Synthesis of plus strands of retroviral DNA in cells infected with avian sarcoma virus and mouse mammary tumor virus

131

The structure of unintegrated human immunodeficiency virus type 1 (HIV-1) DNA from acutely infected human lymphoid cells was analyzed by nuclease S1 cleavage. We observed a unique, discrete single-stranded gap in unintegrated linear DNA molecules, located near the center of the genome. Oligonucleotide primer extension experiments determined that the downstream limit of this gap coincides with the last nucleotide of a central copy of the polypurine tract found in all sequenced lentivirus genomes. Other retroviruses have only one copy of the polypurine tract at the 5' boundary of the 3' long terminal repeat, which has been shown to determine initiation of retroviral DNA plus-strand synthesis. We conclude from our observations that the central repeat of the polypurine tract can create an additional site for plus-strand synthesis initiation in lentiviruses. The central single-stranded gap was not found in circular DNA molecules, the vast majority of them carrying only one long terminal repeat. This finding suggests that the generation of such circular molecules is associated with early DNA ligation events.

Section: Resultssupporting

confidence: 58%

A single-stranded gap in human immunodeficiency virus unintegrated linear DNA defined by a central copy of the polypurine tract

Charneau

Clavel

1991

131

“…In fact, previously published data on simple retroviruses are consistent with the idea that discontinuous reverse transcripts may be the predominant substrates for integration in these systems also. Discontinuous plus strands are the major products of avian sarcoma virus reverse transcription in vivo (3,4,23,26). Mouse mammary tumor virus and Moloney murine leukemia virus also display short heterogeneous plus strands at early times after infection (4 to 10 h) (17,23), but in these cases they display primarily full-length plus strands at later times (24 h).…”

mentioning

confidence: 99%

“…Discontinuous plus strands are the major products of avian sarcoma virus reverse transcription in vivo (3,4,23,26). Mouse mammary tumor virus and Moloney murine leukemia virus also display short heterogeneous plus strands at early times after infection (4 to 10 h) (17,23), but in these cases they display primarily full-length plus strands at later times (24 h). This latter observation led investigators to conclude that the short plus strands apparent at early times were precursors to full-length plus strands, possibly the result of pausing by reverse transcriptase during synthesis (23).…”

mentioning

confidence: 99%

“…Mouse mammary tumor virus and Moloney murine leukemia virus also display short heterogeneous plus strands at early times after infection (4 to 10 h) (17,23), but in these cases they display primarily full-length plus strands at later times (24 h). This latter observation led investigators to conclude that the short plus strands apparent at early times were precursors to full-length plus strands, possibly the result of pausing by reverse transcriptase during synthesis (23). The eventual appearance of full-length plus strands in unintegrated viral cDNAs led to the view that a continuous plus strand is completed prior to integration.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Human immunodeficiency virus type 1 preintegration complexes containing discontinuous plus strands are competent to integrate in vitro

Miller

Wang

Bushman

1995

Despite intensive study, the mechanism by which many retroviruses complete reverse transcription has remained unclear. Most retroviruses and all lentiviruses fail to synthesize a full-length second strand of the viral cDNA (plus strand) efficiently in infected cells. For human immunodeficiency virus type 1, we find in synchronous infection experiments that full-length plus strands are rare (<1% of products) at times when integration is likely taking place. Subviral nucleoprotein complexes containing such discontinuous cDNA can be extracted from infected cells and used to generate integration products in vitro. Analysis of such integration products using two-dimensional gel electrophoresis revealed that the discontinuous viral DNA was efficiently integrated into an added target DNA. These data support a model in which the discontinuities in the plus strand need not be sealed until after integration, potentially by the enzymes that are already thought to repair DNA gaps at the junctions between host and viral DNA.

“…The mechanism by which plus-strand DNA synthesis is primed is unknown. Although multiple priming events might be involved in the synthesis of the complete plus-strand DNA (24,34), studies on the nature of the first plus strand to be synthesized (called plus strong-stop DNA) indicate that the origin for plus-strand initiation is located near the 5' end of the newly synthesized minus-strand DNA (15,16,20). Moreover, for Moloney murine leukemia virus (M-MuLV), the site at which plus strong-stop DNA initiates has been found to be unique and to begin with the sequence 5'-AATGAAAGA (19) (Fig.…”

mentioning

confidence: 99%

RNA-primed initiation of Moloney murine leukemia virus plus strands by reverse transcriptase in vitro

Finston¹,

Champoux²

1984

A 190-base-pair DNA-RNA hybrid containing the Moloney murine leukemia virus origin of plus-strand DNA synthesis was constructed and used as a source of template-primer for the reverse transcriptase in vitro. Synthesis was shown to initiate precisely at the known plus-strand origin. The observation that some of the origin fragments retained ribonucleotide residues on their 5' ends suggests that the primer for chain initiation is an RNA molecule left behind by RNase H during the degradation of the RNA moiety of the DNA-RNA hybrid. If the RNase H is responsible for creating the correct primer terminus, then it must possess a specific endonucleolytic activity capable of recognizing the sequence in the RNA where plus strands are initiated. The 16-base RNase A-resistant fragment which spans the plus-strand origin can also serve as a source of the specific plus-strand primer RNA. Evidence is presented that some of the plus-strand origin fragments synthesized in the endogenous reaction contain 5' ribonucleotides, suggesting that specific RNA primers for plus-strand initiation may be generated during reverse transcription in vivo as well. 26 on July 6, 2020 by guest