Beatriz G.T. Pogo

Biogenesis of vaccinia: Separation of early stages from maturation by means of rifampicin

Abstract The influence of rifampicin on the biogenesis of vaccinia virus was examined by combined biochemical and electron microscopic procedures. Penetration, uncoating, DNA replication and the assembly of membranes and immature particles proceeded in the presence of the antibiotic. However, when wild-type virus was used for inoculation virus maturation as well as the induction of nucleotide phosphohydrolase, RNA polymerase, and two DNases (four virus-associated enzymes), was blocked by rifampicin. By contrast infection with a drug-resistant mutant of vaccinia elicited the synthesis of these enzymes and mature progeny were formed. After washing out rifampicin additional synthesis of RNA and protein were required for the completion of wild-type virus development. These observations indicate that transcription of certain early functions can occur from the parental genome but the integration of late functions within virus membranes is essential for the completion of maturation. The sites of rifampicin action at the synthetic level in the vaccinia and bacterial systems are discussed.

Biogenesis of vaccinia: separation of early stages from maturation by means of hydroxyurea.

Abstract Hydroxyurea (HU), a specific inhibitor of DNA synthesis, arrests morphogenesis of the poxviruses at an immature stage. In this study we examined the biogenesis of vaccinia after reversing prolonged (16 hr) inhibition of DNA replication by HU. After the drug was washed out, DNA was produced in a burst of synthesis which reached a maximum rate within 1 hr and remained at a high level for 2 hr. Among the three virus-associated enzymatic activities studied, induction of the pH 7.8 DNase occurred sooner and rose to almost the highest level within 2 hr after reversal, at the time DNA was being formed most rapidly. With the pH 5.0 DNase and nucleotide phosphohydrolase, induction was slower and highest levels of activity were observed 4 hr after reversal, when the bulk of the DNA had been formed. The morphopoiesis of mature, infectious virus also commenced rapidly, within 2 hr after removal of HU. These combined biochemical and electron microscopic results support the concept that during normal development certain late factors, including the three enzymes studied, are inserted within vaccinia-specified envelopes, formation of which can be regulated by the parental DNA. Close correlation between the appearance of the above enzymes and completion of vaccinia development indicates that maturation is contingent upon the antecedent synthesis of a number of late factors.

Biogenesis of poxviruses: Synthesis and phosphorylation of a basic protein associated with the DNA☆

Abstract The DNA of vaccinia virus was shown to be intimately associated, perhaps by ionic bonds, with an arginine-rich basic polypeptide (AEP) of molecular weight 11,000–12,000. This protein occurs both in the virus cores and the cytoplasmic “factories.” Pulse experiments with [33P]O43− demonstrated that AEP becomes phosphorylated at the time of or soon after its synthesis. The formation of AEP is temporally coupled with synthesis of DNA and can be abolished by suppressing DNA replication. Comparisons of the role in regulation of gene activities by AEP and the basic proteins found in cell nuclei or the T-even bacteriophages are discussed.

The mode of replication of vaccinia virus DNA

Abstract Synthesis of vaccinia virus DNA was studied by following the incorporation of [3H]thymidine in the cytoplasm of infected cells. During short pulses (1–5 min) the radioactive precursor was incorporated into single-stranded DNA fragments that sedimented as 10–12 S in neutral sucrose gradients. After the chase, these fragments were converted into a 34 S intermediate before becoming mature size molecules (68 S). Following longer periods of chase, the labeled DNA sedimented as cross-linked molecules in alkaline sucrose gradients (91–106 S) suggesting that the formation of cross-links is a postreplicative process. The presence of RNA covalently linked to DNA in the 12 S fragments was suggested by a shift in the equilibrium density sedimentation and by sensitivity to spleen phosphodiesterase after alkaline hydrolysis. Determination of the molecular weight of vaccinia DNA synthesized in cultures containing bromodeoxyuridine (BrUdR) demonstrated that replication proceeds in two directions.

Biogenesis of poxviruses: Further evidence for inhibition of host and virus DNA synthesis by a component of the invading inoculum particle☆

Abstract The effect of virion-derived pH 7.8 DNase on the host DNA replication was further studied. DNA polymerase(s) active with exogenous single-stranded (ss) and double-stranded (ds) DNA templates were monitored in nuclear, large particulate and supernatant cytoplasmic fractions from control and infected cells. Infection suppressed the nuclear ds activity appreciably but had little effect on the ss one, regardless of prevailing RNA and protein synthesis. By contrast, both the cytoplasmic supernatant DNA polymerase activities were elevated after infection and this stimulation depended upon ongoing protein synthesis. Kinetics of the polymerizing reaction, followed in pulse-chase experiments revealed that nascent DNA in the controls was conserved while in the infected nuclei it became degraded. The degradation products were characterized by paper chromatography and shown to be predominantly low molecular weight polynucleotide chains less than 2–8 nucleotides long. A DNase activity with characteristics of the virion-associated DNase was detected in infected nuclei at about the same time as host DNA synthesis was affected. Inhibition of DNA synthesis by a factor originating from the virus was also demonstrated on the cytoplasmic vaccinia DNA replication by superinfection with vaccinia under appropriate circumstances. As a whole these observations imply that the ss DNA being formed is the most likely site of action of the nuclease originating from the penetrating virion core and lends further support to the notion that the DNase from the invading virus is responsible for inhibiting host DNA synthesis.

Further characterization of deoxyribonucleases from vaccinia virus

Abstract Further characterization was made of DNases present in vaccinia virus. The nature of the enzymes as they occur within cores and following solubilization was determined. Two activities were identified hydrolyzing single-stranded (ss) DNA but at exclusively a pH optimum of 4.5 or 7.8, respectively. Neither activity has any requirement for ions or cofactors. The pH 7.8 enzyme was activated preferentially by heating cores to 50°. Analysis of the products of hydrolysis by means of DEAE-paper chromatography confirmed that the pH 4.5 activity was an exonuclease and the pH 7.8 enzyme an endonuclease. The exonuclease could act on the 5′-terminus of the DNA. Both nucleases could hydrolyze poly(dT), poly(dA), and poly(dC) to a varying degree but had no effect on poly(dG). Since the oligonucleotides arising as a product of endonuclease action did not serve as a substrate for the exonuclease, it is concluded that the two enzymes probably do not act in concert. Solubilization of both DNases was achieved by treatment of cores with salts or urea. With 0.5 M NaCl, most of the pH 4.5 activity but only 10–20% of the pH 7.8 was released. The presence of 6–8 M urea caused the solubilization of both enzymes. When in their soluble state, the nucleases could be separated by means of isoelectric focusing in either gel or liquid milieu and retain the exo- or endonucleolytic activities. The pH 4.5 DNase had an isoelectric point or pI of approximately 4.5, and the pH 7.8 DNase had a pI of approximately 3.7. Each activity was contained in a single protein. Further analysis of the isolated enzymes, using sodium dodecyl sulfate (SDS)-polyacrylamide-gel electrophoresis, revealed each to be a polypeptide of MW 50,000. Taken together, the evidence indicates that vaccinia cores contain two DNases with independent modes of action associated with separate proteins of similar molecular weights but different pIs.

Biogenesis of vaccinia: Specific inhibition of rapidly labeled host DNA in vaccinia inoculated cells☆

Abstract Because separation of the bulk and nascent (i.e., rapidly labeled) DNA fractions of HeLa cells could be effected satisfactorily it became possible to ascertain the effects of inoculating uv-irradiated vaccinia virus on in vivo host DNA synthesis. In both control and infected cells, labeled 4s fragments, formed during 1-min pulses with [ 3 H]Tdr, existed as single- or double-stranded molecules covalently linked to short stretches of RNA. During the chase period, most of the rapidly labeled DNA within uninfected cells became associated with the bulk DNA component but failed to do so within inoculated cells. Host DNA ligase activity was unaffected by the infection, and there was a rapid breakdown of the 4s DNA fragments, leading to the conclusion that arrest of HeLa nuclear DNA synthesis by vaccinia is, most probably, due to the in vivo hydrolysis of nascent DNA. The present data further substantiate previous studies from this laboratory demonstrating the hydrolysis of in vitro synthesized nascent DNA by a DNAse activity originating from the virus core.

Biogenesis of vaccinia: Effect of rifampicin on transcription☆

Abstract An in vitro assay for RNA polymerase (transcriptase) was developed in which a large particulate fraction containing viroplasmic matrices synthesizes a vaccinia-specific polymer. Transcription is sensitive to rifampicin during the peak of induced RNA polymerase activity, i.e., 6–8 hr after infection, but is refractory during the first 4 hr of infection and again at the termination of the replicative cycle. By contrast, transcription is insensitive at all times in fractions obtained from cells infected with a drug-resistant mutant. Nucleotide incorporation is almost completely abolished following treatment with actinomycin D or RNase, indicating that a DNA template is being transcribed. The ratio of UTP over CTP incorporated into the RNA polymer is 1.5, suggesting that the DNA of vaccinia is being transcribed. These data indicate that in the case of vaccinia either rifampicin binds to a sensitive polypeptide in the manner conceived for bacterial RNA polymerase and prevents the formation of an initiation complex or that the inhibitor interferes with the function of only one out of two or more RNA polymerases that may be induced by the virus.

Early events in lymphocyte transformation by phytohemagglutinin: III. Inhibition of RNA synthesis and transformation by cordycepin

The DNA-dependent RNA polymerase activities of isolated nuclei from lymphocytes were examined after stimulation with phytohemagglutinin (PHA). The nuclear fraction was prepared with Mg++ or Mn++ to distinguish between polymerase I (nucleolar) and polymerase II (nucleoplasmic). Distinction between polymerases II and III was obtained by the addition of α-amanitin to the reaction mixture. The results indicated that within 15 min after exposure to PHA the activity of polymerase I increased. Polymerase II activity increased after 1 hr. The enhancement was linear for 6 hr and then leveled off for the subsequent 48 hr. Small increase in polymerase III activity was observed at 48 hr. Inhibition of protein synthesis at the time of exposure to PHA did not prevent the increase in activities during the initial 6 hr. These results imply that the initial increase in enzymatic activities is dependent upon preexisting polymerase molecules and/or factors.

Formation of Cross-Linked Molecules during Vaccinia DNA Replication

The appearance of cross-linked DNA molecules during the replication of vaccinia virus DNA was investigated. Conversion of full size DNA molecules to cross-linked molecules occurs several hours after completion of DNA synthesis. Inhibition of RNA synthesis by actinomycin D did not prevent this transition; however, interruption of protein synthesis by streptovitacin A resulted in partial inhibition of the process.