Detecting HIV-1 integration by repetitive-sampling Alu-gag PCR

Abstract

In this review, we compare four assays that are currently used to measure HIV integration and discuss their strengths and weaknesses. We then outline advances that have been made toward development of a more robust, more sensitive, quantitative HIV integration assay suitable for clinical use. The assay that we have developed uses repetitive-sampling Alu-gag PCR. The detailed protocol describes our assay step-by-step, the creation of an integration standard cell line and accompanying standard curve, as well as the quantitation of integration and calculation of associated error estimates. Finally, we speculate on fundamental, unresolved issues in HIV latency that can be addressed by measuring HIV integration.

Figures

Fig. 1

Alu-gag PCR integration assay overview. The assay is performed on isolated DNA. First, the Alu-gag master mixture (MM) is added, which contains primers that bind to Alu and gag. Then, the first PCR round is performed, exponentially amplifying integrated HIV DNA, as Alu and gag primers anneal to opposite strands of the same template DNA (panel A). (B) Unintegrated HIV DNA (both circular and linear forms) is only amplified linearly, because the gag primer anneals to only one strand of the HIV template. (C) Alu-Alu segments of human genomic DNA are also exponentially amplified when the elements are sufficiently close together and in opposite orientation. The Alu-Alu amplicons will not be detected, however; only amplicons that contain HIV DNA are detected because a second nested kinetic PCR (kPCR) is performed using a forward primer to R and a reverse primer to U5 and a molecular beacon complementary to the intervening sequence of the HIV LTR. Integrated HIV DNA (panel A) is amplified exponentially and thus more efficiently than unintegrated DNA (panel B). To measure the background signal or the signal expected from unintegrated DNA, a control reaction using only the gag (and not the Alu) primer is included in the first PCR (not shown). This is done in every assay to define the background. The signals from Alu-gag and gag-only are compared to make sure the Alu—gag signal is stronger, and thus that the sample is positive for integration. Then a correlation is developed that relates the kinetic PCR signals to proviral level, which is used to quantify the level of integration in the samples.

Fig. 2

Integration standard curve. Shown is the curve that we developed for use in estimating proviral levels in samples from infected individuals. The natural log (Ln) of HIV DNA copy number is plotted on the y-axis against the Ln of cycle threshold (Ct) on the x-axis.