Abstract
The detection of antisense RNA is hampered by reverse transcription (RT) non-specific priming, due to the ability of RNA secondary structures to prime RT in the absence of specific primers. The detection of antisense RNA by conventional RT-PCR does not allow assessment of the polarity of the initial RNA template, causing the amplification of non-specific cDNAs. In this study we have developed a modified protocol for the detection of human immunodeficiency virus type 1 (HIV-1) antisense protein (ASP) RNA. Using this approach, we have identified ASP transcripts in CD4+ T cells isolated from five HIV-infected individuals, either untreated or under suppressive therapy. We show that ASP RNA can be detected in stimulated CD4+ T cells from both groups of patients, but not in unstimulated cells. We also show that in untreated patients, the patterns of expression of ASP and env are very similar, with the levels of ASP RNA being markedly lower than those of env. Treatment of cells from one viraemic patient with α-amanitin greatly reduces the rate of ASP RNA synthesis, suggesting that it is associated with RNA polymerase II, the central enzyme in the transcription of protein-coding genes. Our data represent the first nucleotide sequences obtained in patients for ASP, demonstrating that its transcription indeed occurs in those HIV-1 lineages in which the ASP open reading frame is present.
Highlights
The ASP gene is an antisense open reading frame (ORF) potentially encoding for the putative human immunodeficiency virus type 1 (HIV-1) antisense protein (ASP) [1]
The tree was inferred using the maximum-likelihood method with 1000 bootstrap replicates using MEGA 7.0 software. Sequences from both cells and serum were aligned with the reference sequences of the HIV-1 M group subtypes, which were obtained from the HIV sequence database
By using a modification of the protocol proposed by Haist et al [17], we detected and sequenced ASP RNA transcripts in CD4+ T lymphocytes in three viraemic patients, who were either naïve to therapy or untreated
Summary
The ASP gene is an antisense open reading frame (ORF) potentially encoding for the putative HIV-1 antisense protein (ASP) [1]. In HTLV-1, antisense transcription results in the production of HTLV-1 basic zipper protein (hbz), which is involved in the regulation of adult T-cell leukaemia oncogenesis and whose highly conserved ORF is located between the env and tax/rex genes [3, 4]. In HIV-1, the ASP ORF is located in the envelope gene at the junction between gp120 and gp, in the antisense reading frame À2, in a similar position to the hbz of HTLV-1 [1]. The putative ASP protein is 189 amino acids long and appears to have two potential transmembrane helices with a cytoplasmic N-terminus, indicating that it could be associated with cell membranes [1, 11, 12]
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