Circular Forms of DNA Synthesized by Rous Sarcoma Virus in Vitro

Clayman, Carol H.; Mosharrafa, E.; Anderson, Dwight L.; Feras, Anthony J.

doi:10.1126/science.91199

Cited by 11 publications

(6 citation statements)

References 24 publications

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“…We tested the total cDNA product synthesized by melittin-activated NY68(B) and monitored cell morphology and reverse transcriptase activity after several passages. At the highest dose tested (50 ng/60-mm plate), both cultures became fully transformed and produced reverse transcriptase-containing particles ( forming unit per 50 ng, the specific infectivity is of a magnitude comparable to that reported by others for cDNA (6,7). Of the lower doses, none showed conclusive signs of transformation and none had detectable levels of reverse transcriptase.…”

Section: Resultssupporting

confidence: 74%

“…In any case, our results indicate that such a critical concentration requirement is not necessary with melittin and suggest that melittin may prove useful for activating reverse transcription in particles obtained from various sources such as the blood of human leukemia patients (28) or released from certain human tumor cell lines (29). Our melittin-stimulated full-length cDNA was assumed to be primarily (-) strand as shown by others (1,3,4,6,14,30) and was demonstrated to be so by comparison of its electrophoretic mobility with the mobilities of separated strands of the 7.7-kb RAV-2 provirus insert (Fig. 3).…”

Section: Discussionmentioning

confidence: 85%

See 1 more Smart Citation

Two species of full-length cDNA are synthesized in high yield by melittin-treated avian retrovirus particles.

Boone¹,

Skalka²

1980

Proc. Natl. Acad. Sci. U.S.A.

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A method of activating endogenous cDNA synthesis in avian retroviruses that results in the formation of two species of full-length cDNA in high yield is described. Tests of biological activity show infectivity of at least the same order of magnitude as for full-length cDNA made by other procedures.Melittin, the major component of bee venom, is used as an alternative to nonionic detergents to make the viral envelope permeable and thus activate the endogenous RNA-dependent DNA polymerase. This compound is a toxic peptide known to interact with phospholipid membranes. It appears to be less disruptive to the viral structure than detergents, resulting in a more efficient transcription of the viral genome. Preliminary tests indicate that this method will also prove useful for studying enzymatic activities associated with other enveloped viruses. The standard method of synthesizing cDNA by the endogenous reverse transcriptase reaction has been to make the virus envelope permeable with a nonionic detergent in the presence of the appropriate salts, buffers, reducing agent, and deoxynucleoside triphosphates. It is clear from several studies that the concentration of some components is critical for the synthesis of a product that contains full-length transcripts of the viral genome. The detergent concentration has a very narrow optimal range for the synthesis of large cDNA molecules from avian sarcoma virus, and no large transcripts are made when this concentration is exceeded (1). The importance of balancing the divalent cation and deoxynucleoside triphosphate concentrations for the synthesis of large transcripts of Moloney murine leukemia virus has also been documented (2, 3). However, high deoxynucleoside triphosphate concentration and long incubation are not required for full-length cDNA synthesis by equine infectious anemia virus (4). Finally, it has been shown that the addition of ribonucleoside triphosphates can increase the yield of full-length transcripts in reactions using purified components (5) and in the endogenous cDNA reaction (6). The full-length in vitro synthesized cDNAs of both murine and avian retroviruses have been shown to be infectious by transfection assays (6, 7).In the present study we examined the use of melittin, the major component in bee (Apis mellifera) venom as an alternative to nonionic detergent for disruption of virions for cDNA synthesis. This cationic peptide of 26 amino acids has an unusual arrangement of largely hydrophobic (positions 1-20) and hydrophilic (positions 21-26) residues which makes it highly surface active. Its lytic activity on both natural and artificial membranes has been well characterized (8-10). We report here that melittin can be used in place of nonionic detergents for the synthesis of cDNA that is the length of in vivo proviral DNA and is infectious. Our data suggest that this compound can make the viral envelope permeable so that substrates for cDNA synthesis can enter, without disruption of structural components that may be required for authentic provi...

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Section: Resultssupporting

confidence: 74%

Section: Discussionmentioning

confidence: 85%

Two species of full-length cDNA are synthesized in high yield by melittin-treated avian retrovirus particles.

Boone¹,

Skalka²

1980

Proc. Natl. Acad. Sci. U.S.A.

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“…on October 3, 2020 by guest http://jvi.asm.org/ Downloaded from virions (3,4). Such studies permit the hypothesis that all of the factors necessary for the synthesis of circular DNA are carried by the virus particles.…”

Section: Pmentioning

confidence: 90%

Effect of aphidicolin on avian sarcoma virus replication

Hsu¹,

Taylor²

1982

J Virol

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We studied the effect of aphidicolin, an inhibitor of eucaryotic DNA polymerase alpha, on viral DNA replication and integration during the first 24 h after infection of quail embryo fibroblasts with avian sarcoma virus. In drug-treated cells, the synthesis of unintegrated linear viral DNA species was not impaired; however, the subsequent accumulation of circular viral DNA species and integrated proviral DNA was reversibly inhibited. After removal of the drug, circular viral DNA species were derived from preexisting linear viral DNA species, instead of being derived by de novo synthesis.

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“…This topoisomerase was detected in a search for an activity which in disrupted virions of RSV may be able to facilitate -in concert with the polymerizing activity of reverse transcriptase -the formation of covalently closed circular DNA (CLAYMAN et al 1979). The topoisomerase is most probably a type I enzyme with a molecular weight similar to that of a nuclear type I topoisomerase.…”

Section: Viral Topoisomerasesmentioning

confidence: 97%