A rapid and efficient system for screening HIV-1 Pol mRNA-specific ribozymes

1997 ◽  
Vol 43 (1) ◽  
pp. 92-96
Author(s):  
A. Ramezani ◽  
W. Marhin ◽  
M. Weerasinghe ◽  
S. Joshi
Keyword(s):  
Reverse Transcriptase ◽  
Hammerhead Ribozyme ◽  
Rapid Screening ◽  
Coding Region ◽  
Efficient System ◽  
Target Sites ◽  
Hiv 1 ◽  
In Cells

Hammerhead ribozymes are potentially important tools for suppressing intracellular expression of unwanted RNAs. However, the reports that exist on their activity against different targets have described mixed success. As an initial step towards developing a rapid and effective system for in vivo testing of ribozymes, two human immunodeficiency virus type-1 (HIV-1) polymerase (Pol) mRNA-specific ribozymes, RzProdirected against the protease (Pro) coding region and RzRTdirected against the reverse transcriptase (RT) coding region, were designed and tested in Escherichia coli. Both ribozymes displayed similar efficiencies in cleaving their target RNAs in vitro. RNA polymerase chain reaction was adapted to demonstrate the in vivo cleavage of RzProand RzRTtarget sites. The resultant drop in HIV-1 RT activity was measured as well. The degree of suppression of RT activity was more apparent in vivo in cells expressing RzRT. The RT activity in cells expressing RzRTwas shown to decrease by up to 96%. This system will be useful for rapid screening of (i) other ribozyme target sites within the Pol mRNA so that multitargeted ribozymes could be designed for use in anti-HIV-1 gene therapy, (ii) ribozymes with improved stability and catalytic activity, and (iii) cofactors, if any, that could enhance ribozyme activity in vivo.Key words: HIV, hammerhead ribozyme, protease, reverse transcriptase.

scholarly journals 1592U89, a novel carbocyclic nucleoside analog with potent, selective anti-human immunodeficiency virus activity.

1997 ◽  
Vol 41 (5) ◽  
pp. 1082-1093 ◽  
Author(s):  
S M Daluge ◽  
S S Good ◽  
M B Faletto ◽  
W H Miller ◽  
M H St Clair ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Oral Bioavailability ◽  
Human Peripheral Blood ◽  
Cross Resistance ◽  
Immunodeficiency Virus ◽  
Carbocyclic Nucleoside ◽  
Hiv 1 ◽  
In Cells

1592U89, (-)-(1S,4R)-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclo pentene-1-methanol, is a carbocyclic nucleoside with a unique biological profile giving potent, selective anti-human immunodeficiency virus (HIV) activity. 1592U89 was selected after evaluation of a wide variety of analogs containing a cyclopentene substitution for the 2'-deoxyriboside of natural deoxynucleosides, optimizing in vitro anti-HIV potency, oral bioavailability, and central nervous system (CNS) penetration. 1592U89 was equivalent in potency to 3'-azido-3'-deoxythymidine (AZT) in human peripheral blood lymphocyte (PBL) cultures against clinical isolates of HIV type 1 (HIV-1) from antiretroviral drug-naive patients (average 50% inhibitory concentration [IC50], 0.26 microM for 1592U89 and 0.23 microM for AZT). 1592U89 showed minimal cross-resistance (approximately twofold) with AZT and other approved HIV reverse transcriptase (RT) inhibitors. 1592U89 was synergistic in combination with AZT, the nonnucleoside RT inhibitor nevirapine, and the protease inhibitor 141W94 in MT4 cells against HIV-1 (IIIB). 1592U89 was anabolized intracellularly to its 5'-monophosphate in CD4+ CEM cells and in PBLs, but the di- and triphosphates of 1592U89 were not detected. The only triphosphate found in cells incubated with 1592U89 was that of the guanine analog (-)-carbovir (CBV). However, the in vivo pharmacokinetic, distribution, and toxicological profiles of 1592U89 were distinct from and improved over those of CBV, probably because CBV itself was not appreciably formed from 1592U89 in cells or animals (<2%). The 5'-triphosphate of CBV was a potent, selective inhibitor of HIV-1 RT, with Ki values for DNA polymerases (alpha, beta, gamma, and epsilon which were 90-, 2,900-, 1,200-, and 1,900-fold greater, respectively, than for RT (Ki, 21 nM). 1592U89 was relatively nontoxic to human bone marrow progenitors erythroid burst-forming unit and granulocyte-macrophage CFU (IC50s, 110 microM) and human leukemic and liver tumor cell lines. 1592U89 had excellent oral bioavailability (105% in the rat) and penetrated the CNS (rat brain and monkey cerebrospinal fluid) as well as AZT. Having demonstrated an excellent preclinical profile, 1592U89 has progressed to clinical evaluation in HIV-infected patients.

scholarly journals Nature, Position, and Frequency of Mutations Made in a Single Cycle of HIV-1 Replication

2010 ◽  
Vol 84 (19) ◽  
pp. 9864-9878 ◽  
Author(s):  
Michael E. Abram ◽  
Andrea L. Ferris ◽  
Wei Shao ◽  
W. Gregory Alvord ◽  
Stephen H. Hughes
Keyword(s):  
Viral Replication ◽  
Mutation Rate ◽  
Hot Spots ◽  
High Rate ◽  
Published Data ◽  
Single Cycle ◽  
Efficient System ◽  
Hiv 1

ABSTRACT There is considerable HIV-1 variation in patients. The extent of the variation is due to the high rate of viral replication, the high viral load, and the errors made during viral replication. Mutations can arise from errors made either by host DNA-dependent RNA polymerase II or by HIV-1 reverse transcriptase (RT), but the relative contributions of these two enzymes to the mutation rate are unknown. In addition, mutations in RT can affect its fidelity, but the effect of mutations in RT on the nature of the mutations that arise in vivo is poorly understood. We have developed an efficient system, based on existing technology, to analyze the mutations that arise in an HIV-1 vector in a single cycle of replication. A lacZα reporter gene is used to identify viral DNAs that contain mutations which are analyzed by DNA sequencing. The forward mutation rate in this system is 1.4 × 10−5 mutations/bp/cycle, equivalent to the retroviral average. This rate is about 3-fold lower than previously reported for HIV-1 in vivo and is much lower than what has been reported for purified HIV-1 RT in vitro. Although the mutation rate was not affected by the orientation of lacZα, the sites favored for mutations (hot spots) in lacZα depended on which strand of lacZα was present in the viral RNA. The pattern of hot spots seen in lacZα in vivo did not match any of the published data obtained when purified RT was used to copy lacZα in vitro.

scholarly journals Impact of Y143 HIV-1 Integrase Mutations on Resistance to Raltegravir In Vitro and In Vivo

2009 ◽  
Vol 54 (1) ◽  
pp. 491-501 ◽  
Author(s):  
Olivier Delelis ◽  
Sylvain Thierry ◽  
Frédéric Subra ◽  
Françoise Simon ◽  
Isabelle Malet ◽  
...  
Keyword(s):  
Viral Genome ◽  
Effective Concentration ◽  
Coding Region ◽  
Delayed Emergence ◽  
Infected Cells ◽  
High Level ◽  
Emergence Of Resistance ◽  
Hiv 1

ABSTRACT Integrase (IN), the HIV-1 enzyme responsible for the integration of the viral genome into the chromosomes of infected cells, is the target of the recently approved antiviral raltegravir (RAL). Despite this drug's activity against viruses resistant to other antiretrovirals, failures of raltegravir therapy were observed, in association with the emergence of resistance due to mutations in the integrase coding region. Two pathways involving primary mutations on residues N155 and Q148 have been characterized. It was suggested that mutations at residue Y143 might constitute a third primary pathway for resistance. The aims of this study were to investigate the susceptibility of HIV-1 Y143R/C mutants to raltegravir and to determine the effects of these mutations on the IN-mediated reactions. Our observations demonstrate that Y143R/C mutants are strongly impaired for both of these activities in vitro. However, Y143R/C activity can be kinetically restored, thereby reproducing the effect of the secondary G140S mutation that rescues the defect associated with the Q148R/H mutants. A molecular modeling study confirmed that Y143R/C mutations play a role similar to that determined for Q148R/H mutations. In the viral replicative context, this defect leads to a partial block of integration responsible for a weak replicative capacity. Nevertheless, the Y143 mutant presented a high level of resistance to raltegravir. Furthermore, the 50% effective concentration (EC50) determined for Y143R/C mutants was significantly higher than that obtained with G140S/Q148R mutants. Altogether our results not only show that the mutation at position Y143 is one of the mechanisms conferring resistance to RAL but also explain the delayed emergence of this mutation.

scholarly journals Strong In Vivo Inhibition of HIV-1 Replication by Nullbasic, a Tat Mutant

mBio ◽  
2019 ◽  
Vol 10 (4) ◽  
Author(s):  
Hongping Jin ◽  
Yifan Sun ◽  
Dongsheng Li ◽  
Min-Hsuan Lin ◽  
Mary Lor ◽  
...  
Keyword(s):  
Mutant Form ◽  
Mrna Levels ◽  
Functional Cure ◽  
Fold Reduction ◽  
Infected Cells ◽  
Hiv 1 ◽  
Transcription And Replication ◽  
In Cells

ABSTRACT Nullbasic is a mutant form of the HIV-1 transcriptional activator protein (Tat) that strongly inhibits HIV-1 transcription and replication in lymphocytes in vitro. To investigate Nullbasic inhibition in vivo, we employed an NSG mouse model where animals were engrafted with primary human CD4+ cells expressing a Nullbasic-ZsGreen1 (NB-ZSG) fusion protein or ZSG. NB-ZSG and ZSG were delivered by using a retroviral vector where CD4+ cells were transduced either prior to (preinfection) or following (postinfection) HIV-1 infection. The transduced cells were analyzed in vitro up to 10 days postinfection (dpi) and in vivo up to 39 dpi. Compared to ZSG, NB-ZSG strongly inhibited HIV-1 replication both in vitro and in vivo using preinfection treatment. In vitro, HIV-1 mRNA levels in cells were reduced by up to 60-fold. In vivo, HIV-1 RNA was undetectable in plasma samples during the course of the experiment, and HIV-1 mRNA levels in resident CD4+ cells in organ tissue were reduced up to 2,800-fold. Postinfection treatment of HIV-1-infected cells with NB-ZSG attenuated HIV-1 infection for up to 14 days. In vitro, a 25-fold reduction of viral mRNA in cells was observed but diminished to a <2-fold reduction by 10 dpi. In vivo, HIV-1 RNA was undetectable in plasma of NB-ZSG mice at 14 dpi but afterwards was not significantly different between NB-ZSG mice and control mice. However, we observed higher levels of CD4+ cells in NB-ZSG mice than in control mice, suggesting that NB-ZSG imparted a survival advantage to HIV-1-infected animals. IMPORTANCE HIV-1 infection is effectively controlled by antiviral therapy that inhibits virus replication and reduces viral loads below detectable levels in patients. However, therapy interruption leads to viral rebound due to latently infected cells, which serve as a source of continued viral infection. Interest in strategies leading to a functional cure for HIV-1 infection by long-term or permanent viral suppression is growing. Here, we show that a mutant form of the HIV-1 Tat protein, referred to as Nullbasic, inhibits HIV-1 transcription in infected CD4+ cells in vivo. Analysis shows that stable expression of Nullbasic in CD4+ cells could lead to durable anti-HIV-1 activity. Nullbasic, as a gene therapy candidate, could be a part of a functional-cure strategy to suppress HIV-1 transcription and replication.

scholarly journals Prevalence of doravirine-associated resistance mutations in HIV-1-infected antiretroviral-experienced patients from two large databases in France and Italy

2020 ◽  
Vol 75 (4) ◽  
pp. 1026-1030 ◽  
Author(s):  
Cathia Soulie ◽  
Maria Mercedes Santoro ◽  
Alexandre Storto ◽  
Basma Abdi ◽  
Charlotte Charpentier ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Resistance Mutation ◽  
Resistance Pattern ◽  
Resistance Mutations ◽  
Large Databases ◽  
The Common ◽  
Hiv 1

Abstract Objectives Doravirine, a novel NNRTI, selects for specific mutations in vitro, including mutations at reverse transcriptase (RT) positions 106, 108, 188, 227, 230 and 234. The aim of this study was to examine the prevalence of doravirine-associated resistance mutations in HIV-1-infected antiretroviral-experienced patients. Methods Doravirine-associated resistance mutations identified in vitro or in vivo were studied in a set of 9199 HIV-1 RT sequences from HIV-1 antiretroviral-experienced patients, including 381 NNRTI-failing patients in France and Italy between 2012 and 2017. The following mutations were considered as resistance mutations: V106A/M, V108I, Y188L, G190S, F227C/L/V, M230I/L, L234I, P236L, K103N + Y181C, K103N + P225H and K103N + L100I. Results The frequencies of doravirine-associated resistance mutations (total dataset versus NNRTI-failing patients) were: V106A/M, 0.8% versus 2.6%; V108I, 3.3% versus 9.2%; Y188L, 1.2% versus 2.6%; G190S, 0.3% versus 2.1%; F227C/L/V, 0.5% versus 1.8%; M230I/L, 2.8% versus 0%; L234I, 0.1% versus 0.5%; K103N + Y181C, 3.9% versus 3.9%; K103N + P225H, 2.9% versus 4.7%; and K103N + L100I, 1.7% versus 3.9%, with a significantly higher proportion of these mutations in the NNRTI-failing group (P &lt; 0.05), except for M230I/L and K103N + Y181C. The overall prevalence of sequences with at least one doravirine-associated resistance mutation was 12.2% and 34.9% in the total dataset and NNRTI-failing patients (P &lt; 0.001), respectively. In comparison, the prevalence of the common NNRTI mutations V90I, K101E/P, K103N/S, E138A/G/K/Q/R/S, Y181C/I/V and G190A/E/S/Q were higher (8.9%, 7.9%, 28.6%, 12.6%, 14.2% and 8.9%, respectively). Conclusions These results suggest that doravirine resistance in antiretroviral-experienced patients generally and specifically among NNRTI-failing patients is lower than resistance to other NNRTIs currently used, confirming its distinguishing resistance pattern.

Mechanism of action of hammerhead ribozymes and their applications in vivo: rapid identification of functional genes in the post-genome era by novel hybrid ribozyme libraries

2002 ◽  
Vol 30 (6) ◽  
pp. 1145-1149 ◽  
Author(s):  
Y. Takagi ◽  
E. Suyama ◽  
H. Kawasaki ◽  
M. Miyagishi ◽  
K. Taira
Keyword(s):  
Metal Binding ◽  
Cancer Metastasis ◽  
Tertiary Structure ◽  
Hammerhead Ribozyme ◽  
Rapid Identification ◽  
Specific Sequence ◽  
Target Sites ◽  
Interfering Rna

A hammerhead ribozyme was demonstrated to be a metalloenzyme. By controlling the metal-binding ability of the hammerhead ribozyme in the presence or absence of a specific sequence of interest, we engineered an allosterically controllable ribozyme, designated the maxizyme. Hybrid ribozymes were then constructed by coupling the site-specific cleavage activity of a hammerhead ribozyme with the unwinding activity of an endogenous RNA helicase. This leads to extremely efficient cleavage of target mRNA, not only in vitro, but also in vivo, and eliminates one of the major problems arising in the application of ribozymes for cleavage of mRNA in vivo: that many target sites on the RNA were previously inaccessible to cleavage owing to secondary and/or tertiary structure formation. Since hybrid ribozymes can efficiently attack target sites within mRNA, libraries were made of hybrid ribozymes with randomized binding arms, which were then introduced into cells. This procedure made it possible to readily identify the relevant genes associated with a specific phenotype, such as in apoptosis and cancer metastasis pathways. This application of a randomized library of hybrid ribozymes represents a simple, yet powerful, method for the identification of genes associated with specific phenotypes in the post-genome era. Moreover, vector-based siRNA (short-interfering RNA for RNA interference, RNAi) can also be used for the creation of the libraries and for the subsequent confirmation of the identified genes, relevant in the examined phenotype.

scholarly journals In VitroandIn VivoActivities of AIC292, a Novel HIV-1 Nonnucleoside Reverse Transcriptase Inhibitor

2013 ◽  
Vol 57 (11) ◽  
pp. 5320-5329 ◽  
Author(s):  
Steffen Wildum ◽  
Daniela Paulsen ◽  
Kai Thede ◽  
Helga Ruebsamen-Schaeff ◽  
Holger Zimmermann
Keyword(s):  
Antiviral Activity ◽  
Reverse Transcriptase ◽  
Highly Active Antiretroviral Therapy ◽  
Xenograft Model ◽  
Wild Type ◽  
Mouse Xenograft ◽  
Mouse Xenograft Model ◽  
Hiv 1

ABSTRACTNonnucleoside reverse transcriptase inhibitors (NNRTIs) are important and frequently used elements of highly active antiretroviral therapy (HAART) for the treatment of human immunodeficiency virus type 1 (HIV-1) infection. However, the development of drug resistance, as well as the side effects of existing drugs, defines a medical need for novel NNRTIs with excellent tolerability, improved activity against NNRTI-resistant viruses, and a low barrier to resistance. Within the chemical class of diarylpyrazole-[imidazolidinone]-carboxamides, AIC292 was identified as a promising novel HIV-1 NNRTI and has successfully completed single-dose clinical phase I studies. Here, we report on the antiviral activity of AIC292, evaluatedin vitroagainst wild-type and NNRTI-resistant HIV-1 isolates andin vivousing an engineered mouse xenograft model. AIC292 inhibited wild-type HIV-1 laboratory strains at low nanomolar concentrations, was well tolerated in different cell lines, and showed excellent selectivity in a lead profiling screen. In addition, activity of AIC292 could be demonstrated against a broad panel of wild-type HIV-1 group M and group O clinical isolates. AIC292 also retained activity against viruses harboring NNRTI resistance-associated mutations (RAMs), including the most prevalent variants, K103N, Y181C, and G190A. Interestingly, viruses bearing the L100I RAM were hypersusceptible to AIC292. Two-drug combination assays showed no antagonistic interactions between AIC292 and representative marketed HIV drugs with regard to antiviral activity. Furthermore, AIC292 displayed potent antiviralin vivoefficacy in a mouse xenograft model when applied once daily. Taken together, these data show that AIC292 represents a molecule with the antiviral properties of a novel NNRTI for the treatment of HIV-1 infection.

scholarly journals Genotypic Correlates of Phenotypic Resistance to Efavirenz in Virus Isolates from Patients Failing Nonnucleoside Reverse Transcriptase Inhibitor Therapy

2001 ◽  
Vol 75 (11) ◽  
pp. 4999-5008 ◽  
Author(s):  
Lee Bacheler ◽  
Susan Jeffrey ◽  
George Hanna ◽  
Richard D'Aquila ◽  
Lany Wallace ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Mononuclear Cells ◽  
Site Directed Mutagenesis ◽  
Cross Resistance ◽  
Peripheral Blood Mononuclear ◽  
In Vitro Susceptibility ◽  
Virus Isolates ◽  
Hiv 1

ABSTRACT Efavirenz (also known as DMP 266 or SUSTIVA) is a potent nonnucleoside inhibitor of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) activity and of HIV-1 replication in vitro and in vivo. Most patients on efavirenz-containing regimens have sustained antiviral responses; however, rebounds in plasma viral load have been observed in some patients in association with the emergence of mutant strains of HIV-1. Virus isolates from the peripheral blood mononuclear cells (PBMCs) of patients with such treatment failures, as well as recombinant viruses incorporating viral sequences derived from patient plasma, show reduced in vitro susceptibility to efavirenz in association with mutations in the RT gene encoding K103N, Y188L, or G190S/E substitutions. Patterns of RT gene mutations and in vitro susceptibility were similar in plasma virus and in viruses isolated from PBMCs. Variant strains of HIV-1 constructed by site-directed mutagenesis confirmed the role of K103N, G190S, and Y188L substitutions in reduced susceptibility to efavirenz. Further, certain secondary mutations (V106I, V108I, Y181C, Y188H, P225H, and F227L) conferred little resistance to efavirenz as single mutations but enhanced the level of resistance of viruses carrying these mutations in combination with K103N or Y188L. Viruses with K103N or Y188L mutations, regardless of the initial selecting nonnucleoside RT inhibitor (NNRTI), exhibited cross-resistance to all of the presently available NNRTIs (efavirenz, nevirapine, and delavirdine). Some virus isolates from nevirapine or delavirdine treatment failures that lacked K103N or Y188L mutations remained susceptible to efavirenz in vitro, although the clinical significance of this finding is presently unclear.

scholarly journals Novel Nucleotide Human Immunodeficiency Virus Reverse Transcriptase Inhibitor GS-9148 with a Low Nephrotoxic Potential: Characterization of Renal Transport and Accumulation

2008 ◽  
Vol 53 (1) ◽  
pp. 150-156 ◽  
Author(s):  
Tomas Cihlar ◽  
Genevieve LaFlamme ◽  
Robyn Fisher ◽  
Anne C. Carey ◽  
Jennifer E. Vela ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Reverse Transcriptase ◽  
Organic Anion ◽  
Transcriptase Inhibitor ◽  
Renal Transport ◽  
Immunodeficiency Virus ◽  
Reverse Transcriptase Inhibitor ◽  
In Cells

ABSTRACT Accumulation of antiviral nucleotides in renal proximal tubules is controlled by their basolateral uptake via the human renal organic anion transporters type 1 (hOAT1) and 3 (hOAT3) and apical efflux via the multidrug resistance protein 4 (MRP4). GS-9148 is a novel ribose-modified nucleotide human immunodeficiency virus (HIV) reverse transcriptase inhibitor, and its oral prodrug GS-9131 is currently being evaluated in the clinic as an anti-HIV agent. To assess the potential of GS-9148 for nephrotoxicity, its mechanism of renal transport, cytotoxicity, and renal accumulation were explored in vitro and in vivo. In comparison with the acyclic nucleotides cidofovir, adefovir, and tenofovir, GS-9148 showed 60- to 100-fold lower efficiency of transport (V max/K m ) by hOAT1 and was 20- to 300-fold less cytotoxic in cells overexpressing hOAT1, indicating its lower hOAT1-mediated intracellular accumulation and reduced intrinsic cytotoxicity. GS-9148 was also relatively inefficiently transported by hOAT3. Similar to acyclic nucleotides, GS-9148 was a substrate for MRP4 as evidenced by its reduced intracellular retention in cells overexpressing the efflux pump. Consistent with these molecular observations, GS-9148 was inefficiently taken up by fresh human renal cortex tissue in vitro and showed a limited accumulation in kidneys in vivo following oral administration of [14C]GS-9131 to dogs. Compared to acyclic nucleotide analogs, GS-9148 was also found to have lower net active tubular secretion in dogs. Collectively, these results suggest that GS-9148 exhibits a low potential for renal accumulation and nephrotoxicity.

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