Purification of HIV‐1 LTR circles from PBMCs to selectively amplify episomal env sequences

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Highly Active Antiretroviral Therapy (HAART) has been effective in suppressing HIV viremia. Two main disadvantages of this regimen are the serious side effects associated with long-term use and its inability to completely eradicate HIV. The source of the virions that emerge during suppressive HAART can be identified by a phylogenetic analysis that compares the virions present in plasma with those undergoing residual replication or in reservoirs. We developed a protocol using an ATP dependent plasmid safe DNAse that selectively digests linear DNA to purify LTR circles from chromosomal DNA. The â-actin gene was amplified to detect contamination from linear DNA. A phylogenetic analysis of the env HIV regions of the LTR circles, PBMCs and free virus in plasma from three patients within a 24 copies to 800 viral copies/ ml viral load range was undertaken to determine if LTR episomes are labile in vivo. No â-actin amplification was observed. Kimura 2-parameter, distances of < 0.01 were observed within each patient group of episomal clones. The mean distances between the episome and free RNA were <0.01 and 0.05 respectively for the 24 viral copies/ml and the 800 viral copies/ ml patients. In contrast, the corresponding mean distances between episomes and PBMCs were 0.03 and 0.07 for these two groups. This method is a rapid and economic alternative to selectively amplify HIV-1 regions from LTR circles. The lower genetic variability in the env region from LTR circles and free RNA may suggest that episomes are surrogate markers of residual replication. This study was supported in part by S06 GM08239 NIH-NIGMS-MBRS grant.ββ