132 Identification of New Patterns of Splice Site Usage by Transcripts of HIV-1 Primary Isolates of Diverse SubtypesDelgado, Elena; Carrera, Cristina; Nebreda, Paloma; Thomson, Michael MJAIDS Journal of Acquired Immune Deficiency Syndromes: April 2011 - Volume 56 - Issue - p 53 doi: 10.1097/01.qai.0000397319.32460.a0 Abstracts Free Author InformationAuthors Article MetricsMetrics Instituto de Salud Carlos III. Centro Nacional de Microbiologia. Ctra. Majadahonda-Pozuelo, Km. 2. 28220 Majadahonda (Madrid). Spain HIV-1 expresses all its RNAs from a single primary transcript which undergoes a complex alternative splicing process, by which more than 40 different spliced transcripts can be generated. Knowledge on HIV-1 splice site usage by primary isolates of non-B subtypes is scarce. Here we analyze HIV-1 splice site usage in acute in vitro infection of peripheral blood mononuclear cells (PBMCs) by 15 primary isolates of subtypes A, B, C, and G. HIV-1 spliced transcripts were amplified by RT-PCR coupled with nested PCR using primers recognizing sequences common to all doubly spliced (DS) or singly spliced (SS) transcripts. Fluorescent labelling of one of the primers in the nested PCR and electrophoresis in an automated sequencer allowed PCR product size determination and quantitation by using the GeneMapper program. PCR products with unexpected sizes, according to known HIV-1 splice site usage, were identified by cloning and sequencing. Wide fluctuations in the relative expression of spliced transcripts within the DS and SS classes following acute infection of PBMCs were frequently observed. In one subtype A virus high levels of expression of tat RNAs were observed, which surpassed rev RNA expression and approached nef transcript levels. All 3 analyzed subtype C viruses utilized a previously unreported splice site for generation of rev transcripts located 7 nucleotides upstream of SA4a, designated SA4d, which was the major splice site used by rev transcripts in two isolates. In one subtype B virus, rev transcripts used predominantly a newly identified splice site (SA4e) located 5 nt upstream of SA4a. In one subtype C virus, a majority of rev transcripts (but not of nef or tat RNAs) incorporated noncoding exon 3. These results show a high diversity of spliced RNA expression in primary HIV-1 isolates of different subtypes.Copyright © 2011 Wolters Kluwer Health, Inc. All rights reserved.