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. 2001 Nov;75(22):11253-60.
doi: 10.1128/JVI.75.22.11253-11260.2001.

Kinetics of human immunodeficiency virus type 1 (HIV) DNA integration in acutely infected cells as determined using a novel assay for detection of integrated HIV DNA

N Vandegraaff  1 , R Kumar, C J Burrell, P Li
Affiliations

Kinetics of human immunodeficiency virus type 1 (HIV) DNA integration in acutely infected cells as determined using a novel assay for detection of integrated HIV DNA

N Vandegraaff et al. J Virol. 2001 Nov.

Abstract

We have developed a novel linker-primer PCR assay for the detection and quantification of integrated human immunodeficiency virus type 1 (HIV) DNA. This assay reproducibly allowed the detection of 10 copies of integrated HIV DNA, in a background of 2 x 10(5) cell equivalents of human chromosomal DNA, without amplifying extrachromosomal HIV DNA. We have used this assay and a near-synchronous one-step T-cell infection model to investigate the kinetics of viral DNA accumulation following HIV infection. We report here that integrated HIV DNA started accumulating 1 h after the first appearance of extrachromosomal viral DNA and accounted for approximately 10% of the total HIV DNA synthesized in the first round of viral replication. These results highlight the efficient nature of integrase-mediated HIV integration in infected T cells.

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Figures

FIG. 1
FIG. 1
LP-PCR method for detection of integrated HIV DNA. (A) LP-PCR-mediated amplification of the integrated HIV DNA forms. The nested PCR product was detected using the U3-106 probe fragment (hatched box) (Table 1). (B) BglII-mediated selection against amplification of the three main extrachromosomal HIV DNA forms.
FIG. 2
FIG. 2
Sensitivity and specificity of LP-PCR and comparison with a nested Alu PCR protocol. (A to C) Viral DNA accumulation following cell-free infection in the presence or absence of inhibitors. HuT-78 T cells were infected using the centrifugal enhancement protocol at 0.5 TCID50 per cell and cellular DNA prepared from infected cells harvested at 26 h p.i. 3TC and L-731,988 were used as specific inhibitors of reverse transcription and integration, respectively. (A) Sensitivity of LP-PCR (as measured by amplification of the HA8 integrated HIV DNA standard) and integrated HIV DNA accumulation following infection as measured by LP-PCR performed on 100 cell equivalents of Hirt pellet (chromosomal) DNA preparations. (B) Total reverse-transcribed DNA as measured by GAG-PCR performed on combined Hirt supernatant (extrachromosomal) and Hirt pellet (chromosomal) DNA samples. (C) Graphical representation of the accumulation of integrated HIV DNA. Data were obtained by PhosphorImager analysis of the bands in panel A. (D) Comparison of PCR detection of integrated HIV DNA by LP-PCR and Alu PCR. Chromosomal DNA was isolated from ACH-2 or 8E5 cells and shown to contain equivalent amounts of total HIV DNA by GAG-PCR (314-bp band). Sizes of expected bands for LP-PCR (measuring integrated HIV DNA) are given on the left (104-bp fragment), while the expected size of the product obtained following Alu PCR (also measuring integrated HIV DNA) is indicated on the right (351-bp fragment).
FIG. 3
FIG. 3
Accumulation kinetics of total, integrated, and 2-LTR viral DNA forms following high-multiplicity infection of HuT-78 T cells. Infections were performed using 1 TCID50 of HIVHXB2 per cell with centrifugal enhancement. All PCRs were confirmed to amplify DNA in a linear fashion by quantification of standards (A to D) or dilution sets (E) (dilutions not shown). DNA markers (pUC19/HpaII) are indicated (M). (A) Total HIV DNA forms as measured by GAG-PCR using 500 cell equivalents of total DNA (combined Hirt supernatant and Hirt pellet). (B.i) Integrated HIV DNA levels as measured by LP-PCR on 100 cell equivalents of chromosomal DNA (Hirt pellet). (B.ii) Integrated HIV DNA levels as measured by the modified nested Alu PCR method performed on 1,000 cell equivalents of chromosomal DNA. (C.i) 2-LTR HIV DNA levels as measured by 2-LTR PCR on 500 cell equivalents of total DNA. (C.ii) Reanalysis of later time points for the 2-LTR DNA forms using 1,000 cell equivalents of total DNA. (D) β-Globin levels assayed by PCR on 50 cell equivalents of chromosomal DNA. Standards represent amplification of various amounts (based on cell counts) of HA8 chromosomal DNA. (E) Mitochondrial DNA levels assayed by PCR on 50 cell equivalents of Hirt supernatant (extrachromosomal) fraction.
FIG. 4
FIG. 4
HIV replication parameters following high-multiplicity infection of HuT-78 T cells. Cells were infected with HIVHXB2 at 1 TCID50 per cell using a centrifugally enhanced protocol. (A) P24 levels in culture supernatants were measured at various time points. (B) Comparison of the total (▴), integrated (■), and 2-LTR (×) HIV DNA forms. Data were determined by PhosphorImager quantification of bands in Fig. 3. The levels of total, integrated, and 2-LTR DNA at 26 h p.i. and total HIV DNA at 14 h p.i. are shown. The dashed line indicates one DNA copy/cell.
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