scholarly journals Impact of the Central Polypurine Tract on the Kinetics of Human Immunodeficiency Virus Type 1 Vector Transduction

2003 ◽  
Vol 77 (8) ◽  
pp. 4685-4694 ◽  
Author(s):  
Bénédicte Van Maele ◽  
Jan De Rijck ◽  
Erik De Clercq ◽  
Zeger Debyser
Keyword(s):  
Nuclear Import ◽  
Transcriptase Inhibitor ◽  
Immunodeficiency Virus ◽  
Central Polypurine Tract ◽  
Reverse Transcriptase Inhibitor ◽  
The Impact ◽  
Kinetics Of ◽  
Hiv 1 ◽  
Vector Transduction

ABSTRACT Lentiviral vectors derived from human immunodeficiency virus type 1 (HIV-1) show great promise as gene carriers for future gene therapy. Insertion of a fragment containing the central polypurine tract (cPPT) in HIV-1 vector constructs is known to enhance transduction efficiency drastically, reportedly by facilitating the nuclear import of HIV-1 cDNA through a central DNA flap. We have studied the impact of the cPPT on the kinetics of HIV-1 vector transduction by real-time PCR. The kinetics of total HIV-1 DNA, two-long-terminal-repeat (2-LTR) circles, and, by an Alu-PCR, integrated proviral DNA were monitored. About 6 to 12 h after transduction, the total HIV-1 DNA reached a maximum level, followed by a steep decrease. The 2-LTR circles peaked after 24 to 48 h and were diluted upon cell division. Integration of HIV-1 DNA was first detected at 12 h postinfection. When HIV-1 vectors that contained the cPPT were used, DNA synthesis was similar but a threefold higher amount of 2-LTR circles was detected, confirming the impact on nuclear import. Moreover, a 10-fold increase in the amount of integrated DNA was observed in the presence of the cPPT. Only in the absence of the cPPT was a saturation in 2-LTR circle formation seen at a high multiplicity of infection, suggesting a role for the cPPT in overcoming a barrier to the nuclear import of HIV-1 DNA. A major effect of the central DNA flap on the juxtaposition of both LTRs is unlikely, since transduction with HIV-1 vectors containing ectopic cPPT fragments resulted in increased amounts of 2-LTR circles as well as integrated DNA. Inhibitors of transduction by cPPT-containing HIV vectors were also studied by real-time PCR. The reverse transcriptase inhibitor azidothymidine (AZT) and the nonnucleoside reverse transcriptase inhibitor α-APA clearly inhibited viral DNA synthesis, whereas integrase inhibitors such as the diketo acid L-708,906 and the pyranodipyrimidine V-165 specifically inhibited integration.

scholarly journals Activity against Human Immunodeficiency Virus Type 1, Intracellular Metabolism, and Effects on Human DNA Polymerases of 4′-Ethynyl-2-Fluoro-2′-Deoxyadenosine

2007 ◽  
Vol 51 (8) ◽  
pp. 2701-2708 ◽  
Author(s):  
Hirotomo Nakata ◽  
Masayuki Amano ◽  
Yasuhiro Koh ◽  
Eiichi Kodama ◽  
Guangwei Yang ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Transcriptase Inhibitor ◽  
Multidrug Resistant ◽  
Wild Type ◽  
Immunodeficiency Virus ◽  
Reverse Transcriptase Inhibitor ◽  
Hiv 1

ABSTRACT We examined the intracytoplasmic anabolism and kinetics of antiviral activity against human immunodeficiency virus type 1 (HIV-1) of a nucleoside reverse transcriptase inhibitor, 4′-ethynyl-2-fluoro-2′-deoxyadenosine (EFdA), which has potent activity against wild-type and multidrug-resistant HIV-1 strains. When CEM cells were exposed to 0.1 μM [3H]EFdA or [3H]3′-azido-2′,3′-dideoxythymidine (AZT) for 6 h, the intracellular EFdA-triphosphate (TP) level was 91.6 pmol/109 cells, while that of AZT was 396.5 pmol/109 cells. When CEM cells were exposed to 10 μM [3H]EFdA, the amount of EFdA-TP increased by 22-fold (2,090 pmol/109 cells), while the amount of [3H]AZT-TP increased only moderately by 2.4-fold (970 pmol/109 cells). The intracellular half-life values of EFdA-TP and AZT-TP were ∼17 and ∼3 h, respectively. When MT-4 cells were cultured with 0.01 μM EFdA for 24 h, thoroughly washed to remove EFdA, further cultured without EFdA for various periods of time, exposed to HIV-1NL4-3, and cultured for an additional 5 days, the protection values were 75 and 47%, respectively, after 24 and 48 h with no drug incubation, while those with 1 μM AZT were 55 and 9.2%, respectively. The 50% inhibitory concentration values of EFdA-TP against human polymerases α, β, and γ were >100 μM, >100 μM, and 10 μM, respectively, while those of ddA-TP were >100 μM, 0.2 μM, and 0.2 μM, respectively. These data warrant further development of EFdA as a potential therapeutic agent for those patients who harbor wild-type HIV-1 and/or multidrug-resistant variants.

scholarly journals Reassessment of the Roles of Integrase and the Central DNA Flap in Human Immunodeficiency Virus Type 1 Nuclear Import

2002 ◽  
Vol 76 (23) ◽  
pp. 12087-12096 ◽  
Author(s):  
Jeffrey D. Dvorin ◽  
Peter Bell ◽  
Gerd G. Maul ◽  
Masahiro Yamashita ◽  
Michael Emerman ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
In Situ Hybridization ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Nuclear Import ◽  
Immunodeficiency Virus ◽  
Central Polypurine Tract ◽  
Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) can infect nondividing cells productively because the nuclear import of viral nucleic acids occurs in the absence of cell division. A number of viral factors that are present in HIV-1 preintegration complexes (PICs) have been assigned functions in nuclear import, including an essential valine at position 165 in integrase (IN-V165) and the central polypurine tract (cPPT). In this article, we report a comparison of the replication and infection characteristics of viruses with disruptions in the cPPT and IN-V165. We found that viruses with cPPT mutations still replicated productively in both dividing and nondividing cells, while viruses with a mutation at IN-V165 did not. Direct observation of the subcellular localization of HIV-1 cDNAs by fluorescence in situ hybridization revealed that cDNAs synthesized by both mutant viruses were readily detected in the nucleus. Thus, neither the cPPT nor the valine residue at position 165 of integrase is essential for the nuclear import of HIV-1 PICs.

scholarly journals Insertions in the Reverse Transcriptase Increase both Drug Resistance and Viral Fitness in a Human Immunodeficiency Virus Type 1 Isolate Harboring the Multi-Nucleoside Reverse Transcriptase Inhibitor Resistance 69 Insertion Complex Mutation

2002 ◽  
Vol 76 (20) ◽  
pp. 10546-10552 ◽  
Author(s):  
Miguel E. Quiñones-Mateu ◽  
Mahlet Tadele ◽  
Mariona Parera ◽  
Antonio Mas ◽  
Jan Weber ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Transcriptase Inhibitor ◽  
Viral Fitness ◽  
Wild Type ◽  
Sequence Context ◽  
Recombinant Viruses ◽  
Immunodeficiency Virus ◽  
Reverse Transcriptase Inhibitor ◽  
Hiv 1

ABSTRACT Recent studies have shown that the accumulation of multiple mutations associated with nucleoside reverse transcriptase inhibitor (NRTI) resistance may be grouped as multi-NRTI resistance (MNR) complexes. In this study, we have examined the viral fitness of recombinant viruses carrying the reverse transcriptase (RT) of a human immunodeficiency virus type 1 (HIV-1) primary isolate harboring mutations comprising the MNR 69 insertion complex. Different RT mutants were prepared in the sequence context of either the wild-type RT sequence of the HIV-1BH10 isolate or the sequence found in a clinical HIV-1 isolate with the MNR 69 insertion mutation. As expected, in the presence of zidovudine, recombinant viruses harboring the MNR RT from the patient were more fit than wild-type viruses. However, in the absence of drug, the virus with the RT from the original clinical isolate (SS) was more fit than (i) the wild-type virus with an engineered serine insertion between residues 69 and 70 (T69SSS) and (ii) the recombinant virus with the MNR RT where the insertion was removed (2S0S). These results suggest that RT insertions, in the right sequence context (i.e., additional mutations contained in the MNR 69 insertion complex), enhance NRTI resistance and may improve viral fitness. Thus, comparing complex mutation patterns with viral fitness may help to elucidate the role of uncharacterized drug resistance mutations in antiretroviral treatment failure.

scholarly journals Potent Activity of a Nucleoside Reverse Transcriptase Inhibitor, 4′-Ethynyl-2-Fluoro-2′-Deoxyadenosine, against Human Immunodeficiency Virus Type 1 Infection in a Model Using Human Peripheral Blood Mononuclear Cell-Transplanted NOD/SCID Janus Kinase 3 Knockout Mice

2009 ◽  
Vol 53 (9) ◽  
pp. 3887-3893 ◽  
Author(s):  
Shinichiro Hattori ◽  
Kazuhiko Ide ◽  
Hirotomo Nakata ◽  
Hideki Harada ◽  
Shinya Suzu ◽  
...  
Keyword(s):  
Nucleoside Reverse Transcriptase Inhibitor ◽  
Human Peripheral Blood ◽  
Transcriptase Inhibitor ◽  
Multidrug Resistant ◽  
Janus Kinase ◽  
Peripheral Blood Mononuclear ◽  
Immunodeficiency Virus ◽  
Reverse Transcriptase Inhibitor ◽  
Hiv 1

ABSTRACT 4′-Ethynyl-2-fluoro-2′-deoxyadenosine (EFdA), a recently discovered nucleoside reverse transcriptase inhibitor, exhibits activity against a wide spectrum of wild-type and multidrug-resistant clinical human immunodeficiency virus type 1 (HIV-1) isolates (50% effective concentration, 0.0001 to 0.001 μM). In the present study, we used human peripheral blood mononuclear cell-transplanted, HIV-1-infected NOD/SCID/Janus kinase 3 knockout mice for in vivo evaluation of the anti-HIV activity of EFdA. Administration of EFdA decreased the replication and cytopathic effects of HIV-1 without identifiable adverse effects. In phosphate-buffered saline (PBS)-treated mice, the CD4+/CD8+ cell ratio in the spleen was low (median, 0.04; range, 0.02 to 0.49), while that in mice receiving EFdA was increased (median, 0.65; range, 0.57 to 1.43). EFdA treatment significantly suppressed the amount of HIV-1 RNA (median of 9.0 × 102 copies/ml [range, 8.1 × 102 to 1.1 × 103 copies/ml] versus median of 9.9 × 104 copies/ml [range, 8.1 × 102 to 1.1 × 103 copies/ml]; P < 0.001), the p24 level in plasma (2.5 × 103 pg/ml [range, 8.2 × 102 to 5.6 × 103 pg/ml] versus 2.8 × 102 pg/ml [range, 8.2 × 101 to 6.3 × 102 pg/ml]; P < 0.001), and the percentage of p24-expressing cells in the spleen (median of 1.90% [range, 0.33% to 3.68%] versus median of 0.11% [range, 0.00% to 1.00%]; P = 0.003) in comparison with PBS-treated mice. These data suggest that EFdA is a promising candidate for a new age of HIV-1 chemotherapy and should be developed further as a potential therapy for individuals with multidrug-resistant HIV-1 variants.

scholarly journals N-[2-(4-Methylphenyl)Ethyl]-N′-[2-(5-Bromopyridyl)]-Thiourea as a Potent Inhibitor of NNRTI-Resistant and Multidrug-Resistant Human Immunodeficiency Virus Type 1

2000 ◽  
Vol 11 (2) ◽  
pp. 135-140 ◽  
Author(s):  
Fatih M Uckun ◽  
Chen Mao ◽  
Sharon Pendergrass ◽  
Danielle Maher ◽  
Dan Zhu ◽  
...  
Keyword(s):  
Mononuclear Cells ◽  
Human Peripheral Blood ◽  
Binding Pocket ◽  
Transcriptase Inhibitor ◽  
Multidrug Resistant ◽  
Peripheral Blood Mononuclear ◽  
Immunodeficiency Virus ◽  
Reverse Transcriptase Inhibitor ◽  
Hiv 1

The composite non-nucleoside reverse transcriptase inhibitor (NNRTI) binding pocket model was used to study a number of thiourea analogues with different substitutions at the 4-phenyl position including N-[2-(4-methylphenyl)ethyl]-N′-[2-(5-bromopyridyl)]-thiourea (compound HI-244), which inhibited recombinant RT better than trovirdine or compound HI-275 with an unsubstituted phenyl ring. HI-244 effectively inhibited the replication of HIV-1 strain HTLVIIIB in human peripheral blood mononuclear cells with an IC50 value of 0.007 μM, which is equal to the IC50 value of trovirdine. Notably, HI-244 was 20 times more effective than trovirdine against the multidrug-resistant HIV-1 strain RT-MDR with a V106A mutation (as well as additional mutations involving the RT residues 74 V, 41L and 215Y) and seven times more potent than trovirdine against the NNRTI- resistant HIV-1 strain A17 with a Y181C mutation.

scholarly journals In Vitro Cross-Resistance Profiles of Rilpivirine, Dapivirine, and MIV-150, Nonnucleoside Reverse Transcriptase Inhibitor Microbicides in Clinical Development for the Prevention of HIV-1 Infection

2017 ◽  
Vol 61 (7) ◽  
Author(s):  
Nicholas S. Giacobbi ◽  
Nicolas Sluis-Cremer
Keyword(s):  
Reverse Transcriptase ◽  
Genital Tract ◽  
Transcriptase Inhibitor ◽  
Cross Resistance ◽  
Nonnucleoside Reverse Transcriptase Inhibitor ◽  
Immunodeficiency Virus ◽  
Reverse Transcriptase Inhibitor ◽  
Hiv 1

ABSTRACT Rilpivirine (RPV), dapivirine (DPV), and MIV-150 are in development as microbicides. It is not known whether they will block infection of circulating nonnucleoside reverse transcriptase inhibitor (NNRTI)-resistant human immunodeficiency virus type 1 (HIV-1) variants. Here, we demonstrate that the activity of DPV and MIV-150 is compromised by many resistant viruses containing single or double substitutions. High DPV genital tract concentrations from DPV ring use may block replication of resistant viruses. However, MIV-150 genital tract concentrations may be insufficient to inhibit many resistant viruses, including those harboring K103N or Y181C.

scholarly journals Identification and Characterization of Persistent Intracellular Human Immunodeficiency Virus Type 1 Integrase Strand Transfer Inhibitor Activity

2010 ◽  
Vol 55 (1) ◽  
pp. 42-49 ◽  
Author(s):  
Yasuhiro Koh ◽  
Hillel Haim ◽  
Alan Engelman
Keyword(s):  
Human Immunodeficiency Virus ◽  
Reverse Transcriptase ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Transcriptase Inhibitor ◽  
Strand Transfer ◽  
Immunodeficiency Virus ◽  
Reverse Transcriptase Inhibitor ◽  
Hiv 1

ABSTRACTPharmacokinetic and pharmacodynamic considerations significantly impact infectious disease treatment options. One aspect of pharmacodynamics is the postantibiotic effect, classically defined as delayed bacterial growth after antibiotic removal. The same principle can apply to antiviral drugs. For example, significant delays in human immunodeficiency virus type 1 (HIV-1) replication can be observed after nucleoside/nucleotide reverse transcriptase inhibitor (N/NtRTI) removal from culture medium, because these prodrugs must be anabolized into active, phosphorylated forms once internalized into cells. A relatively new class of anti-HIV-1 drugs is the integrase strand transfer inhibitors (INSTIs), and the INSTIs raltegravir (RAL) and elvitegravir (EVG) were tested here alongside positive N/NtRTI controls tenofovir disoproxil fumarate (TDF) and azidothymidine (AZT), as well as the nonnucleoside reverse transcriptase inhibitor negative control nevirapine (NVP), to assess potential postantiviral effects. Transformed and primary CD4-positive cells pretreated with INSTIs significantly resisted subsequent challenge by HIV-1, revealing the following hierarchy of persistent intracellular drug strength: TDF > EVG ∼ AZT > RAL > NVP. A modified time-of-addition assay was moreover developed to assess residual drug activity levels. Approximately 0.8% of RAL and 2% of initial EVG and TDF 1-h pulse drug levels persisted during the acute phase of HIV-1 infection. EVG furthermore displayed significant virucidal activity. Although there is no reason to suspect obligate intracellular modification, this study nevertheless defines significant intracellular persistence of prototype INSTIs. Ongoing second-generation formulations should therefore consider the potential for significant postantiviral effects among this drug class. Combined intracellular persistence and virucidal activities suggest potential pre-exposure prophylaxis applications for EVG.

scholarly journals In Vitro Microbicidal Activity of the Nonnucleoside Reverse Transcriptase Inhibitor (NNRTI) UC781 against NNRTI-Resistant Human Immunodeficiency Virus Type 1

2006 ◽  
Vol 80 (9) ◽  
pp. 4440-4446 ◽  
Author(s):  
Mohammad M. Hossain ◽  
Michael A. Parniak
Keyword(s):  
Human Immunodeficiency Virus ◽  
Reverse Transcriptase ◽  
Transcriptase Inhibitor ◽  
Resistant Virus ◽  
Microbicidal Activity ◽  
Nonnucleoside Reverse Transcriptase Inhibitor ◽  
Immunodeficiency Virus ◽  
Reverse Transcriptase Inhibitor ◽  
Hiv 1

ABSTRACT The nonnucleoside reverse transcriptase inhibitor (NNRTI) UC781 is under development as a microbicide to prevent sexual transmission of the human immunodeficiency virus type 1 (HIV-1). However, NNRTI-resistant HIV-1 is increasingly prevalent in the infected population, and one of the concerns for NNRTI-based microbicides is that they will be ineffective against drug-resistant virus and may in fact selectively transmit NNRTI-resistant virus. We evaluated the microbicidal activity of UC781 against UC781-resistant (UCR), efavirenz-resistant (EFVR), and nevirapine-resistant (NVPR) strains in a variety of microbicide-relevant tests, including inactivation of cell-free virus, inhibition of cell-to-cell HIV-1 transmission, and the ability of UC781 pretreatment to protect cells from subsequent infection in the absence of exogenous drug. UC781 was 10- to 100-fold less effective against NNRTI-resistant HIV-1 compared to wild-type (wt) virus in each of these tests, with UC781 microbicidal activity against the various virus strains being wt ≥ NVPR > UCR ≥ EFVR. Breakthrough experiments using UC781-pretreated cells and mixtures of wt and NNRTI-resistant HIV-1 showed that UC781-pretreatment selected for NNRTI-resistant HIV-1. However, the efficacy of UC781 was dose dependent, and 25 μM UC781 provided essentially equivalent microbicidal activity against NNRTI-resistant and wt virus. The amount of UC781 in topical microbicide formulations under current development is approximately 100-fold greater than this concentration, so transmission of NNRTI-resistant virus may not be an issue at these microbicide formulation levels of UC781. Nonetheless, the reduced microbicidal activity of UC781 against NNRTI-resistant HIV-1 suggests that additional antiviral agents should be included in NNRTI-based microbicide formulations.

scholarly journals Stampidine Is a Potent Inhibitor of Zidovudine- and Nucleoside Analog Reverse Transcriptase Inhibitor-Resistant Primary Clinical Human Immunodeficiency Virus Type 1 Isolates with Thymidine Analog Mutations

2002 ◽  
Vol 46 (11) ◽  
pp. 3613-3616 ◽  
Author(s):  
Fatih M. Uckun ◽  
Sharon Pendergrass ◽  
T. K. Venkatachalam ◽  
Sanjive Qazi ◽  
Douglas Richman
Keyword(s):  
Human Immunodeficiency Virus ◽  
Reverse Transcriptase ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Transcriptase Inhibitor ◽  
Nucleoside Analog ◽  
Immunodeficiency Virus ◽  
Reverse Transcriptase Inhibitor ◽  
Hiv 1

ABSTRACT We report the antiretroviral activity of stavudine-5′-(p-bromophenyl methoxyalaninyl phosphate) (stampidine [STAMP]), a novel aryl phosphate derivative of stavudine, against primary clinical human immunodeficiency virus type 1 (HIV-1) isolates. STAMP inhibited each one of nine clinical HIV-1 isolates of non-B envelope subtype and 20 genotypically and phenotypically nucleoside analog reverse transcriptase inhibitor-resistant HIV-1 isolates at subnanomolar to low-nanomolar concentrations.

scholarly journals Single-Dose Escalation and Multiple-Dose Safety, Tolerability, and Pharmacokinetics of IDX899, a Candidate Human Immunodeficiency Virus Type 1 Nonnucleoside Reverse Transcriptase Inhibitor, in Healthy Subjects

2009 ◽  
Vol 53 (5) ◽  
pp. 1739-1746 ◽  
Author(s):  
Xiao-Jian Zhou ◽  
Keith Pietropaolo ◽  
David Damphousse ◽  
Bruce Belanger ◽  
Jie Chen ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Single Dose ◽  
Adverse Events ◽  
Transcriptase Inhibitor ◽  
Nonnucleoside Reverse Transcriptase Inhibitor ◽  
Immunodeficiency Virus ◽  
Reverse Transcriptase Inhibitor ◽  
Hiv 1

ABSTRACT IDX899 is a novel nonnucleoside reverse transcriptase inhibitor (NNRTI) with potent in vitro activity against wild-type and NNRTI-resistant strains of human immunodeficiency virus type 1 (HIV-1) and with a high genetic barrier to resistance. Single rising doses of 50 and 100 (given by use of a 50-mg capsule) and 200, 400, 800, and 1,200 mg (given by use of a 200-mg capsule) of IDX899 or matching placebo were administered sequentially to cohorts of healthy male subjects, followed by the administration of multiple doses of 800 mg once daily (QD) or 400 mg twice daily (BID) for 7 days. A single dose of 400 mg was also administered to a cohort of females. IDX899 was administered orally under fasted (50- to 400-mg doses) and then fed (≥200-mg doses) conditions. Exposure to IDX899 was dose proportional and comparable in males and females. With a different drug-to-excipient ratio, the 50-mg capsule led to a higher exposure but a shorter mean terminal half-life (t 1/2) of 6.2 to 6.8 h. The 200-mg capsule resulted in a more sustained exposure with a longer mean t 1/2 of 7.9 to 14.6 h. Food enhanced absorption by approximately twofold, while it delayed the time to the maximum concentration. The mean concentration at 24 h following the administration of a single 200-mg dose under fed conditions exceeded the in vitro protein binding-adjusted 90% inhibitory concentration by fourfold. The levels of plasma exposure were similar between the single dosing and the repeat dosing with 800 mg QD and was approximately twofold higher with 400 mg BID. Mean steady-state trough levels were 0.9 μg/ml (range, 0.2 to 2.5 μg/ml) and 2.1 μg/ml (range, 0.5 to 4.5 μg/ml) for the 800-mg QD and 400-mg BID regimens, respectively. The level of excretion of unchanged drug in urine was negligible. IDX899 was well tolerated; and no serious adverse events, dose-dependent adverse events, or laboratory abnormalities were detected. These favorable safety and pharmacokinetic results support further clinical studies with patients with HIV-1 infection by the use of a QD regimen.

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