Analysis of Human Immunodeficiency Virus Type 1 Integration by Using A Specific, Sensitive and Quantitative Assay Based on Real-time Polymerase Chain Reaction

Abstract

A novel real-time nested-PCR assay was developed to quantify integrated human immunodeficiency virus type-1 (HIV-1) DNA with high specificity and sensitivity. This assay reproducibly allowed the detection of three copies of integrated HIV DNA in a background of 100,000 cell equivalents of human chromosomal DNA. The non-specific amplification of unintegrated HIV-1 DNA was significantly inhibited in this assay and the specificity of this assay was much higher than the previously reported method. This assay showed that kinetics in viral DNA synthesis was cell-type dependent and that the kinetics of HIV-1 DNA integration was very rapid in Jurkat T cell line. This method may provide new insights into the integration processes and be useful in evaluating future integrase inhibitors.

Inhibiting HIV-1 integrase by shifting its oligomerization equilibrium

“…Real-time PCR experiments were performed as described in ref. 45. The second round of PCR was performed on 1/25 of the first-round PCR product in a mixture containing 300 nM each primer, 12.5 l of 2ϫ SYBR green master mix (Applied Biosystems) at a final volume of 25 l, run on an ABI PRIZM 7700 (Applied Biosystems).…”

Nucleocytoplasmic shuttling of HIV-1 integrase is controlled by the viral Rev protein

“…21 Briefly, Integrated HIV-1 sequences were amplified by two PCR replication steps using the HIV-1 LTR-specific primer (LTR-TAG-F 5'-ATG CCA CGT AAG CGA AAC TCT GGC TAA CTA GGG AAC CCA CTG-3') and Alu-targeting primers (first-Alu-F 5'-AGC CTC CCG AGT AGC TGG GA-3' and first-Alu-R 5'-TTA CAG GCA TGA GCC ACC G-3'). 52 Alu-LTR fragments were amplified from 10 ng of total cell DNA in a 25-µl reaction mixture containing 1X PCR buffer, 3.5 mM MgCl 2 , 200 µM dNTPs, 300 nM primers, and 0.025 units/µl of Taq polymerase. The first-round PCR cycle conditions were as follows: a DNA denaturation and polymerase activation step of 10 min at 95°C and then 12 cycles of amplification (95°C five washes with PBS + 0.05% Tween 20 between antibodies.…”

Adenovirus Vectors Block Human Immunodeficiency Virus–1 Replication in Human Alveolar Macrophages by Inhibition of the Long Terminal Repeat

“…AMs were infected with HIV-1 JRFL, as described previously here, and DNA was extracted from the AMs after 72 hours with the Dneasy Tissue Kit (Qiagen). A two-step nested amplification was performed to initially amplify sequences exclusively containing only human 300-base pair repetitive DNA sequences (Alu) adjacent to HIV-1 envelope, based on a recently published method (34). Primers and probes were developed to quantify human Alu sequences and the JRFL envelope gene with the PrimerExpress software (Perkin Elmer).…”