Shreesh Saurya1, Zelman Lichtenstein, Abraham Karpas. 1. Department of Haematology, University of Cambridge Clinical School, MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK. ssaurya@hammer.imm.ox.ac.uk
Abstract
BACKGROUND: Highly Active Antiretroviral Therapy (HAART) has emerged as one the most effective method for treating AIDS patients. However, after variable period of treatment, many AIDS patients on HAART show elevated PCR viral load without a corresponding decline in CD4+ T-cells. Our earlier studies have revealed that the viruses present in the plasma of such patients are not infectious. OBJECTIVE: The aim of this study is to characterize the changes in the regulatory genes of HIV-1, namely tat, rev and rev response element (RRE) isolated from the plasma of such AIDS patients and to assess their role in role in affecting viral infectivity, hence its contribution, in the 'contradictory phenomenon' of high viral load and high CD4+ T-cell counts. STUDY DESIGN: The viral RNA was isolated from the plasma of HAART patients when they exhibited high plasma viral load and high CD4+ T-cell counts. The target regulatory genes were amplified by RT-PCR and sequenced. Sequences were also obtained from the proviral DNA from the peripheral blood mononuclear cells (PBMCs) of the study subjects. The sequences were compared with the wild type viral sequence to look for the changes induced in them due to HAART regime. RESULTS AND CONCLUSION: Our data revealed that RRE was missing in the viral particles isolated from the plasma of all study subjects. In two patients, the second exon of the rev gene was missing thereby leading to defective Rev protein. In another patients, Rev synthesis was prematurely stopped due to G135T substitution in the amino terminal domain. No such changes were observed in the corresponding proviral DNA. These changes are likely to result in the assembly of non-infectious virus due to lack of envelope proteins. Absence of RRE and Rev protein also leads to transport and packaging of multiply spliced transcripts into the virions instead of complete genomic RNA.
BACKGROUND: Highly Active Antiretroviral Therapy (HAART) has emerged as one the most effective method for treating AIDSpatients. However, after variable period of treatment, many AIDSpatients on HAART show elevated PCR viral load without a corresponding decline in CD4+ T-cells. Our earlier studies have revealed that the viruses present in the plasma of such patients are not infectious. OBJECTIVE: The aim of this study is to characterize the changes in the regulatory genes of HIV-1, namely tat, rev and rev response element (RRE) isolated from the plasma of such AIDSpatients and to assess their role in role in affecting viral infectivity, hence its contribution, in the 'contradictory phenomenon' of high viral load and high CD4+ T-cell counts. STUDY DESIGN: The viral RNA was isolated from the plasma of HAART patients when they exhibited high plasma viral load and high CD4+ T-cell counts. The target regulatory genes were amplified by RT-PCR and sequenced. Sequences were also obtained from the proviral DNA from the peripheral blood mononuclear cells (PBMCs) of the study subjects. The sequences were compared with the wild type viral sequence to look for the changes induced in them due to HAART regime. RESULTS AND CONCLUSION: Our data revealed that RRE was missing in the viral particles isolated from the plasma of all study subjects. In two patients, the second exon of the rev gene was missing thereby leading to defective Rev protein. In another patients, Rev synthesis was prematurely stopped due to G135T substitution in the amino terminal domain. No such changes were observed in the corresponding proviral DNA. These changes are likely to result in the assembly of non-infectious virus due to lack of envelope proteins. Absence of RRE and Rev protein also leads to transport and packaging of multiply spliced transcripts into the virions instead of complete genomic RNA.