AEGiS-14IAC: Identification of HIV-1 spliced transcripts expressed in vivo.

14th International AIDS Conference

Barcelona, Spain - July 7-12, 2002

Identification of HIV-1 spliced transcripts expressed in vivo.

Int Conf AIDS 2002 Jul 7-12; 14:(abstract no. A10017)

Sierra M, Thomson MM, Delgado E, Colomo C, Bru F, Carmona R, Cuevas L, Cuevas MT, Medrano L, Perez-Alvarez L, Vazquez-de Parga E, Villahermosa ML, Najera R
Instituto de Salud Carlos III, Majadahonda (Madrid), Spain

BACKGROUND: The expression of the multiple HIV-1 alternatively spliced transcripts has been generally examined in vitro, and knowledge on their in vivo expression is scarce.

METHODS: RNA was extracted from 24 uncultured PBMC samples from 16 HIV-1-infected individuals. Short transcripts, coding for nef, rev, tat, and vpr, were coamplified by RT-PCR and nested PCR, using primers binding at the 5' and 3' exons common to all RNAs. PCR products were identified by electrophoresis in a high resolution agarose gel and by highly accurate size determination by using fluorescently-labeled primer and size standards, with electrophoresis in an automated sequencer, and analysis by Genescan software. The identity of the products was confirmed by PCR with primers specific for individual transcripts or for transcripts incorporating alternatively spliced exons, and also by direct sequencing or by sequencing of cloned PCR products.

RESULTS: 14 different transcript classes coding for Nef (n=5), Rev (n=5), Tat (n=2) and Vpr (n=2) were identified, using splice sites previously identified in vitro. The number of samples in which transcripts coding for each protein were detected was: Nef: 18; Rev: 6; Tat: 14; Vpr: 13. Transcripts potentially coding for a 34 amino acid peptide at the C-terminus of Nef were detected in 5 samples. The detection of transcripts by size determination using Genescan was confirmed by PCR amplification with primers specific for individual RNAs.

CONCLUSIONS: the patterns of in vivo expression of HIV-1 early transcripts are similar to those found in vitro. Nef-coding transcripts are the most frequently detected, followed by those coding for Tat, Vpr and Rev. Transcripts potentially coding for a 34 amino acid peptide at the C-terminus of nef were identified in some subjects. Identification and quantitation of individual HIV-1 transcripts may be useful for examining the involvement of the expression of viral genes in pathogenesis.

Keywords: AEGIS, HIV-1, Polymerase Chain Reaction, Genes, Viral, Reverse Transcriptase Polymerase Chain Reaction, In Vitro, genetics

020707
A10017