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

Abstract

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.

Human immunodeficiency virus type 1 integrase: effects of mutations on viral ability to integrate, direct viral gene expression from unintegrated viral DNA templates, and sustain viral propagation in primary cells

“…One of these exhibited a temperaturesensitive phenotype and is described in the accompanying paper (57). The mutations were designed so as not to interrupt the following regions of integrase: the DNA sequence of the central polypurine tract (11), the resident splice acceptor and splice donor sites (40), and the region coding for the amino terminus of the Vif protein (40). In some cases aberrant mutagenesis occurred, resulting in single alanine substitutions (H51A and D167A), substitutions with amino acids other than alanine (R199T/D202A, K51A/D55V), and an in-frame deletion of 69 amino acids near the carboxy terminus (E85A/ E87A⌬181-250).…”

Multiple but dissectible functions of FEN‐1 nucleases in nucleic acid processing, genome stability and diseases

“…The HIV-1 central DNA flap (CDF) is formed during reverse transcription of HIV-1 RNA genome. (93) The 99-nucleotide long CDF is a substrate for human FEN-1 regardless of the presence of secondary structure within the CDF. (63) Furthermore, HIV-1 integrase has been reported to interact with hFEN-1 and mutual stimulation of their respective activities has been (6) observed.…”

Identification of conserved lentiviral sequences as landmarks of genomic flexibility

“…It is worth mentioning that CLSs 16 and 2 have a very close function. CLS 16 was a part of the cPPT involved in the initiation of lentivirus reverse transcription [53] where DNA synthesis enhances DNA/DNA recombination [54]. CLS 2 represented the well-conserved part of the distal PPT, and perhaps the action site, while the neighbouring sequences which varied highly from a virus to another one might constitute a specific recognition site for the reverse transcrip-tase associated with a given virus.…”

“…CLS 2 represented the well-conserved part of the distal PPT, and perhaps the action site, while the neighbouring sequences which varied highly from a virus to another one might constitute a specific recognition site for the reverse transcrip-tase associated with a given virus. HIV-1s displayed two CLS 2 at positions shown to be the cPPT (pol gene) [44] and the distal PPT (nef gene) [53]. Also, HIV-2s revealed a slightly different organization, since CLS 16 was part of the cPPT (pol gene) and CLS 2 part of the distal PPT when the nef gene overlaps the 3 LTR.…”