DETECTION AND QUANTITATION OF CELLULAR AND HIV-1 MESSENGER RNAs UTILIZING RT-PCR

Abstract

Effective methods are needed to identify and quantitate differential gene expression in various cell types and viruses. The lab in which I work and study, under the direction of Dr. George Pavlakis, has made improvements to the reverse transcription-polymerase chain reaction (RTPCR) methodology. Two different sets of HIV-1 specific oligonucleotides were designed for the amplification of 1) unspliced, singly and multiply-spliced RNA transcripts and 2) Rev dependent and independent transcripts. Other noteworthy modifications to the standard RT-PCR protocol include, the use of sets of HIV-1 cDNA mixes to determine size and quantity of mRNA products and the use of denaturing acrylamide gels to run the amplification products. By using the set of oligonucleotides which determine multiply-spliced messages I was able to identify a novel rev/nef splice acceptor in HIV-1 from a asymptomatic HIV-1 infected individual as well as in the LAI laboratory strain of HIV-1. Finally, these modifications in RT-PCR methodology facilitated the better quantitation HIV-1 gene expression in response to interferon alpha (IFN-α.) It has been shown that IFN inhibits HIV-1 expression in vitro in cell lines. To demonstrate inhibition in vivo ,in a more natural system, donor peripheral blood mononuclear cells (PBMC), were treated with IFN α over a time course of 12-14 days in culture. Upon infection with HIV-1 LAI , we collected samples every 48 hours for p24 antigen capture assay and RNA analysis. RNA was extracted and PCR was performed on the cDNA products of the reverse transcription reactions. IFN α dramatically down regulates the levels of p24 Gag antigen and all HIV-1 transcripts. The effect of IFNs is more prominent on the Rev dependent mRNA transcripts. DNA PCR was also performed at early time points in the first 24 hours post infection. By 24 hours, the levels of gag DNA in untreated cells were six times higher than in cells treated with IFN α. Lower proviral DNA levels may directly translate into the reduction of RNA observed. Alternatively, IFN α could inhibit HIV-1 expression both at an early stage of infection and later through interfering with a posttranscriptional pathway. IFN a is known to induce a variety of cellular factors including one which was recently identified in our laboratory. RNA binding protein (RBP) 9-27 was identified through the screening of a U937 cDNA library with an HIV-1 Rev Responsive Element (RRE) RNA probe. The protein binds specifically to the highly ordered RAE RNA structure, and in transfection experiments, inhibits the expression of transcripts that require Rev/RAE interaction (mainly gag/pol and vpu/env.) The 9-27 gene is a member of the 1-8 family of interferon-inducible genes. The role of two additional members of the family (1-8U and 1-8D) in regulating HIV-1 expression are also being investigated in our laboratory. The basal and interferon-induced levels of 9-27,1-8D and 1-8U RNAs were determined in our HIV-1 infection experiments using specific oligonucleotide primers. Infection (without IFN α treatment) results in a very low level increase in 9-27 over baseline. The other two members are not induced by infection alone. IFN α treatment induces 1-8D and 1-8U and further induces 9-27 expression. There may be a correlation between the rise in gene expression in the 1-8 family and the inhibition of HIV-1 gene expression by IFN α. Our studies attempt to investigate such a correlation and examine the possible post transcriptional inhibitory effects of these interferon-inducible genes on HIV-1 expression in vivo.