Phenotypic characterization of temperature-sensitive mutants of vaccinia virus with mutations in a 135,000-Mr subunit of the virion-associated DNA-dependent RNA polymerase.

The phenotypic defects of three temperature-sensitive (ts) mutants of vaccinia virus, the ts mutations of which were mapped to the gene for one of the high-molecular-weight subunits of the virion-associated DNA-dependent RNA polymerase, were characterized. Because the virion RNA polymerase is required for the initiation of the viral replication cycle, it has been predicted that this type of mutant is defective in viral DNA replication and the synthesis of early viral proteins at the nonpermissive temperature. However, all three mutants synthesized both DNA and early proteins, and two of the three synthesized late proteins as well. RNA synthesis in vitro by permeabilized mutant virions was not more ts than that by the wild type. Furthermore, only one of three RNA polymerase activities that was partially purified from virions assembled at the permissive temperature displayed altered biochemical properties in vitro that could be correlated with its ts mutation: the ts13 activity had reduced specific activity, increased temperature sensitivity, and increased thermolability under a variety of preincubation conditions. Although the partially purified polymerase activity of a second mutant, ts72, was also more thermolabile than the wild-type activity, the thermolability was shown to be the result of a second mutation within the RNA polymerase gene. These results suggest that the defects in these mutants affect the assembly of newly synthesized polymerase subunits into active enzyme or the incorporation of RNA polymerase into maturing virions; once synthesized at the permissive temperature, the mutant polymerases are able to function in the initiation of subsequent rounds of infection at the nonpermissive temperature.

Fine structure marker rescue of temperature-sensitive mutations of vaccinia virus within a central conserved region of the genome.

Fine structure marker rescue involving the use of subfragments of vaccinia virus HindIII DNA fragments L, J, and H has been used to map the mutations in eight temperature-sensitive mutants of vaccinia virus representing four complementation groups. Comparison of their map locations with the positions of the open reading frames and RNA transcripts that have been mapped within this region has allowed the identification of one or two polypeptides as the temperature-sensitive gene product of each mutant.

Marker rescue of temperature-sensitive mutations of vaccinia virus WR: correlation of genetic and physical maps.

The physical map locations of 62 temperature-sensitive mutations of vaccinia virus WR have been determined by marker rescue experiments, using cloned HindIII fragments of wild-type DNA. Since vaccinia virus DNA is not infectious, marker rescue was performed by infecting monolayers of cells at the nonpermissive temperature with a low multiplicity of the mutant to be rescued and transfecting with calcium phosphate-precipitated recombinant DNA. Wild-type recombinants were measured by using either a direct plaque assay technique or a two-step procedure in which the final yield of virus from the transfected cells was assayed at the permissive and nonpermissive temperatures. Mutants that had been previously assigned to the same complementation-recombination group were rescued by the same HindIII fragment, with the exception of three mutants in one group that were rescued by either one of two adjacent fragments. A comparison between the genetic linkage map of the temperature-sensitive mutations in 30 mutants with their physical locations demonstrated that not only was the order of the genetic map correct but also recombination frequencies generally reflected actual physical distances.

Isolation and genetic characterization of temperature-sensitive mutants of vaccinia virus WR.

One hundred temperature-sensitive mutants of vaccinia virus WR were isolated from virus that had been mutagenized with 5-bromodeoxyuridine or N-methyl-N'-nitro-N-nitrosoguanidine. A rapid screening procedure based on the ability of vaccinia virus to form plaques under liquid overlay medium was used to identify potential mutants among randomly picked plaque isolates or plaques preselected for their small size after temperature shift-up. The preselection technique resulted in a sixfold increase in the number of successful mutant isolations relative to the number of plaques picked. All of the mutants had efficiencies of plating at 39.5 degrees C relative to that at 33 degrees C of 10(-4) or less, and 33 of 40 produced 10% or less of the amount of virus at the nonpermissive temperature (39.5 degrees C) relative to that at the permissive temperature (33 degrees C). Experiments with the fluorescent DNA binding dye Hoechst 33258 demonstrated that 6 of the 100 mutants failed to form characteristic cytoplasmic DNA factories at 39.5 degrees C. To facilitate the functional grouping of such a large number of mutants, a rapid infectious center assay was developed. Thirty of the mutants were assigned to 16 or 17 complementation-recombination groups by using this assay. Recombination experiments have allowed the construction of a genetic map representing 22 mutants in 12 of these groups.

HeLa cell RNA (2'-O-methyladenosine-N6-)-methyltransferase specific for the capped 5'-end of messenger RNA.

A novel enzyme involved in the post-transcriptional modification of the 5'-end of mRNA has been partially purified from HeLa cells. Termed an S-adenosyl-L-methionine:RNA(2'-O-methyladenosine-N4)-methyltransferase, the enzyme specifically catalyzes the transfer of a methyl group from S-adenosylmethionine to the N6 position of a 2'-O-methyladenosine residue located within the "capped" 5'-end of mRNA. The dimethylated nucleoside, N6,2'-O-dimethyladenosine, is formed as indicated by the following reaction in which m7G(5')pppAm- represents the capped and methylated 5'-end of mRNA: AdoMet + m7G(5')pppAm- leads to AdoHcy + m7G(5')pppm6A7- Greatest activity is obtained with RNA acceptors ending in m7G(5')pppAm-; less activity is found with RNA ending in m7G(5')pppA-; and barely detectable and no activity is found with RNA ending in G(5')pppA- and ppA-, respectively. Furthermore, no activity is found with oligonucleotides such as m7G(5')pppA, m7G(5')pppAm, and m7G(5')pppAmpN indicating that a longer polymer is required. It can be concluded from the substrate specificity of the enzyme that the formation of N6,2'-O-dimethyladenosine follows the biosynthesis of molecules containing m7G(5')pppAm-N-. The molecular weight of the methyltransferase, as determined by sedimentation in sucrose gradients, is approximately 65,000.

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.

Modification of the 5' terminus of mRNA by an RNA (guanine-7-)-methyltransferase from HeLa cells.

The 5' termini of many viral and cellular mRNAs contain sequences of the type m7G(5")pppNm. An RNA (guanine-7-)-methyltransferase that specifically methylates the 5'-terminal guanosine residue of RNAs ending in the dinucleoside triphosphate G(5')pppN- has been purified from the cytoplasm of HeLa cells. Approximately two-thirds of the methyltransferase activity detected in an assay employing umnethylated vaccinia virus mRNA as acceptor was located in the cytoplasm when cells were disrupted by Dounce homogenization; 30% of the cytoplasmic activity was associated with ribosomes but was removed by washing with 0.5 M KCl. The enzyme was purified 165-fold from the cytoplasm by removing nucleic acid by phase partition followed by ammonium sulfate precipitation and column chromatography on DEAE-cellulose, denatured DNA-agarose, and CM-Sephadex. The partially purified enzyme preparation methylated heterologous tRNAs as well as vaccinia mRNA, but the tRNA methyltransferases could be separated from the mRNA activity by sucrose gradient sedimentation and gel filtration on Sephadex G-200. The product of the partially purified enzyme using vaccinia mRNA as substrate was exclusively 7-methylguanosine located in the terminal dinucleoside triphosphate. In addition to RNAs and synthetic polyribonucleotides terminating in a dinucleoside triphosphate, free G(5')pppG could be methylated but GTP, GDP, and G(5')pppG could not. The enzyme also methylated the dinucleoside diphosphate G(5')pppG but much less efficiently than G(5')pppG. An S20, W of 3.8, a Stokes radius of 3.6 nm, and a molecular weight of 56,000 were obtained from sucrose gradient sedimentation and Sephadex G-200 column chromatography.

Modification of the 5'-terminus of mRNA by soluble guanylyl and methyl transferases from vaccinia virus.

RNA guanylyl and methyl transferases have been solubilized from vaccinia virus cores. The guanylyl transferase specifically adds a GMP residue to the 5'-terminus of unmethylated vaccinia virus mRNA to form the structures G(5')ppp(5')Gp- and G(5')ppp(5')Ap-. Studies with [alpha-32P]GTP and [beta, gamma-32P]GTP indicated that only the alpha-phosphate is transferred. In the presence of S-adenosylmethionine, the methyl transferases convert the blocked 5'-termini to m7G(5')ppp(5')Gmp- and m7G(5')ppp(5')Amp-. Similarly, the enzymes can modify synthetic poly(A) to form the structure m7G(5')ppp(5')Amp-.

Thermolabile DNA binding proteins from cells infected with a temperature-sensitive mutant of adenovrius defective in viral DNA synthesis.

Infection of African green monkey kidney cells with type 5 adenovirus leads to the synthesis of two infected, cell-specific proteins with approximate molecular weights of 72,000 and 48,000, that bind specifically to single-stranded but not double-stranded DNA. The production of these two proteins was studied after infection with two DNA-negative adenovirus mutants belonging to different complementation groups (H5 ts36 and H5 ts 125). Both DNA binding proteins were detected in cells infected with either mutant at the permissive temperature (32 C) AND ALSO IN H5 ts36-infected cells at the nonpermissive temperature (39.5 C). In H5 ts125-infected cells at 39.5 C, however, less than 5% of the normal wild-type level of these DNA binding proteins was detectable. When H5 ts125-infected cells were labeled with radioactive leucine at 32 C and subsequently shifted to 39.5 C in the presence of unlabeled leucine (chase), the level of DNA binding proteins found in these infected cells was markedly reduced compared to cultures not shifted to 39.5 C. These data suggest that the DNA binding proteins themselves were temperature sensitive. This conclusion was confirmed by experiments in which the DNA binding proteins were eluted from DNA cellulose with buffers of increasing temperatures (thermal elution). The H5 ts 125 proteins were shown to elute at lower temperatures than either wild-type or H5 ts36 proteins. These results are taken to indicate that the H5 ts125 mutant codes for a DNA binding protein that is thermolabile for continued binding to single-stranded DNA.

Selection and preliminary characterization of temperature-sensitive mutants of type 5 adenovirus.

Eight temperature-sensitive (ts) mutants that replicate normally at 32 C but poorly, if at all, at 39.5 C have been isolated from mutagenized stocks of a wild-type strain of type 5 adenovirus. Three mutagens were employed: nitrous acid, hydroxylamine, and nitrosoguanidine. Ts mutants were isolated from mutagenized viral stocks with frequencies between 0.01 and 0.1%. All eight mutants had reversion frequencies of 10(-5) or less. Complementation experiments in doubly infected cultures at the nonpermissive temperature separated the mutants into three nonoverlapping complementation groups. Complementation yields ranged from a 2.3- to a 3,000-fold increase over the sums of the yields from the two singly infected controls. Genetic recombination was also demonstrated; approximate recombination frequencies ranged from 0.1 to 15%. Preliminary biochemical and immunological characterization of the mutants indicated that: (i) the single mutant in complementation group I did not replicate its deoxyribonucleic acid (DNA) or synthesize late proteins at the nonpermissive temperature but did inhibit host DNA synthesis to 25% of an uninfected control; (ii) the four group II mutants replicated viral DNA, shut off host DNA synthesis, synthesized penton base and fiber, but did not synthesize immunologically detectable hexon; the three mutants in complementation group III synthesized viral DNA, shut off host DNA synthesis, and made immunologically reactive capsid proteins (hexon, penton base, and fiber).