Quantitative and discriminative detection of individual HIV-1 mRNA subspecies by an RNAse mapping assay.
HIV-1 genes are expressed through the complex splicing of a single mRNA precursor leading to three mRNA classes: unspliced, singly-spliced and multiply-spliced. Each class may include several mRNA species specifically encoding one or two HIV-1 proteins. Northern blotting and RT-PCR are the techniques currently used to analyse HIV-1 mRNA expression. Northern blotting allows quantitative detection of these three classes of viral RNA but does not discriminate between individual RNA species. 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 discriminative HIV-1 RNA detection. A radiolabeled probe overlapping the major splicing sites of HIV-1 used for the generation of HIV-1 mRNA subspecies was synthesized. This probe protects differential sizes of these species, allowing discrimination between them. We investigated the RNA expression 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 discriminative and quantitative properties, this application is particularly convenient for the investigation of HIV-1 mRNA expression during the course of HIV-1 infections.
Mary
C
C
Unité mixte CNRS/Biomérieux, Lyon, France.
Telles
J N
JN
Cheynet
V
V
Oriol
G
G
Mallet
F
F
Mandrand
B
B
Verrier
B
B
D001483:Base Sequence; D015152:Blotting, Northern; D002460:Cell Line; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics; D005814:Genes, Viral; D005822:Genetic Vectors; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics*; D012367:RNA, Viral / Q000032:analysis* / Q000235:genetics*; D012260:Ribonucleases; D014773:Virology / Q000379:methods*
D017931:DNA Primers; D004279:DNA, Viral; D012333:RNA, Messenger; D012367:RNA, Viral; D012260:Ribonucleases