QUANTITATIVE AND DISCRIMINATIVE DETECTION OF INDIVIDUAL HIV-1 MESSENGER-RNA SUBSPECIES BY AN RNASE MAPPING ASSAY

HIV-1 genes are expressed through the complex splicing of a single mRN A precursor leading to three mRNA classes: unspliced, singly-spliced a nd multiply-spliced. Each class may include several mRNA species speci fically encoding one or two HIV-1 proteins. Northern blotting and RT-P CR are the techniques currently used to analyse HIV-1 mRNA expression. Northern blotting allows quantitative detection of these three classe s of viral RNA but does not discriminate between individual RNA specie s. RT-PCR allows discrimination between different species but does not provide a quantitative analysis. Here, we describe an application of an RNAse mapping assay which gives both quantitative and discriminativ e HIV-1 RNA detection. A radiolabeled probe overlapping the major spli cing sites of HIV-1 used for the generation of HIV-1 mRNA subspecies w as synthesized. This probe protects differential sizes of these specie s, allowing discrimination between them. We investigated the RNA expre ssion pattern in high titer HIV-1 producing cells. The HIV-1-specific probe allowed the detection of multiply-spliced vpr, rev and nef mRNAs , singly-spliced env mRNA and unspliced genomic RNA, With its discrimi native and quantitative properties, this application is particularly c onvenient for the investigation of HIV-1 mRNA expression during the co urse of HIV-1 infections.