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.

scholarly journals CCR5 Expression Correlates with Susceptibility of Maturing Monocytes to Human Immunodeficiency Virus Type 1 Infection

1998 ◽  
Vol 72 (1) ◽  
pp. 830-836 ◽  
Author(s):  
Hassan M. Naif ◽  
Shan Li ◽  
Mohammed Alali ◽  
Andrew Sloane ◽  
Lijun Wu ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Chemokine Receptors ◽  
Ccr5 Expression ◽  
Hiv Dna ◽  
Immunodeficiency Virus ◽  
P24 Antigen ◽  
Hiv 1

ABSTRACT The chemokine receptor CCR5 and to a lesser extent CCR3 and CCR2b have been shown to serve as coreceptors for human immunodeficiency virus type 1 (HIV-1) entry into blood- or tissue-derived macrophages. Therefore, we examined the expression of the chemokine receptors CCR1, CCR2b, CCR3, CCR5, and CXCR4 as RNAs or as membrane-expressed antigens in monocytes maturing into macrophages and correlated these results with the susceptibility of macrophages to HIV-1 infection, as measured by their concentrations of extracellular p24 antigen and levels of intracellular HIV DNA by quantitative PCR. There was little change in levels of CCR1, CCR2b, and CCR5 RNAs. CCR3 RNA and surface antigen were undetectable throughout maturation of adherent monocytes over 10 days. CXCR4 RNA and membrane antigen were strongly expressed in newly adherent monocytes, but their levels declined at day 7. The amounts of CCR5 RNA remained stable, but the amounts of CCR5 antigen increased from undetectable to peak levels at day 7 and then declined slightly at day 10. Levels of susceptibility to laboratory (HIV-1BaL) and clinical strains of HIV-1 showed parallel kinetics, peaking at day 7 and then decreasing at days 10 to 14. The concordance of levels of HIV DNA and p24 antigen suggested that the changes in susceptibility with monocyte maturation were at or immediately after entry and correlated well with CCR5 expression and inversely with CXCR4 expression.

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 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 Natural Polymorphisms of Human Immunodeficiency Virus Type 1 Integrase and Inherent Susceptibilities to a Panel of Integrase Inhibitors

2009 ◽  
Vol 53 (10) ◽  
pp. 4275-4282 ◽  
Author(s):  
Andrea Low ◽  
Nicole Prada ◽  
Michael Topper ◽  
Florin Vaida ◽  
Delivette Castor ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Early Infection ◽  
Integrase Inhibitors ◽  
Coding Region ◽  
Recombinant Viruses ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT We evaluated the human immunodeficiency virus type 1 (HIV-1) integrase coding region of the pol gene for the presence of natural polymorphisms in patients during early infection (AHI) and with triple-class drug-resistant HIV-1 (MDR). We analyzed selected recombinant viruses containing patient-derived HIV-1 integrase for susceptibility to a panel of strand transfer integrase inhibitors (InSTI). A pretreatment sequence analysis of the integrase coding region was performed for 112 patients identified during acute or early infection and 15 patients with triple-class resistance. A phenotypic analysis was done on 10 recombinant viruses derived from nine patients against a panel of six diverse InSTI. Few of the polymorphisms associated with in vitro InSTI resistance were identified in the samples from newly infected individuals or those patients with MDR HIV-1. We identified polymorphisms V72I, L74I, T97A, V151I, M154I/L, E157Q, V165I, V201I, I203M, T206S, and S230N. V72I was the most common, seen in 63 (56.3%) of the AHI samples. E157Q was the only naturally occurring mutation thought to contribute to resistance to elvitegravir, raltegravir, and L-870,810. None of the patient-derived viruses demonstrated any significant decrease in susceptibility to the drugs tested. In summary, the integrase coding region contains as much natural variation as that seen in protease, but mutations associated with high-level resistance to existing InSTI are rarely, if ever, present in integrase naïve patients, especially those being used clinically. Most of the highly prevalent polymorphisms have little effect on InSTI susceptibility in the absence of specific primary mutations. Baseline testing for integrase susceptibility in InSTI-naïve patients is not currently warranted.

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