Quantitative dynamics of HIV type 1 expression.
A competitive PCR and RT-PCR procedure was developed for the quantification of HIV-1 nucleic acids in infected biological samples, with particular reference to the study of the kinetics of production of differently processed viral transcripts. The procedure entails the utilization of a competitor plasmid DNA (on DNA samples) or of an in vitro transcription product obtained from this plasmid (on RNA samples) and allows the quantification of proviral DNA, viral genomic RNA, and viral single- and multispliced mRNAs. Furthermore, it permits the direct standardization of these measurements to the amount of a reference cellular gene (for DNA quantification) or of a reference cellular transcript (for RNA quantification). This quantification procedure was used to monitor the dynamics of HIV-1 transcriptional activation in the latently infected U1 monocytic cell line after stimulation with phorbol-12-myristate-13-acetate, and in experimentally infected peripheral blood lymphocytes. Despite the biological differences between the two experimental systems, in both cases production of infectious virus is accompanied by a remarkable increase in the levels of unspliced viral mRNAs (rising up to 20,000 fold in U1 cells) and by a consequent switch in the abundance of the differently spliced transcript classes. These observations reinforce the notion that the control of infection is subjected also to posttranscriptional events and prompts for quantitative evaluation of HIV-1 transcript class abundance in infected individuals to define potential markers for disease progression.
Comar
M
M
International Centre for Genetic Engineering and Biotechnology, Trieste, Italy.
Marzio
G
G
D'Agaro
P
P
Giacca
M
M
D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers; D004279:DNA, Viral; D015967:Gene Expression Regulation, Viral; D015497:HIV-1 / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D008969:Molecular Sequence Data; D012367:RNA, Viral; D013698:Templates, Genetic; D015533:Transcriptional Activation
D017931:DNA Primers; D004279:DNA, Viral; D012367:RNA, Viral