Quantitative and Discriminative Detection of Individual HIV-1 mRNA Subspecies by an RNase Mapping Assay
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In addition to the Gag, Pol, and Env structural proteins, HIV-1 encodes at least six regulatory proteins: Tat, Rev, Nef, Vif, Vpr, and Vpu. All HIV-I proteins are encoded by overlapping reading frames and are expressed through the complex alternative splicing of a single precursor RNA leading to three major RNA classes (8 -8 ): an unspliced class which includes both genomic RNA and gag-pol mRNA; a singly spliced class that includes mRNAs coding for Env, Vpu, Vif, Vpr, and a truncated form of Tat protein; and a multiply-spliced class that includes mRNAs coding for the regulatory proteins Tat, Rev, and Nef (Fig. 1 ). The function of the regulatory proteins Tat, Rev, Nef, Vif, Vpr, and Vpu has not yet been fully elucidated. Nevertheless, they seem to play specific roles during the different steps of the HIV-1 replication cycle (9 -11 ). For this reason, the detection of mRNA species encoding these proteins at different steps of the virus replication cycle will provide important information about the function of these proteins. In order to obtain both a quantitative and qualitative detection of these mRNAs, we used a ribonuclease protection assay. Ribonuclease protection assays are commonly used for the detection and quantification of mRNAs (12 ,13 ) and are well-suited for mapping the position of internal and external junctions in mRNAs (14 ,15 ). In addition, as the hybridization takes place in liquid conditions, this technique is more sensitive than other quantitative methods of detection of RNAs such as Northern blotting (16 ). Our assay uses a DNA template specific of the HIV-1 strain to be investigated.
Fig. 1. (A ) HIV-1 mRNA splicing pattern: HIV-1 genomic organization and the different exons are shown at the top. HIV-1 major splice sites are indicated and the three mRNA classes (unspliced, singly spliced, and multiply spliced) are represented. Both singly spliced and multiply spliced mRNA species contain a splice from the 5′ splice site d1 to one of the 3′ splice sites a1-a6. Multiply spliced mRNAs contain an additional splice from the 5′ splice site d4 to the 3′ splice site a10. Correspondence of each coding exon to its encoded protein(s) is given at the right of the panel referring to Schwartz’s nomenclature. (B ) Enlargement of the HIV-1 genome central region amplified for T7 polymerase template construction. Major splice acceptors used for singly spliced vif, vpr, tat, vpu/env , and multiply spliced vpr, tat, rev, nef, tev mRNA coding exons are indicated at the top. Horizontal arrows indicate RT-PCR primer pair positions used for vpr cDNA amplification. (C ) vpr cDNA template. This region was obtained by RT-PCR amplification of vpr mRNA with the primers pair 619-512. Vertical arrows indicate restriction enzyme site positions used for template cloning or vector linearization. Restriction enzyme sites provided by RT-PCR primers sequence are indicated by an asterisk ~undefined). Other sites are internal in the HIV-1/HXB2c sequence. The box above the scheme represents sequences from the pGEM vector. Numbers in brackets indicate nucleotide positions referring to the HXB2 CAP site.
