Synthesis of polynucleotide 5'-triphosphatase in vaccinia virus-infected HeLa cells.

Synthesis of polynucleotide 5'-triphosphatase, which is presumably involved in the initial modification in the series of reactions by which 5'-termini of vaccinia mRNA become capped and methylated, has been demonstrated in vaccinia virus infected HeLa cells. Synthesis of the enzyme is prevented by actinomycin D and cycloheximide, suggesting that both de novo DNA-dependent RNA and protein syntheses are required. On the other hand, cytosine arabinoside, an inhibitor of viral DNA replication, does not prevent induction of the enzyme. The latter observation, together with the kinetics of synthesis of the enzyme in vaccinia virus-infected HeLa cells, suggests that polynucleotide 5'-triphosphatase is an "early" or prereplicative viral protein. Immunologlobulin produced against the purified virion-associated polynucleotide 5'-triphosphatase as antigen neutralized the activity of the induced polynucleotide 5'-triphosphatase, thus indicating the identity of the two enzymes.

Purification and characterization of core-associated polynucleotide 5'-triphosphatase from Vaccinia virus.

A core-associated enzyme, designated as polynucleotide 5'-triphosphatase, has been purified from vaccinia. Fractionation on ADP-agarose of the soluble extract from detergent-disrupted cores followed by chromatography on poly(U)-agarose produced an 80-fold purification of the enzyme. The enzyme has an approximate molecular weight of 113,000 and is composed of two polypeptides with approximate molecular weights of 90,000 and 26,000. Divalent metal ions are necessary for enzymatic activity, which occurs optimally at pH 8.4. The enzyme acts upon 5'-ATP- and 5'-GTP-terminated RNA and also on 5'-ATP-terminated poly(A), catalyzing the hydrolysis of only the gamma-phosphate. The presumed biological role of the enzyme based upon this specificity is the participation in the initial step in the sequence of reactions through which the primary 5' termini of vaccinia messenger RNA are capped with the groups m7G(5')ppp(5')NmpN-.

Identification and preliminary characterization of a mutant defective in the bacteriophage T4-induced unfolding of the Escherichia coli nucleoid.

The nucleoids of Escherichia coli S/6/5 cells are rapidly unfolded at about 3 min after infection with wild-type T4 bacteriophage or with nuclear disruption deficient, host DNA degradation-deficient multiple mutants of phage T4. Unfolding does not occur after infection with T4 phage ghosts. Experiments using chloramphenicol to inhibit protein synthesis indicate that the T4-induced unfolding of the E. coli chromosomes is dependent on the presence of one or more protein synthesized between 2 and 3 min after infection. A mutant of phage T4 has been isolated which fails to induce this early unfolding of the host nucleoids. This mutant has been termed "unfoldase deficient" (unf-) despite the fact that the function of the gene product defective in this strain is not yet known. Mapping experiments indicate that the unf- mutation is located near gene 63 between genes 31 and 63. The folded genomes of E. coli S/6/5 cells remain essentially intact (2,000-3,000S) at 5 min after infection with unfoldase-, nuclear disruption-, and host DNA degradation-deficient T4 phage. Nuclear disruption occurs normally after infection with unfoldase- and host DNA degradation-deficient but nuclear disruption-proficient (ndd+), T4 phage. The host chromosomes remain partially folded (1,200-1,800S) at 5 min after infection with the unfoldase single mutant unf39 x 5 or an unfoldase- and host DNA degradation-deficient, but nuclear disruption-proficient, T4 strain. The presence of the unfoldase mutation causes a slight delay in host DNA degradation in the presence of nuclear disruption but has no effect on the rate of host DNA degradation in the absence of nuclear disruption. Its presence in nuclear disruption- and host DNA degradation-deficient multiple mutants does not alter the shutoff to host DNA or protein synthesis.

Unfolding of the host genome after infection of Escherichia coli with bacteriophage T4.

The folded genome of Escherichia coli is converted to a slower-sedimenting form within 5 min after infection with bacteriophage T4 or T4nd28(den A)-amN82(44). Chloramphenicol sensitivity and response to UV-irradiation of the phage suggest participation of viral-induced functions.