Analysis of early human immunodeficiency virus type 1 DNA synthesis by use of a new sensitive assay for quantifying integrated provirus

Abstract

A novel Alu-long terminal repeat (LTR)-based real-time nested-PCR assay was developed to quantify integrated human immunodeficiency virus type 1 (HIV-1) DNA in infected cells with both accuracy and high sensitivity (six proviruses within 50,000 cell equivalents). Parallel assays for total HIV-1 DNA and two-LTR HIV-1 DNA circles allowed the synthesis and fate of the different HIV-1 DNA species to be monitored upon a single round of viral replication.

FIG. 1.

Real-time PCR strategies and location of primers and probes for the quantification of integrated HIV-1 DNA (a), total HIV-1 DNA (b), and two-LTR circles (c). Thin arrows, primers; thick arrows, probes.

FIG. 2.

Characteristics of Alu-LTR nested PCR. (a) Fluorescence curves generated by two-step amplification of serial dilutions of HeLa R7 Neo cell DNA. The copy numbers for each standard dilution are shown over the corresponding fluorescence curve. (b) Linear regression for quantifying integrated HIV-1 DNA. (c) Specificity of the Alu-LTR nested PCR. Fluorescence curves generated by amplification of viral DNA from CEM cells infected with the VSV-G-pseudotyped HIV-1 R7 Neo virus and collected in the presence of Alu primers (pink, 8 h p.i.; green, 48 h p.i.), in the absence of Alu primers (blue, 8 h p.i.; red, 48 h p.i.), or without a preamplification step (purple, 8 h p.i.; orange, 48 h p.i.) are shown.

FIG. 3.

Quantification of total HIV-1 DNA (a), integrated HIV-1 DNA (b), and two-LTR circles (c) during a single round of replication of the VSV-G-pseudotyped HIV-1 R7 Neo virus in CEM cells. Values are means ± standard errors of the means. The depicted results were obtained from a representative experiment.