SPLICING VARIABILITY IN HIV TYPE-1 REVEALED BY QUANTITATIVE POLYMERASE CHAIN-REACTION

A quantitative RNA-polymerase chain reaction (PCR) method able to dete ct 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 condition s and eliminated the generation of artifactual PCR bands. The use of R NA and cDNA standards demonstrated that the RNA amplification is linea r within the tested range and suggested that it can be used to quantit ate 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 de terminant of the biological characteristics of the different HIV-1 clo nes, and demonstrates a great plasticity of the HIV-1 genome. The desc ribed RNA-PCR methodology was used for the study of HIV-1 expression i n unstimulated peripheral blood mononuclear cells (PBMCs) of infected individuals. In general, the same mRNAs were identified in HIV-infecte d cultured cell lines and in unstimulated PBMCs. Analysis of a variant band found after amplification of PBMC RNA from an HIV-infected indiv idual revealed a new splice site for the generation of Rev/Nef-encodin g mRNAs. The availability of a sensitive, rapid, and essentially quant itative method to examine the major HIV-1 mRNAs will facilitate the de tailed analysis of HIV-1 expression in human cells.