus-infected HeLa cells.

DNA synthesis in chromatin isolated from herpes simplex virus type 1-infected HeLa cells (HSV chromatin) was examined in vitro. The HSV chromatin was found to carry out an initial limited synthesis of DNA in vitro, 50 to 64 pmol of dTMP incorporated in 10(6) nuclei per 10 min, which is comparable to that found in nuclei isolated from HSV-infected cells. DNA synthesis in vitro proceeded for only 30 min, and both HSV DNA and host DNA were synthesized in significant amounts. The HSV and host DNA synthesis in isolated chromatin were inhibited to the same extent by anti-HSV antiserum or by phosphonoacetic acid. The results indicate that the HSV-induced DNA polymerase is most likely involved in the synthesis of host and HSV DNA in isolated chromatin, even though this chromatin contains small amounts of the host gamma-polymerase in addition to the HSV-induced DNA polymerase. The HSV chromatin contains no detectable levels of DNA polymerases alpha and beta, even though infected cells have normal, or increased, levels of these enzymes.     

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.     

Cellular integrity is required for inhibition of initiation of cellular DNA synthesis by reovirus type 3.

Synchronized HeLa cells, primed for entry into the synthesis phase by amethopterin, were prevented from initiating DNA synthesis 9 h after infection with reovirus type 3. However, nuclei isolated from synchronized cells infected with reovirus for 9 or 16 h demonstrated a restored ability to synthesize DNA. The addition of enucleated cytoplasmic extracts from infected or uninfected cells did not affect this restored capacity for synthesis. The addition of ribonucleotide triphosphates to nuclei isolated from infected cells stimulated additional DNA synthesis, suggesting that these nuclei were competent to initiate new rounds of DNA replication. Permeabilization of infected cells did not restore the ability of these cells to synthesize DNA. Nucleoids isolated from intact or permeabilized cells, infected for 9 or 16 h displayed an increased rate of sedimentation when compared with nucleoids isolated from uninfected cells. Nucleoids isolated from the nuclei of infected cells demonstrated a rate of sedimentation similar to that of nucleoids isolated from the nuclei of uninfected cells. The inhibition of initiation of cellular DNA synthesis by reovirus type 3 appears not to have been due to a permanent alteration of the replication complex, but this inhibition could be reversed by the removal of that complex from factors unique to the structural or metabolic integrity of the infected cell.     

DNA polymerase in nuclei isolated from herpes simplex virus type-2-infected cells. Characterization of the reaction product and inhibition by substrate analogs

Nuclei isolated from herpes simplex virus (HSV) type 2-infected KB cells were examined for their capacity to serve as an in situ source of herpes DNA polymerase. In contrast to purified enzymes with added template, approx. 80% of the DNA synthesized in isolated nuclei was viral. The average size of DNA fragments labeled in vitro was 3.2 X 10(6) Da. Based on an increase in DNA density when nuclei were incubated in the presence of BrdUTP rather than dTTP, 16% of the nucleotides were added during the in vitro reaction. Sucrose gradient analysis of DNA polymerase activity in extracts of isolated nuclei demonstrated the nearly exclusive presence of herpes DNA polymerase. Km concentrations for the four dNTPs were from 0.14 to 0.55 microM. DNA synthesis was inhibited competitively by the 5'-triphosphates of ara-A and ara-C (Ki = 0.03 and 0.22 microM, respectively) but not by the 5'-triphosphate of dideoxythymidine. aATP also served as a substrate (Km = 0.014 microM) for the reaction. We conclude that nuclei from HSV-infected cells have significant advantages for the detailed study of inhibitors of herpesvirus replication.     

Synthesis of mRNA guanylyltransferase and mRNA methyltransferases in cells infected with vaccinia virus.

Guanylyltransferase and methyltransferases that modify the 5'-terminals of viral mRNA's to form the structures m7G(5')pppAm- and m7G(5')pppGm- appear to be synthesized afte- vaccinia virus infection of HeLa cells. Elevations in these enzyme activities were detected within 1 h after virus inoculation and increased 15- to 30-fold by 4 to 10 h. Increases in the guanylyl- and methyltransferase activities were prevented by cycloheximide, an inhibitor of protein synthesis, but not by cytosine arabinoside, an inhibitor of DNA synthesis. The latter results suggest that the mRNA guanylyl- and methyltransferases are "early" or prereplicative viral gene products. The guanylyltransferase and two methyltransferases, a guanine-7-methyltransferase and nucleoside-2'-methyltransferase, were isolated by column chromatography from infected cell extracts and found to have properties similar or identical to those of the corresponding enzyme previously isolated from vaccinia virus cores. In contrast, enzymes with these properties could not be isolated from uninfected cells.     

Regulation of Synthesis of Two Immunologically Distinct Nucleic Acid-Dependent Nucleoside Triphosphate Phosphohydrolases in Vaccinia Virus-Infected HeLa Cells

The two nucleic acid-dependent nucleoside triphosphate phosphohydrolases, previously purified from vaccinia virus cores, were shown to be immunologically distinct enzymes. Antiserum prepared against purified phosphohydrolase I and antiserum prepared against purified phosphohydrolase II only neutralized the activity of that enzyme used as antigen. Both enzymes were induced in HeLa cells after vaccinia infection. DNA-cellulose chromatography was used to purify the two phosphohydrolases from the cytoplasms of infected cells. The enzymes were identified by their different substrate specificities, nucleic acid dependence, and neutralization with specific antiserum. A third chromatographically separable nucleic acid-dependent phosphohydrolase similar to phosphohydrolase I in substrate specificity but not neutralizable by antiserum to either phosphohydrolase I or II, was also isolated from infected cells. No nucleic acid-dependent nucleoside triphosphate phosphohydrolase activity was detected by similar methods from uninfected HeLa cells. Formation of these virus-induced enzymes was prevented by actinomycin D and cycloheximide, indicating a requirement for de novo RNA and protein synthesis, respectively. The kinetics of induction and inhibition by cytosine arabinoside, an inhibitor of DNA synthesis, suggested that synthesis of the phosphohydrolases is a late viral function. Rifampin, an inhibitor of vaccinia virus growth which prevents virion assembly, had no inhibitory effect on the induction of the phosphohydrolases. This result was consistent with the finding that these enzymes exist in a soluble as well as in a particulate form in the cytoplasm of infected cells. Addition of another specific anti-poxviral drug, isatin-beta-thiosemicarbazone, to vaccinia-infected cells partially inhibited induction of the phosphohydrolases.     

High expression of functional adenovirus DNA polymerase and precursor terminal protein using recombinant vaccinia virus.

Initiation of Adenovirus (Ad) DNA replication occurs by a protein-priming mechanism in which the viral precursor terminal protein (pTP) and DNA polymerase (pol) as well as two nuclear DNA-binding proteins from uninfected HeLa cells are required. Biochemical studies on the pTP and DNA polymerase proteins separately have been hampered due to their low abundance and their presence as a pTP-pol complex in Ad infected cells. We have constructed a genomic sequence containing the large open reading frame from the Ad5 pol gene to which 9 basepairs from a putative exon were ligated. When inserted behind a modified late promoter of vaccinia virus the resulting recombinant virus produced enzymatically active 140 kDa Ad DNA polymerase. The same strategy was applied to express the 80 kDa pTP gene in a functional form. Both proteins were overexpressed at least 30-fold compared to extracts from Adenovirus infected cells and, when combined, were fully active for initiation in an in vitro Adenovirus DNA replication system.     

Identification and characterization of a DNA primase activity present in herpes simplex virus type 1-infected HeLa cells.

A novel DNA primase activity has been identified in HeLa cells infected with herpes simplex virus type 1 (HSV-1). Such an activity has not been detected in mock-infected cells. The primase activity coeluted with a portion of HSV-1 DNA polymerase from single-stranded DNA agarose columns loaded with high-salt extracts derived from infected cells. This DNA primase activity could be distinguished from host HeLa cell DNA primase by several criteria. First, the pH optimum of the HSV primase was relatively broad and peaked at 8.2 to 8.7 pH units. In contrast, the pH optimum of the HeLa DNA primase was very sharp and fell between pH 7.9 and 8.2. Second, freshly isolated HSV DNA primase was less salt sensitive than the HeLa primase and was eluted from single-stranded DNA agarose at higher salt concentrations than the host primase. Third, antibodies raised against individual peptides of the calf thymus DNA polymerase:primase complex cross-reacted with the HeLa primase but did not react with the HSV DNA primase. Fourth, freshly prepared HSV DNA primase appeared to be associated with the HSV polymerase, but after storage at 4 degrees C for several weeks, the DNA primase separated from the viral DNA polymerase. Separation or decoupling could also be achieved by gel filtration of the HSV polymerase:primase. This free DNA primase had an apparent molecular size of approximately 40 kilodaltons, whereas free HeLa DNA primase had an apparent molecular size of approximately 110 kilodaltons. On the basis of these data, we believe that the novel DNA primase activity in HSV-infected cells may be virus coded and that this enzyme represents a new and important function involved in the replication of HSV DNA.     

Synthesis of herpes simplex virus DNA in isolated chromatin.

Herpes simplex virus DNA synthesis was studied in isolated chromatin (HSV chromatin) of African green monkey kidney (RC-37) cells after HSV type 1 infection. After optimizing the in vitro system, HSV chromatin was shown to synthesize both viral and cellular DNA at ratios identical with those seen in vivo. After 30 min of DNA synthesis in vitro, the DNA products were identical in size to the prelabeled parental DNA. More than 60% of the newly synthesized single-stranded DNA fragments sedimented with a sedimentation constant of greater than 10 S. HSV DNA polymerase was found to be responsible for the synthesis of 80% of all in vitro made viral and most likely also cellular DNA sequences.     

Mode of action of phosphonoformate as an anti-herpes simplex virus agent

Phosphonoformate inhibited the replication of Herpes simplex virus (HSV) type 1 and type 2 in culture. The concentration required to inhibit the replication of both types of virus by 2 logs at 28 h post-infection was approximately 150 microM. It was more potent than phosphonoacetate against the growth of both virus types. A virus mutant which is resistant to phosphonoacetate was cross-resistant to phosphonoformate. Arsonoacetate, at 300 microM, had no antivirus activity. Phosphonoformate also inhibited HeLa and KB cell growth; at a concentration of about 500 microM, cell growth was inhibited by 50%. The anti-cell growth effects of the drug were completely reversible. The antivirus effect of phosphonoformate was partially reversible, depending on the time and duration of exposure of infected cultures to the drug. To obtain the maximum antivirus effect, phosphonoformate had to be added within the first 3 h post-virus-infection and be continuously present for at least 18 h. Phosphonoformate, added at 0 h post-infection, suppressed the induction of virus-specific DNA polymerase and DNAase activities. dTMP incorporation into DNA was preferentially inhibited in nuclei isolated from infected cells compared to uninfected cells, and the degree of inhibition varied with the ionic strength of the assay. Phosphonoformate was a potent inhibitor of the purified HSV-1 and HSV-2 DNA polymerases, inhibiting DNA polymerase activity by 50% at a concentration of 3 microM and ionic strength of 0.2.     

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.     

Nonstructural proteins of herpes simplex virus. I. Purification of the induced DNA polymerase.

Herpes simplex virus-induced DNA polymerase purified by published methods was found to be contaminated with many others proteins, including virus structural proteins. Thus, DEAE-cellulose and phosphocellulose chromatography were used in combination with affinity chromatography to purify DNA polymerase from herpes simplex virus type 1- and type 2-infected cells. The purified enzyme retained unique features of the herpesvirus-induced DNA polymerase, including a requirement for high salt concentrations for maximal activity, a sensitivity to low phosphonoacetate concentrations, and the capacity to be neutralized by rabbit antiserum to herpesvirus-infected cells. By polyacrylamide gel electrophoresis, the purified DNA polymerase was associated with a virus-induced polypeptide of about 150,000 molecular weight.     

Inhibitory effect of various analogs of nucleoside-5'-triphosphates on DNA synthesis catalyzed by DNA polymerase from herpes simplex virus type I

The effect of nucleoside-5-triphosphates analogues on the DNA polymerase of herpes simplex virus (HSV) has been investigated. Evidence is obtained that 3-amino-2,3-dideoxythymidine triphosphate selectively inhibits the DNA synthesis, catalyzed by HSV DNA polymerase. 3-amino-2,3-dideoxythymidine exhibits antiherpetic effect in single cells cultures. It may be phosphorylated by cellular thymidine kinase. The nuclei of Vero cells infected by HSV are an adequate system for antiherpetic compounds screening.     

In vitro synthesis of DNA in nuclei isolated from human lung cells infected with herpes simplex type II virus.

An isolated nuclei system prepared from herpes type II- and mock-infected human embryonic lung cells is able to synthesize cellular and viral DNA in the same proportion as in vivo at various times after infection. Incorporation of (3H)TTP in the in vitro reaction mixture requires Mg2 plus and ATP. Overall in vitro DNA synthesis in nuclei isolated from herpes-infected cells is semiconservative as demonstrated by bromodeoxyuridine-substituted DNA density-transfer experiments, but exhibits a significant fraction of repair-type replication. Relative rates of total DNA synthesis in vitro and in vivo are the same any time after infection. Isolated nuclei synthesize cell and viral DNA for a length of time and at a rate dependent upon the incubation temperature, but there are differences in the length of time of linear in vitro DNA synthesis between herpes- and mock-infected cells. The temperature optima for in vitro DNA synthesis differ significantly for herpes- and mock-infected cells, and are the same for cells abortively infected with herpes type II as for mock-infected cells.     

Reduced yield of infectious pseudorabies virus and herpes simplex virus from cell lines producing viral glycoprotein gp50.

Pseudorabies virus (PRV) glycoprotein gp50 is the homolog of herpes simplex virus (HSV) glycoprotein D. Several cell lines that constitutively synthesize gp50 were constructed. Vero cells, HeLa cells, and pig kidney (MVPK) cells that produce gp50 all gave reduced yields of PRV and HSV progeny viruses when compared with the parent cell line or the same cell line transfected to produce a different protein. The reduction in virus yield was greatest at low multiplicities of infection. The Vero and HeLa cells that produce gp50 showed an even greater reduction in HSV yield than in PRV yield. This phenomenon may be an example in a herpesvirus of the interference observed in retroviruses or cross-protection in plant virus systems.