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E Paoletti

Publications and source records attributed to E Paoletti.

At least 163 records · Page 9Linked to original sources
PubMed1984

Genetically engineered poxviruses: a novel approach to the construction of live vaccines.

Gene splicing techniques have been used to modify the smallpox vaccine virus thus providing a generic approach for the construction of live vaccines directed against a variety of heterologous infectious disease agents. The technique involves translocating a particular gene from an infectious agent into the genetic material of the smallpox vaccine virus. This unique foreign gene, selected because it contains the information essential for the synthesis of an antigen important in immunity to that particular infectious disease agent, is now expressed under the regulation of the engineered smallpox vaccine virus. On immunization with this live recombinant vaccine, the body is fooled into thinking that it was infected by the foreign infectious disease agent and mounts a defensive attack resulting in immunity to that particular infectious agent. Three examples of this approach are provided. Thus, smallpox vaccine viruses were engineered to express genes encoding the hepatitis B virus surface antigen (HBsAg), the herpes simplex virus glycoprotein D (HSV-gD) and the haemagglutinin (HA) from influenza virus. These foreign gene products when synthesized in vitro under vaccinia virus regulation were shown to be antigenic by a variety of serological tests. When these recombinant vaccinia viruses were inoculated into laboratory animals, the heterologous gene products elicited the production of specific antibodies thus demonstrating that they were immunogenic. Serum neutralizing antibodies were demonstrated to be present for both influenza and herpes simplex viruses. Additional studies in mice showed that a recombinant smallpox vaccine virus expressing a gene from herpes simplex virus effectively protected the mice when subsequently challenged with what would normally be lethal doses of infectious herpes simplex virus.

Animals↗
PubMed1984

Construction of live vaccines using genetically engineered poxviruses: biological activity of vaccinia virus recombinants expressing the hepatitis B virus surface antigen and the herpes simplex virus glycoprotein D.

Potential live vaccines using recombinant vaccinia viruses have been constructed for both hepatitis B and herpes simplex. These recombinant vaccinia viruses express cloned genes of the hepatitis B virus surface antigen (HBsAg) or the glycoprotein D from herpes simplex virus (HSV-gD). The HBsAg synthesized in vitro under the regulation of vaccinia virus is secreted from infected cells as a particle of approximately equal to 22 nm diameter with a density of 1.2 g/ml as determined on CsCl gradients. Inoculation of rabbits with the recombinant vaccinia virus that expresses the HBsAg elicits the production of high-titered antibodies. Synthesis of the HSV-gD was detected in tissue culture by radioimmunoassay on unfixed cells, suggesting that the HSV-gD synthesized by the recombinant vaccinia virus is membrane associated. Inoculation of rabbits with the recombinant vaccinia virus expressing HSV-gD resulted in the production of antibodies that reacted with authentic HSV-gD as detected by radioimmunoassay. Furthermore, the anti-serum was shown by plaque-reduction assay to neutralize the infectivity of herpes simplex virus. Immunization of mice with the vaccinia recombinant expressing HSV-gD gave complete protection on subsequent challenge with lethal doses of live herpes simplex virus.

Animals↗
PubMed1983

Construction of live vaccines by using genetically engineered poxviruses: biological activity of recombinant vaccinia virus expressing influenza virus hemagglutinin.

Recombinant vaccinia viruses containing the cloned hemagglutinin (HA) gene from influenza virus were constructed. The biological activity of these poxvirus vectors was demonstrated both in vitro and in vivo. Expression of HA in cells infected with recombinant vaccinia was detected by using specific anti-HA antiserum and 125I-labeled protein A, showing that HA synthesized under the regulation of vaccinia virus was antigenic. Immunization of rabbits with these recombinant poxviruses resulted in the production of antibodies reactive with authentic influenza HA as detected by radioimmunoassay, by inhibition of HA erythrocyte agglutination, and by neutralization of influenza virus infectivity. The production of antibodies directed against influenza HA suggested that the HA gene expressed in vaccinia is immunogenic. These data indicate the potential of genetically engineered poxviruses for use as generic live vaccine vehicles that have both human and veterinary applications.

Animals↗
PubMed1982

Construction of poxviruses as cloning vectors: insertion of the thymidine kinase gene from herpes simplex virus into the DNA of infectious vaccinia virus.

We have constructed recombinant vaccinia viruses containing the thymidine kinase gene from herpes simplex virus. The gene was inserted into the genome of a variant of vaccinia virus that had undergone spontaneous deletion as well as into the 120-megadalton genome of the large prototypic vaccinia variant. This was accomplished via in vivo recombination by cotransfection of eukaryotic tissue culture cells with cloned BamHI-digested thymidine kinase gene from herpes simplex virus containing flanking vaccinia virus DNA sequences and infectious rescuing vaccinia virus. Pure populations of the recombinant viruses were obtained by replica filter techniques or by growth of the recombinant virus in biochemically selective medium. The herpes simplex virus thymidine kinase gene, as an insert in vaccinia virus, is transcribed in vivo and in vitro, and the fidelity of in vivo transcription into a functional gene product was detected by the phosphorylation of 5-[125I]iodo-2'-deoxycytidine.

Base Sequence↗
PubMed1982

Molecular genetics of vaccinia virus: demonstration of marker rescue.

Two genomic variants of vaccinia virus isolated from serially propagated stocks were used to demonstrate marker rescue. The smaller (S variant) virus contains a 6.3 megadalton (MDal) deletion of unique DNA sequences present in the 123-MDal larger (L variant) virus. The deletion was mapped at 6.85 MDal from the left terminus of the genome, just outside of the inverted terminal repetition. Rescue of the unique deleted DNA sequences by infectious S variant virus was obtained in CV-1 cells by using the calcium orthophosphate precipitation technique of intact or restriction endonuclease-treated L-variant DNA. Restriction fragments that overlapped the deletion allowed marker rescue, but restriction of the L-variant DNA within the unique deleted sequences gave negative results. Restriction endonuclease analysis of the DNA obtained from twice-plaque-purified recombinant virus derived from the rescue of overlap donor fragments gave a restriction pattern identical to that of L-variant virus, indicating that the donor DNA was inserted into the rescuing virus by double recombination. No amplification of the unique sequences was observed from intact L-variant DNA in the absence of infectious S-variant virus, suggesting that deproteinized vaccinia DNA is noninfectious and that the donor DNA was neither integrated into the host DNA nor present as an episomal structure. By using 1 microgram of intact L-variant DNA per CV-1 monolayer in a 6-cm Petri dish, approximately 1--5% of the plaques contained the L-variant genotype, and the dose--response curve was essentially linear from 0.1 to 2 microgram of DNA.

Chromosome Deletion↗
PubMed1982

Analysis of vaccinia virus transcriptional complexity in vitro and in vivo: characterization of RNase T1-resistant 5'-terminal oligonucleotides.

Vaccinia virus mRNAs synthesized in vitro and in vivo, polyadenylated leader sequences synthesized in vitro in the absence of added GTP, CTP, or UTP or in the presence of 20 micrograms of actinomycin D per ml, and high-molecular-weight RNA synthesized in vitro under limiting ATP concentrations were labeled specifically in the cap structure using [alpha-32P]GTP and vaccinia-soluble enzyme extracts. The complexity of RNase T1-resistant 5'-terminal oligonucleotides was analyzed by two-dimensional polyacrylamide gel electrophoresis. Approximately 190 unique T1-resistant 5'-terminal oligonucleotides were observed from vaccinia virus 8 to 12S RNA synthesized in vitro. A somewhat greater complexity was observed with polyadenylated leader sequences and actinomycin D RNAs where unique T1-resistant oligonucleotides ranged from approximately 210 to 280 5'-terminal fragments. On a composite fingerprint of the above RNAs, more than 300 identifiable unique T1-resistant 5'-terminal oligonucleotides were observed. Significantly, close to 300 T1-resistant fragments were derived from RNA sedimenting faster than 18S on denaturing sucrose gradients. Analysis of vaccinia RNAs synthesized in vivo in the absence of either de novo protein synthesis or DNA replication or in the presence of actinomycin D gave essentially similar profiles of 5'-terminal T1-resistant oligonucleotide fingerprints consisting of approximately 200 fragments. Analysis of the 5'-terminal T1-resistant oligonucleotides of vaccinia RNAs present after DNA replication showed essentially the same pattern of early T1-fragments albeit in reduced amounts but in addition revealed a complex pattern of T1-resistant oligonucleotides unique to this class of vaccinia RNA.

Adenosine Triphosphate↗
PubMed1981

Two major DNA variants present in serially propagated stocks of the WR strain of vaccinia virus.

Two major DNA variants have been isolated from serially propagated stocks of the WR strain of vaccinia virus. Restriction enzyme mapping of the two variants with HindIII, AvaI, XhoI, SstII, and SmaI revealed a 6.3-megadalton deletion in the smaller DNA variant. The deletion was mapped at ca. 6.8 megadaltons in from the left terminus, just beyond the inverted terminal repeat. The additional DNA present in the larger variant was found to represent unique viral sequences that were transcribed both in vitro and in vivo. One-step growth curves in HeLa cells revealed no difference in rat of replication or burst size when progeny was scored on CV-1 monolayers.

Base Sequence↗
PubMed1980

Capped and polyadenylated low-molecular-weight RNA synthesized by vaccinia virus in vitro.

In the presence of ATP plus two other ribonucleoside triphosphates or in reactions containing all four ribonucleoside triphosphates and actinomycin D, vaccinia virus synthesizes in vitro discrete low-molecular-weight RNA molecules ranging in size from about 20 to several hundred bases. A novel feature of these small RNA molecules is that they are capped and methylated at the 5' terminus, containing both mGpppGm and mGpppAm type cap structures, and in addition these molecules are polyadenylated at the 3' terminus. Hybridization of these RNAs to restriction fragments derived from vaccinia virus DNA indicates a considerable degree of complexity, suggesting the presence of a large number of promoters throughout the genome. However, measurable sensitivity to pancreatic RNase of the 5' capped end of these RNAs while in hybrid form to the DNA suggests other possible roles for these small RNAs in vaccinia virus mRNA biogenesis.

Adenosine Triphosphate↗
PubMed1978

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.

Cell-Free System↗
PubMed1978

Effect of UV irradiation on the expression of vaccinia virus gene products synthesized in a cell-free system coupling transcription and translation.

The effect of UV irradiation on the expression of the vaccinia virus genome was investigated in a cell-free system coupling transcription with translation. Exposure of vaccinia virus to an increasing dose of irradiation resulted in differential reduction in the syntheses of virus-specified polypeptides in the coupled system, with sensitivity being proportional to the size of the gene pro duct. This suggests that each translationally functional mRNA species produced in vitro by vaccinia virus cores is synthesized from an individual promoter site.

Cell-Free System↗
PubMed1978

Soluble endoribonuclease activity from vaccinia virus: specific cleavage of virion-associated high-molecular-weight RNA.

A soluble endoribonuclease activity was extracted from purified vaccinia virus cores by treatment with sodium-deoxycholate and dithiothreitol. The soluble enzyme readily cleaved purified virion-associated high-molecular-weight RNA to limit-sized fragments sedimenting at 8 to 12S. Purified virion-released 8 to 12S polyadenylated mRNA was not degraded by the enzyme extract. The soluble endoribonuclease did not require the presence of ribonucleoside triphosphates for activity.

Endonucleases↗
PubMed1978

Cell-free translation of purified virion-associated high-molecular-weight RNA synthesized in vitro by vaccinia virus.

Virion-associated high-molecular-weight (HMW) RNA synthesized in vitro by purified vaccinia virus particles has been translated in a wheat germ cell-free protein synthesizing system. Purified HMW RNA directs the synthesis of translation products which are identical to the translation products made in response to in vitro-synthesized, virion-released 8 to 12S mRNA. The translation of HMW RNA proceeds exclusively through a 5'-terminal cap-mediated initiation step. Furthermore, only one coding sequence is translated per HMW RNA molecule, and that sequence is probably located near the 5' end of the molecule. These conclusions are based on the following results. (i) Sodium dodecyl sulfate--polyacrylamide gel electrophoresis patterns of translation products synthesized in response to HMW RNA and in response to 8 to 12S mRNA were qualitatively identical. (ii) On an equal weight basis, HMW RNA was 25 to 30% as active as 8 to 12S mRNA in stimulating in vitro protein synthesis. (iii) Unmethylated HMW RNA was translated at 10% the efficiency of the methylated form of this RNA. (iv) m7pG inhibited the translation of fully methylated HMW RNA by 90%. (v) After the initiation step of translation was blocked by aurintricarboxylic acid, the rate with which amino acids were incorporated into individual polypeptides decreased in a similar manner for the translation of both HMW RNA and 8 to 12S mRNA. Virion-released 8 to 12S mRNA derived from virion-associated HMW RNA during a chase in the presence of ATP, GTP, and S-adenosylmethionine was also translated. At low RNA concentrations, the derived RNA appeared to stimulate amino acid incorporation more efficiently than the HMW RNA precursor. However, at higher concentrations of this RNA, protein synthesis was severely inhibited.

Aurintricarboxylic Acid↗
PubMed1977

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-.

Chemical Phenomena↗
PubMed1977

In vitro synthesis of a high molecular weight virion-associated RNA by vaccinia.

Although the bulk of RNA synthesized in vitro by vaccinia virus is 8 to 12 S, a small amount of high molecular weight RNA can be detected. This RNA is virion-associated and is not extruded from the virus as high molecular weight RNA. It is sensitive to pancreatic RNase digestion in high salt, has a density in neutral CS2SO4 of 1.68 g ml-1 and remains large after digestion with DNase or denaturation in dimethyl sulfoxide. In the presence of high concentrations of virus in the in vitro RNA polymerase reaction, pulse-labeling experiments indicate an RNA sedimenting heterogeneously between 20 and 30 S. Pulse-chase experiments indicate that a fraction of this high molecular weight RNA can be chased into RNA sedimenting at 8 to 12 S. Cleavage into smaller fragments is not dependent on continued RNA synthesis but does require ribonucleoside triphosphates. In the presence of ethidium bromide, the RNA is not cleaved.

Centrifugation, Density Gradient↗
PubMed1977

High molecular weight virion-associated RNA of vaccinia. A possible precursor to 8 to 12 S mRNA.

The high molecular weight virion-associated RNA synthesized by vaccinia in vitro can be cleaved into smaller components, some of which are extruded from the virus as 8 to 12 S RNA. The high molecular weight virion-associated RNA fails to bind appreciably (5%) to poly(U) filters indicating that it is not polyadenylated. Its cleavage products will, however, bind to poly(U) (40 to 50%) after processing in the presence of ATP. The high molecular weight virion-associated RNA is methylated by the virus, and purified unmethylated RNA can be methylated by detergent-solubilized extracts of vaccinia virus cores. In the presence of GTP, methylation is stimulated 3-fold. The level of methylation of purified unmethylated high molecular weight RNA achieved by soluble core extracts is approximately 80% of the level of methylation achieved with purified unmethylated 8 to 12 S viral RNA, suggesting that more residues than the primary 5' termini became methylated. Approximately 85% of the methylated RNA binds to poly(U) when purified high molecular weight RNA is processed with soluble core extracts in the presence of S-adenosyl[methyl-3H]methionine, GTP, and ATP, conditions which also cleave the RNA. Nucleic acid hybridization-competition studies indicate that virion-extruded 8 to 12 S mRNA contains sequences found in the high molecular weight virion-associated RNA.

Centrifugation, Density Gradient↗