Vaccinia Virus Cores Research Articles

Characterization of a polyriboadenylate polymerase from vaccinia virions.

A poly(A) polymerase with a molecular weight of approximately 80,000 containing 51,000 and 35,000 molecular weight subunits, was purified by affinity chromatography from vaccinia virus cores. The enzyme had a pH optimum of about 8.6, was dependent on divalent cations, and had considerably more activity with Mn-2+ than Mg-2+. At equimolar concentrations, other ribonucleoside triphosphates inhibited poly(A) polymerase activity by less than 10%; NaCl was extremely inhibitory at concentrations above 0.1 M. Under standard assay conditions, poly(A) polymerase activity was stimulated more than 10-fold by poly(C), but to small extent or not at all by other homopolyribonucleotides or natural RNA species unless they were first subjected to partial hydrolysis and alkaline phosphatase treatment. The ineffectiveness of most long polyribonucleotides was attributed to enzyme binding to internal regions. Short poly- or oligoribonucleotides prepared from natural or synthetic RNAs, except poly(G), exhibited similar priming abilities, and isotope transfer experiments indicated the covalent attachment of poly(A) to cytidylate, uridylate, and inosinate residues. Experiments with a series of uridylate oligomers indicated that the minimum effective primer length was four to six nucleotides. Partially digested DNA and short poly- and oligodeoxyribonucleotides of dT, dC, and dI, but not of dA and dG, also acted as effective primers for the poly(A) polymerase.

Messenger activity of RNA transcribed in vitro by DNA-RNA polymerase associated to vaccinia virus cores.

The coding properties of RNA transcribed in vitro by purified vaccinia cores have been investigated using Krebs ascites tumor cells, L cells, and reticulocyte lysates. Six to 10 proteins synthesized in vitro are separated on polyacrylamide gels by electrophoresis in the presence of sodium dodecyl sulfate. Their molecular weights vary from 10,000 to 44,000. The electrophoretic behavior of these proteins is similar to that of early proteins isolated from infected L cells. The tryptic peptide analysis of one of these proteins indicates similarity in amino acid sequences. These results show fidelity of both in vitro transcription and molecular weight above 44,000 are synthesized in vitro does not seem due to a competition between 12S mRNA synthesized in excess and RNA of a higher sedimentation coefficient present in a lower amount.

Characterization of a protein kinase and two phosphate acceptor proteins from vaccinia virions.

The phosphorylation of two purified vaccinia virus proteins (Acceptors I and II) by a protein kinase isolated from vaccinia virus cores has been studied. Phosphorylation of viral acceptor proteins by the purified enzyme was dependent on the presence of ATP, Mg2+, and protamine or other basic proteins, and was maximal at alkaline pH values. Cyclic mononucleotides did not stimulate the vaccinia protein kinase under a variety of conditions. Protamine, however, was shown to function as an enzyme activator. In its presence, the purified vaccinia protein kinase phosphorylated mainly serine residues in Acceptor I, and predominantly threonine residues in Acceptor II. Phosphorylation of protamine accounted for less than 1% of the total 23P incorporation. Tryptic peptide maps prepared from 32P-labeled Acceptors I and II demonstrated that they contained different labeled peptide sequences and were, therefore, distinct protein species. From additional studies on both purified and virus-associated protein kinase it was concluded that various proteins affected the protein kinase reaction in one of three ways. One class of proteins served as phosphate acceptors, but only when another activator protein was present. A second class consisted of proteins that were strong activators but poor phosphate acceptors. The third class contained proteins that were fair phosphate acceptors, but which also activated the phosphorylation of other acceptor proteins.

Poly(A) sequences of vaccinia virus messenger RNA: Nature, mode of addition and function during translation in vitro and in vivo

Studies were undertaken of the nature and mode of addition of the poly(A) sequences at the 3′-termini of vaccinia virus messenger RNA, as well as of their function during translation in vivo and in vitro. The median length of poly(A) sequences on messenger RNA synthesized in vivo, both “early” and “late” in infection, and on RNA transcribed by vaccinia virus cores in vitro was the same, namely about 100 residues. Poly(A) on messenger RNA synthesized in vivo was slightly more homogeneous in size than that on RNA transcribed in vitro. No nucleic acid bases other than adenine were present. Base composition analysis of poly(A) liberated by pancreatic and T1 ribonuclease indicated that the average 3′-terminal sequences of vaccinia virus RNA were G(C2 U4 A?)A100 Aoh.When vaccinia virus messenger RNA was hybridized to DNA, the poly(A) sequences remained free, and at least 95% of them could be removed by treatment with pancreatic and/or T1 ribonuclease, with no detectable diminution in size. This was interpreted as evidence that they were added posttranscriptionally, rather than copied from sequences of poly(dT) in the viral genome. In fact, the vaccina virus strain WR genome was found to contain essentially no poly(dT) sequences (less than 0.015% of the virus genome) as judged by poly(A) binding.In the presence of cordycepin, the formation of both early and late vaccinia messenger RNA in infected cells was markedly inhibited (90% inhibition by 50 μg/ml). The residual 10% of messenger RNA that was still formed under these conditions contained poly(A) sequences that were only about one-half their normal length, but gave rise to about one-half of the amount of virus-specified protein that was synthesized in the absence of cordycepin. Halving the length of poly(A) sequences on vaccinia virus messenger RNA therefore appears to have no adverse effects on its ability to be translated in vivo.Not all vaccinia virus messenger RNA molecules possessed poly(A); both early and late messenger RNA synthesized in vivo, as well as RNA transcribed in vitro, contained about 10% of molecules that did not bind to poly(U)-cellulose and contained no poly(A). The poly(A) (−) RNA molecules that were transcribed in vitro had the same size as poly(A) (+) RNA, and possessed the same base sequences as judged by hybridization analysis. Further, they were translated as efficiently as poly(A) (+) RNA molecules in an in vitro Krebs II ascites cell-free protein synthesizing system, yielding the same polypeptides. The presence of poly(A) was therefore not essential for translation in vitro. The pattern of polypeptides that was obtained was complex; this was no doubt due to the fact that, as determined by hybridization analysis, the messenger RNA synthesized by vaccinia virus cores in vitro was transcribed from 50% of the viral genome. This degree of complexity was quantitatively comparable to that of early messenger RNA synthesized in vivo.

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.

Vaccinia virus polyriboadenylate polymerase: convalent linkage of the product with polyribonucleotide and polydeoxyribonucleotide primers.

A POLYRIBOADENYLATE [POLY(A)] POLYMERASE, PURIFIED FROM VACCINIA VIRUS CORES, WAS STIMULATED BY POLYDEOXYRIBOADENYLATE: polydeoxyribothymidylate [poly(dA:dT)] and by polyribocytidylate [poly(C)] primers suggesting mechanisms of either transcription or terminal addition. Evidence for the latter was obtained by the demonstration of covalent linkages between the poly(A) products and both primers. In 99% dimethylsulfoxide-sucrose gradients, the sedimentation of poly(A) formed with poly(dA: dT) primer was reduced after DNase I treatment and the sedimentation of poly(A) formed with poly(C) primer was reduced by RNase A treatment, whereas the sedimentation of poly(A) formed without primer was not affected by either. Formation of a phosphodiester bond between primer and product was demonstrated by means of isotope transfer experiments. (32)P from alpha-[(32)P]ATP was transferred to 2'(3')-CMP after alkaline or enzymatic hydrolysis of the poly(C)-primed polymerase reaction product. Transfer primarily or exclusively to 3'-dTMP was found after enzymatic hydrolysis of the poly(dA: dT)-primed polymerase reaction product. The elution pattern of the poly(A) polymerase from DNA-cellulose suggested that a single enzyme catalyzes the attachment of adenylate residues to both polyribonucleotide and polydeoxyribonucleotide primers; nevertheless the purest enzyme preparations contain two bands resolved by polyacrylamide gel electrophoresis in sodium dodecyl sulfate.

Single-Stranded Deoxyribonucleic Acid-specific Nuclease from Vaccinia Virus: PURIFICATION AND CHARACTERIZATION

Abstract A deoxyribonuclease was extracted from vaccinia virus cores and purified 200-fold by chromatography on DEAE-cellulose, DNA-cellulose, and hydroxyapatite. Approximately 0.35 mg of enzyme was isolated from 63 mg of cores. Preparations of [3H]leucine-labeled deoxyribonuclease appeared nearly homogeneous as judged by gel filtration and had a molecular weight of 50,000 as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The molecular weight of the native enzyme determined by sucrose gradient sedimentation and filtration through Sephadex G-200 was 105,000, suggesting that it may exist as a dimer. The enzyme hydrolyzed single-stranded DNA most actively at pH 4.4 and did not require divalent cations.

Two Nucleic Acid-dependent Nucleoside Triphosphate Phosphohydrolases from Vaccinia Virus: PURIFICATION AND CHARACTERIZATION

Abstract Two nucleic acid-dependent nucleoside triphosphate phosphohydrolases have been found in vaccinia virus cores. Both enzymes hydrolyze ATP to ADP and Pi in the presence of nucleic acid. The enzymes were solubilized from purified viral cores by treatment with sulfhydryl-reducing agent, salt, and sodium deoxycholate in a Tris buffer at alkaline pH. DNA-free viral extracts were prepared by adsorption of viral DNA to DEAE-cellulose columns in the presence of high salt, and the two enzymes were purified by DNA-cellulose chromatography. Phosphohydrolase I was purified 300- to 400-fold with yields of 80 to 90 % and is approximately 95 % pure. A molecular weight of approximately 61,000 was calculated for the native enzyme using Sephadex gel chromatography and sucrose gradient sedimentation. A molecular weight value of 68,000 was obtained under denaturing conditions of sodium dodecyl sulfate polyacrylamide gel electrophoresis, indicating that phosphohydrolase I is a monomeric enzyme. The smaller amount of phosphohydrolase II prevented accurate determinations of specific activity or purity. The molecular weight of the native enzyme was calculated to be 68,000 and it may also consist of a single polypeptide. Nucleic acids were required by phosphohydrolase I and II for the hydrolysis of ATP, and optimal activities were obtained at neutral pH in the presence of divalent cations. The hydrolysis of ATP resulted in the production of stoichiometric amounts of ADP and Pi. The Km values of phosphohydrolases I and II were 1.4 x 10-4 m and 6.4 x 10-4 m, respectively.

Polyadenylate polymerase from vaccinia virions.

HETEROGENEOUS nuclear, messenger1–9 and viral1,10–16 RNAs from eukaryotic organisms contain long polyadenylate (poly (A)) sequences of unknown function. The poly (A) is located at the 3′ terminus17–21 and may be added after transcription is completed11,22. Although enzymes that can add adenylate residues to RNA have been obtained from eukaryotic organisms28–29, they have not yet been coupled with RNA synthesis in vitro and it is not known whether specific sequences in DNA or nascent RNA act as signals for the addition of adenylate residues. Vaccinia virions seem to be a uniquely suitable system for studying the biosynthesis de novo of poly (A) and its attachment to RNA. Kates and Beeson1,10 showed that isolated vaccinia virus cores synthesized poly (A) in the presence of ATP and that RNA with terminal poly (A) sequences was made in the presence of all four ribonucleoside triphosphates. They suggested1,10 that in this system the adenylate residues were added by a transcriptional mechanism, possibly by the viral DNA-dependent RNA polymerase30,31. Evidence for such a mechanism was indirect and included the finding that poly (A) synthesis was inhibited by ethidium bromide and proflavine, drugs that bind to double stranded nucleic acids by intercalation1,10. The possibility that vaccinia DNA contains poly (dA>poly (dT) tracts that could serve as templates was consistent with hybridization data and with the transcription of poly (A) sequences by E. coli RNA polymerase1,10. The potential usefulness of vaccinia virus enzymes, for the study of RNA and poly (A) synthesis, has not been fully realized because of the particulate nature of the viral core. Of the five enzymatic activities known to be present in vaccinia cores30–35, only the DNA-dependent nucleotide phosphohydrolase (ATPase) has been solubilized36. We now describe the solubilization, isolation and some interesting properties of the poly (A) polymerase from vaccinia virions.

Deoxyribonucleic acid-dependent nucleotide phosphohydrolase activity in purified vaccinia virus.

A nucleotide phosphohydrolase (adenosine triphosphatase), which is associated with vaccinia virus cores, has been solubilized and shown to be deoxyribonucleic acid dependent.

Protein kinase and specific phosphate acceptor proteins associated with vaccinia virus cores.

Incubation of purified vaccinia virus with gamma-(32)P-adenosine triphosphate resulted in the incorporation of (32)P into hot trichloroacetic acid-insoluble material. Enzymatic activity was completely dependent on the addition of divalent cations and was stimulated by nonionic detergents and dithiothreitol. Chemical studies demonstrated that serine and threonine residues of 15,000 molecular weight viral polypeptides were phosphorylated. In contrast, the major structural proteins were not phosphorylated or were phosphorylated to a much lesser extent. Added histones and protamine, but not serum albumin, casein, or phosvitin were phosphorylated by the partially disrupted vaccinia virus preparations. The protein kinase was tightly associated with vaccinia virus particles since the specific enzymatic activity remained constant during the final steps of virus purification, the specific activities of many different preparations of virus were similar, and the enzymatic activity cosedimented with vaccinia virus during rate zonal sucrose gradient and potassium tartrate gradient equilibrium centrifugations. Controlled degradation of vaccinia virus, with nonionic detergents and dithiothreitol, indicated that both the protein kinase and the specific phosphate acceptor proteins were located in the virus core.

Formation of a vaccinia virus structural polypeptide from a higher molecular weight precursor: inhibition by rifampicin.

A vaccinia virus core polypeptide, with a molecular weight of 76,000 and a relative deficiency in tryptophan, was shown by pulse-chase experiments to form from a precursor. The latter may be a rapidly labeled, 125,000-molecular weight, tryptophan-deficient, virus-induced polypeptide, which diminished in quantity during the chase period and was barely detectable after two to three hours. Rifampicin completely prevented the formation of the core polypeptide without inhibiting the synthesis of the precursor. A rifampicin-resistant vaccinia mutant was used to demonstrate the specificity of this effect. The sequence of events after the removal of the drug suggested that cleavage of the precursor occurs during the formation of the virus core. Rifampicin appears to act by interrupting earlier maturational events which precede the formation of the core polypeptide.

Ribonucleic acid synthesis in vaccinia virus: II. Synthesis of polyriboadenylic acid

Vaccinia virus cores catalyze the incorporation of ATP into polyriboadenylic acid sequences of approximately 150 nucleotides in length. This incorporation of ATP occurs also in the presence of all four natural ribonucleotides in the reaction mixture. Poly A sequences are found in high molecular weight RNA synthesized by cores and evidence is presented for a covalent association between the poly A sequences and the regular viral RNA moiety.The transcription of poly dT sequences in vaccinia DNA appears to be the likely source of poly A, since about 1% of vaccinia DNA contains regions which hybridize with synthetic poly A and poly U. In addition, the synthesis of poly A is inhibited by proflavin sulfate and ethidium bromide, which complex with DNA. Poly A sequences are also synthesized in vivo during the growth cycle of vaccinia virus in HeLa cells.