Splicing Variability in HIV Type 1 Revealed by Quantitative RNA Polymerase Chain Reaction

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Abstract
A quantitative RNA-polymerase chain reaction (PCR) method able to detect the majority of mRNAs produced by human immunodeficiency virus type 1 (HIV-1) was developed and used to study expression of different HIV-1 clones in human cells. Amplified mRNAs were compared to known cDNA standards. This comparison permitted the optimization of PCR conditions and eliminated the generation of artifactual PCR bands. The use of RNA and cDNA standards demonstrated that the RNA amplification is linear within the tested range and suggested that it can be used to quantitate individual mRNAs. The results demonstrate the overall conservation of splicing in different HIV-1 clones. Although, in general, splicing was conserved, extensive qualitative and quantitative variability was observed in different HIV-1 clones. This variability is likely one determinant of the biological characteristics of the different HIV-1 clones, and demonstrates a great plasticity of the HIV-1 genome. The described RNA-PCR methodology was used for the study of HIV-1 expression in unstimulated peripheral blood mononuclear cells (PBMCs) of infected individuals. In general, the same mRNAs were identified in HIV-infected cultured cell lines and in unstimulated PBMCs. Analysis of a variant band found after amplification of PBMC RNA from an HIV-infected individual revealed a new splice site for the generation of Rev/Nef-encoding mRNAs. The availability of a sensitive, rapid, and essentially quantitative method to examine the major HIV-1 mRNAs will facilitate the detailed analysis of HIV-1 expression in human cells.