In vitro synthesis of infectious transforming DNA by the avian sarcoma virus reverse transcriptase

The endogenous reverse transcriptase reaction of equine infectious anemia virus (EIAV) has been studied, and conditions allowing synthesis of full-length minus-strand DNA have been determined. In contrast to results reported for other retroviruses, synthesis of EIAV full-length minus-strand DNA was not impaired by high concentrations of Nonidet P-40, a nonionic detergent used to make the virion envelope permeable. All components of the reaction were titrated for maximum synthesis of complete minus strands, and a time course under the standardized conditions was determined. Minor subgenomic bands were observed in some cases, and both the size and proportion varied with reaction conditions. Conditions established for full-length EIAV DNA synthesis also allowed full-genome-length human immunodeficiency virus type 1 DNA synthesis. The human immunodeficiency virus type 1 DNA product contained a greater proportion of reverse transcripts that were shorter than the complete virus genome. Also in contrast to EIAV, the endogenous synthesis of high-molecularweight human immunodeficiency virus type 1 DNA was drastically reduced at Nonidet P-40 concentrations above 0.02%. These results indicated that a detergent-stable core is not a property shared by all lentiviruses. The EIAV virion synthetic machinery is unusually stable and provides a convenient system for further in vitro study of reverse transcription.

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

Equine infectious anemia virus and human immunodeficiency virus DNA synthesis in vitro: characterization of the endogenous reverse transcriptase reaction

Borroto–Esoda

Boone

1991

“…Our studies raise the question of the role of early viral proteins in the infectious cycle of retroviruses. Recent reports (6,29) demonstrated that viral cDNA prepared in vitro is infectious. However, the amount used in these experiments was severalfold higher than that anticipated to participate in a normal infection (about 100 equivalents of viral genome RNA, which equals about 5 x 10'4 pg [32]).…”

Section: --I F T -I ---mentioning

confidence: 99%

Viral genome RNA serves as messenger early in the infectious cycle of murine leukemia virus

Shurtz¹,

Dolev²,

Aboud³

et al. 1979

When NIH/3T3 mouse fibroblasts were infected with the Moloney strain of murine leukemia virus, part of the viral genome RNA molecules were detected in polyribosomes of the infected cells early in the infectious cycle. The binding appears to be specific, since we could demonstrate the release of viral RNA from polyribosomes with EDTA. Moreover, when infection occurred in the presence of cycloheximide, most viral RNA molecules were detected in the free cytoplasm. Size analysis on polyribosomal viral RNA molecules indicated that two size class molecules, 38S and 23S, are present in polyribosomes at 3 h after infection. Analysis of the polyriboadenylate [poly(rA)] content of viral RNA extracted from infected polyribosomes demonstrated that such molecules bind with greatest abundance at 3 h after infection, as has been detected with total viral RNA. No molecules lacking poly(rA) stretches could be detected in polyribosomes. Furthermore, when a similar analysis was performed on unbound molecules present in the free cytoplasm, identical results were obtained. We conclude that no selection towards poly(rA)-containing viral molecules is evident on binding to polyribosomes. These findings suggest that the incoming viral genome of the Moloney strain of murine leukemia virus may serve as a messenger for the synthesis of one or more virus-specific proteins early after infection of mouse fibroblasts. MATERIALS AND METHODS Cells and viruses. NIH/3T3 mouse fibroblasts were used as control cells throughout this study. NIH/ 3T3 cells chronically infected with MLV [NIH/ 3T3(MLV)] were used as a source for infecting virus particles. Unless otherwise indicated, the cells were grown in 100-mm dishes (Nunc, Roskilde, Denmark)

“…This may be due to a large excess of enzyme molecules present in reconstituted reactions, allowing an extensive action of RNase H in spite of nonoptimal conditions. DNA made in vitro in endogenous reactions can be largely converted into double-stranded and infectious forms (7,22,35). Since synthesis of the second DNA strand (plus strand) requires removal of the RNA in the RNA-DNA hybrid, a role of RNase H in the synthesis of plus-strand DNA has been postulated (27,31,32,46).…”

Section: Discussionmentioning

confidence: 99%

Effect of viral RNase H on the avian sarcoma viral genome during early transcription in vitro

Friedrich¹,

Moelling²

1979

We investigated the influence of viral RNase H on the transcription of the avian sarcoma virus RNA in a virion-associated reaction. The ability of RNase H to degrade the RNA moiety of the initially formed RNA-DNA hybrid at the 5' end of the viral genome was found to be greatly dependent on the exact concentration of nonionic detergent used to activate the reaction. At a detergent concentration optimal for extensive and faithful in vitro transcription of avian sarcoma virus RNA by the virion-associated RNA-dependent DNA polymerase, most of the 5' terminus of the RNA was digested in 30 min at 41°C. At higher than optimal detergent concentrations, however, little of that RNA was digested. We conclude that removal of the 5'-terminal redundancy in the RNA after its transcription into DNA is a prerequisite for base pairing of the DNA to the 3'terminal redundant sequence. Lack of removal of this sequence leads to incorrect elongation and substantial reduction of DNA synthesis. When tested with a synthetic RNA-DNA hybrid, virion-associated RNase H did not reveal a detergent dependence.