scholarly journals A Sensitive, Quantitative Assay for Human Immunodeficiency Virus Type 1 Integration

2002 ◽  
Vol 76 (21) ◽  
pp. 10942-10950 ◽  
Author(s):  
Una O'Doherty ◽  
William J. Swiggard ◽  
Deepa Jeyakumar ◽  
David McGain ◽  
Michael H. Malim
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Quantitative Methods ◽  
Integrase Inhibitor ◽  
Pcr Assay ◽  
Immunodeficiency Virus ◽  
Latent Hiv ◽  
Hiv 1

ABSTRACT Quantitative methods to measure human immunodeficiency virus type 1 (HIV-1) integration promise to be important tools in dissecting the mechanisms whereby latent reservoirs of provirus are established, most notably in the resting T cells of patients receiving antiretroviral therapy. Here we describe a fluorescence-monitored, nested PCR assay that is able to quantify the relatively rare integration events that occur within these cells. Following DNA extraction, a nonkinetic preamplification step is performed with primers that bind genomic Alu elements and HIV-1 gag sequences, under conditions where primers, deoxynucleoside triphosphates, and enzyme are not limiting. This is followed by a kinetic PCR that quantitates HIV-1 long terminal repeat sequences. A T-cell-based integration standard which reflects the randomness of HIV-1 integration is also described. The assay is 10 to 100 times more sensitive than previously reported quantitative Alu PCR-based integration assays. It is specific for integration events, since no proviruses are detected in cells infected either in the presence of an integrase inhibitor or with an integrase-deficient virus. This method promises to provide important new insights into the processes underlying the accumulation and persistence of latent HIV-1 reservoirs and may eventually be useful clinically in monitoring the eradication of latent virus by novel therapies.

scholarly journals Fluorescence-Based Quantitative Methods for Detecting Human Immunodeficiency Virus Type 1-Induced Syncytia

2000 ◽  
Vol 38 (8) ◽  
pp. 3055-3060 ◽  
Author(s):  
Sabina Wünschmann ◽  
Jack T. Stapleton
Keyword(s):  
Human Immunodeficiency Virus ◽  
Cell Fusion ◽  
Cell Lines ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Quantitative Methods ◽  
Cytoplasmic Staining ◽  
Immunodeficiency Virus ◽  
Hiv 1

Cell fusion induced by human immunodeficiency virus type 1 (HIV-1) is usually assessed by counting multinucleated giant cells (syncytia) visualized by light microscopy. Currently used methods do not allow quantification of syncytia, nor do they estimate the number of cells involved in cell fusion. We describe two fluorescence-based methods for the detection and quantification of HIV-1-induced in vitro syncytium formation. The lymphoblastoid cell lines MT-2 and SupT1 were infected with syncytium-inducing (SI) HIV-1 isolates. Syncytia were detected by DNA staining with propidium iodide using flow cytometry to determine cell size or by two-color cytoplasmic staining of infected cell populations by using fluorescence microscopy. Both methods were able to detect and quantify HIV-induced syncytia. The methods could distinguish between SI and non-SI HIV isolates and could be used with at least two separate types of CD4+ T-cell lines. Small syncytia can be readily identified by the two-color cytoplasmic staining method. Both methods were also shown to be useful for evaluating antiretroviral compounds, as demonstrated by the accurate assessment of HIV inhibition by azidothymidine (zidovudine), dideoxycytidine (zalcytibine), and hydroxyurea. These fluorescence-based assays allow a rapid and practical method for measuring HIV replication and anti-HIV activity of potential inhibitory compounds.

scholarly journals Human Immunodeficiency Virus Type 1 Can Establish Latent Infection in Resting CD4+ T Cells in the Absence of Activating Stimuli

2005 ◽  
Vol 79 (22) ◽  
pp. 14179-14188 ◽  
Author(s):  
William J. Swiggard ◽  
Clifford Baytop ◽  
Jianqing J. Yu ◽  
Jihong Dai ◽  
Chuanzhao Li ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
T Cells ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Immunodeficiency Virus ◽  
Latently Infected ◽  
Latent Hiv ◽  
Hiv 1

ABSTRACT Resting CD4+ T cells are the best-defined reservoir of latent human immunodeficiency virus type 1 (HIV-1) infection, but how the reservoir is formed is unclear. Understanding how the reservoir of latently infected cells forms is critical because it is a major barrier to curing HIV infection. The system described here may provide an in vitro model of latent HIV-1 infection in resting CD4+ T cells. We demonstrated that HIV-1 integrates into the genomes of in vitro-inoculated resting CD4+ T cells that have not received activating stimuli and have not entered cell cycle stage G1b. A percentage of the resting CD4+ T cells that contain integrated DNA produce virus upon stimulation, i.e., are latently infected. Our results show that latent HIV-1 infection occurs in unstimulated resting CD4+ T cells and suggest a new route for HIV-1 reservoir formation.

scholarly journals Analysis of Early Human Immunodeficiency Virus Type 1 DNA Synthesis by Use of a New Sensitive Assay for Quantifying Integrated Provirus

2003 ◽  
Vol 77 (18) ◽  
pp. 10119-10124 ◽  
Author(s):  
Audrey Brussel ◽  
Pierre Sonigo
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
High Sensitivity ◽  
Pcr Assay ◽  
Immunodeficiency Virus ◽  
Infected Cells ◽  
Early Human ◽  
Hiv 1

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.

scholarly journals Pharmacovirological Impact of an Integrase Inhibitor on Human Immunodeficiency Virus Type 1 cDNA Species In Vivo

2009 ◽  
Vol 83 (15) ◽  
pp. 7706-7717 ◽  
Author(s):  
Christine Goffinet ◽  
Ina Allespach ◽  
Lena Oberbremer ◽  
Pamela L. Golden ◽  
Scott A. Foster ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Integrase Inhibitor ◽  
Strand Transfer ◽  
Immunodeficiency Virus ◽  
The Impact ◽  
Hiv 1

ABSTRACT Clinical trials of the first approved integrase inhibitor (INI), raltegravir, have demonstrated a drop in the human immunodeficiency virus type 1 (HIV-1) RNA loads of infected patients that was unexpectedly more rapid than that with a potent reverse transcriptase inhibitor, and apparently dose independent. These clinical outcomes are not understood. In tissue culture, although their inhibition of integration is well documented, the effects of INIs on levels of unintegrated HIV-1 cDNAs have been variable. Furthermore, there has been no report to date on an INI's effect on these episomal species in vivo. Here, we show that prophylactic treatment of transgenic rats with the strand transfer INI GSK501015 reduced levels of viral integrants in the spleen by up to 99.7%. Episomal two-long-terminal-repeat (LTR) circles accumulated up to sevenfold in this secondary lymphoid organ, and this inversely correlated with the impact on the proviral burden. Contrasting raltegravir's dose-ranging study with HIV patients, titration of GSK501015 in HIV-infected animals demonstrated dependence of the INI's antiviral effect on its serum concentration. Furthermore, the in vivo 50% effective concentration calculated from these data best matched GSK501015's in vitro potency when serum protein binding was accounted for. Collectively, this study demonstrates a titratable, antipodal impact of an INI on integrated and episomal HIV-1 cDNAs in vivo. Based on these findings and known biological characteristics of viral episomes, we discuss how integrase inhibition may result in additional indirect antiviral effects that contribute to more rapid HIV-1 decay in HIV/AIDS patients.

scholarly journals Specific Inhibition of Human Immunodeficiency Virus Type 1 (HIV-1) Integration in Cell Culture: Putative Inhibitors of HIV-1 Integrase

2001 ◽  
Vol 45 (9) ◽  
pp. 2510-2516 ◽  
Author(s):  
Nick Vandegraaff ◽  
Raman Kumar ◽  
Helen Hocking ◽  
Terrence R. Burke ◽  
John Mills ◽  
...  
Keyword(s):  
Cell Culture ◽  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Integrase Inhibitors ◽  
Pcr Assay ◽  
Hiv Dna ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT To study the effect of potential human immunodeficiency virus type 1 (HIV-1) integrase inhibitors during virus replication in cell culture, we used a modified nested Alu-PCR assay to quantify integrated HIV DNA in combination with the quantitative analysis of extrachromosomal HIV DNA. The two diketo acid integrase inhibitors (L-708,906 and L-731,988) blocked the accumulation of integrated HIV-1 DNA in T cells following infection but did not alter levels of newly synthesized extrachromosomal HIV DNA. In contrast, we demonstrated that L17 (a member of the bisaroyl hydrazine family of integrase inhibitors) and AR177 (an oligonucleotide inhibitor) blocked the HIV replication cycle at, or prior to, reverse transcription, although both drugs inhibited integrase activity in cell-free assays. Quercetin dihydrate (a flavone) was shown to not have any antiviral activity in our system despite reported anti-integration properties in cell-free assays. This refined Alu-PCR assay for HIV provirus is a useful tool for screening anti-integration compounds identified in biochemical assays for their ability to inhibit the accumulation of integrated HIV DNA in cell culture, and it may be useful for studying the effects of these inhibitors in clinical trials.

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