Abstract
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.
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Mary, C., Akaoka, H., Verrier, B. (1998). Quantitative and Discriminative Detection of Individual HIV-1 mRNA Subspecies by an RNase Mapping Assay. In: Meltzer, S.J. (eds) PCR in Bioanalysis. Methods In Molecular Medicine™, vol 92. Humana Press. https://doi.org/10.1385/0-89603-497-6:89
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DOI: https://doi.org/10.1385/0-89603-497-6:89
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