M. Neumann et al., SPLICING VARIABILITY IN HIV TYPE-1 REVEALED BY QUANTITATIVE POLYMERASE CHAIN-REACTION, AIDS research and human retroviruses, 10(11), 1994, pp. 1531-1542
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.