scholarly journals Structure–function analysis of the ribosomal frameshifting signal of two human immunodeficiency virus type 1 isolates with increased resistance to viral protease inhibitors

2007 ◽  
Vol 88 (1) ◽  
pp. 226-235 ◽  
Author(s):  
Roseanne Girnary ◽  
Louise King ◽  
Laurence Robinson ◽  
Robert Elston ◽  
Ian Brierley
Keyword(s):  
Human Immunodeficiency Virus ◽  
Protease Inhibitors ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Ribosomal Frameshift ◽  
Viral Protease ◽  
Immunodeficiency Virus ◽  
Slippery Sequence ◽  
Hiv 1

Expression of the pol-encoded proteins of human immunodeficiency virus type 1 (HIV-1) requires a programmed –1 ribosomal frameshift at the junction of the gag and pol coding sequences. Frameshifting takes place at a heptanucleotide slippery sequence, UUUUUUA, and is enhanced by a stimulatory RNA structure located immediately downstream. In patients undergoing viral protease (PR) inhibitor therapy, a p1/p6gag L449F cleavage site (CS) mutation is often observed in resistant isolates and frequently generates, at the nucleotide sequence level, a homopolymeric and potentially slippery sequence (UUUUCUU to UUUUUUU). The mutation is located within the stimulatory RNA downstream of the authentic slippery sequence and could act to augment levels of pol-encoded enzymes to counteract the PR deficit. Here, RNA secondary structure probing was employed to investigate the structure of a CS-containing frameshift signal, and the effect of this mutation on ribosomal frameshift efficiency in vitro and in tissue culture cells was determined. A second mutation, a GGG insertion in the loop of the stimulatory RNA that could conceivably lead to resistance by enhancing the activity of the structure, was also tested. It was found, however, that the CS and GGG mutations had only a very modest effect on the structure and activity of the HIV-1 frameshift signal. Thus the increased resistance to viral protease inhibitors seen with HIV-1 isolates containing mutations in the frameshifting signal is unlikely to be accounted for solely by enhancement of frameshift efficiency.

scholarly journals Human immunodeficiency virus type 1 (HIV-1) protease inhibitors block cell-to-cell HIV-1 endocytosis in dendritic cells

2009 ◽  
Vol 90 (11) ◽  
pp. 2777-2787 ◽  
Author(s):  
Claudia Muratori ◽  
Eliana Ruggiero ◽  
Antonella Sistigu ◽  
Roberta Bona ◽  
Maurizio Federico
Keyword(s):  
Human Immunodeficiency Virus ◽  
Dendritic Cells ◽  
Protease Inhibitors ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Hiv Protease ◽  
Donor Cells ◽  
Immunodeficiency Virus ◽  
Hiv 1

Sexual transmission is now the most frequent means of diffusion of human immunodeficiency virus type 1 (HIV-1). Even if the underlying mechanism is still largely unknown, there is a consensus regarding the key role played by mucosal dendritic cells (DCs) in capturing HIV through contact with infected subepithelial lymphocytes, and their capacity to spread HIV by trans-infection. We found that HIV protease inhibitors (PIs) reduced virion endocytosis strongly in monocyte-derived immature (i) DCs contacting HIV-1-infected cells, and that this phenomenon led to dramatically impaired trans-infection activity. This inhibitory effect was not mediated by the block of viral protease activity, as it was also operative when donor cells were infected with a PI-resistant HIV-1 strain. The block of virus maturation imposed by PIs did not correlate with significant variations in the levels of virus expression in donor cells or of Gag/Env virion incorporation. Also, PIs did not affect the endocytosis activity of DCs. In contrast, we noticed that PI treatment inhibited the formation of cell–cell conjugates whilst reducing the expression of ICAM-1 in target iDCs. Our results contribute to a better delineation of the mechanisms underlying HIV-1 trans-infection activity in DCs, whilst having implications for the development of new anti-HIV microbicide strategies.

Effects of Human Immunodeficiency Virus Type 1 Resistance to Protease Inhibitors on Reverse Transcriptase Processing, Activity, and Drug Sensitivity

1999 ◽  
Vol 73 (4) ◽  
pp. 3455-3459 ◽  
Author(s):  
Laurence Carron de la Carrière ◽  
Sylvie Paulous ◽  
François Clavel ◽  
Fabrizio Mammano
Keyword(s):  
Human Immunodeficiency Virus ◽  
Reverse Transcriptase ◽  
Protease Inhibitors ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Resistance Mutations ◽  
Immunodeficiency Virus ◽  
Rt Inhibitors ◽  
Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) variants resistant to protease inhibitors often display a reduced replicative capacity as a result of an impairment of protease function. Such fitness-impaired viruses display Gag precursor maturation defects. Here, we report that some protease inhibitor-resistant viruses also display abnormalities in the processing of reverse transcriptase (RT) by the protease. In three recombinant viruses carrying resistant protease sequences from patient plasma, we observed a marked decrease in the amount of mature RT subunits and of particle-associated RT activity compared to their parental pretherapy counterparts. We investigated the possibility that a decrease in the amount of particle-associated mature RT could affect the sensitivity of the corresponding virus to RT inhibitors. We observed a twofold increase of sensitivity to zidovudine (AZT) when a virus which carried AZT mutations was processed by a resistant protease. Interestingly, the presence of AZT-resistance mutations partially rescued the replication defect associated with the mutated protease. The interplay between resistance to protease inhibitors and to RT inhibitors described here may be relevant to the therapeutic control of HIV-1 infection.

scholarly journals Identification of a Key Target Sequence To Block Human Immunodeficiency Virus Type 1 Replication within thegag-pol Transframe Domain

2000 ◽  
Vol 74 (10) ◽  
pp. 4621-4633 ◽  
Author(s):  
Shizuko Sei ◽  
Quan-en Yang ◽  
Dennis O'Neill ◽  
Kazuhisa Yoshimura ◽  
Kunio Nagashima ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Binding Motif ◽  
Target Sequence ◽  
Viral Protease ◽  
Conserved Sequence ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Although the full sequence of the human immunodeficiency virus type 1 (HIV-1) genome has been known for more than a decade, effective genetic antivirals have yet to be developed. Here we show that, of 22 regions examined, one highly conserved sequence (ACTCTTTGGCAACGA) near the 3′ end of the HIV-1 gag-poltransframe region, encoding viral protease residues 4 to 8 and a C-terminal Vpr-binding motif of p6Gag protein in two different reading frames, can be successfully targeted by an antisense peptide nucleic acid oligomer named PNAPR2. A disrupted translation of gag-pol mRNA induced at the PNAPR2-annealing site resulted in a decreased synthesis of Pr160Gag-Pol polyprotein, hence the viral protease, a predominant expression of Pr55Gag devoid of a fully functional p6Gag protein, and the excessive intracellular cleavage of Gag precursor proteins, hindering the processes of virion assembly. Treatment with PNAPR2abolished virion production by up to 99% in chronically HIV-1-infected H9 cells and in peripheral blood mononuclear cells infected with clinical HIV-1 isolates with the multidrug-resistant phenotype. This particular segment of the gag-pol transframe gene appears to offer a distinctive advantage over other regions in invading viral structural genes and restraining HIV-1 replication in infected cells and may potentially be exploited as a novel antiviral genetic target.

scholarly journals Retracing the Evolutionary Pathways of Human Immunodeficiency Virus Type 1 Resistance to Protease Inhibitors: Virus Fitness in the Absence and in the Presence of Drug

2000 ◽  
Vol 74 (18) ◽  
pp. 8524-8531 ◽  
Author(s):  
Fabrizio Mammano ◽  
Virginie Trouplin ◽  
Veronique Zennou ◽  
Francois Clavel
Keyword(s):  
Human Immunodeficiency Virus ◽  
Protease Inhibitors ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Drug Concentration ◽  
Resistance Mutations ◽  
Drug Concentrations ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) resistance to protease inhibitors (PI) is a major obstacle to the full success of combined antiretroviral therapy. High-level resistance to these compounds is the consequence of stepwise accumulation of amino acid substitutions in the HIV-1 protease (PR), following pathways that usually differ from one inhibitor to another. The selective advantage conferred by resistance mutations may depend upon several parameters: the impact of the mutation on virus infectivity in the presence or absence of drug, the nature of the drug, and its local concentration. Because drug concentrations in vivo are subject to extensive variation over time and display a markedly uneven tissue distribution, the parameters of selection for HIV-1 resistance to PI in treated patients are complex and poorly understood. In this study, we have reconstructed a large series of HIV-1 mutants that carry single or combined mutations in the PR, retracing the accumulation pathways observed in ritonavir-, indinavir-, and saquinavir-treated patients. We have then measured the phenotypic resistance and the drug-free infectivity of these mutant viruses. A deeper insight into the evolutionary value of HIV-1 PR mutants came from a novel assay system designed to measure the replicative advantage of mutant viruses as a function of drug concentration. By tracing the resultant fitness profiles, we determined the range of drug concentrations for which mutant viruses displayed a replicative advantage over the wild type and the extent of this advantage. Fitness profiles were fully consistent with the order of accumulation of resistance mutations observed in treated patients and further emphasise the key importance of local drug concentration in the patterns of selection of drug-resistant HIV-1 mutants.

scholarly journals Susceptibility to PNU-140690 (Tipranavir) of Human Immunodeficiency Virus Type 1 Isolates Derived from Patients with Multidrug Resistance to Other Protease Inhibitors

2000 ◽  
Vol 44 (5) ◽  
pp. 1328-1332 ◽  
Author(s):  
Stefano Rusconi ◽  
Simona La Seta Catamancio ◽  
Paola Citterio ◽  
Semir Kurtagic ◽  
Michela Violin ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Drug Resistance ◽  
Amino Acid ◽  
Protease Inhibitors ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Entire Group ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT In our study we examined the anti-human immunodeficiency virus type 1 (anti-HIV-1) activity of a novel HIV-1 protease inhibitor, PNU-140690 (tipranavir), against patient-derived isolates resistant to multiple other protease inhibitors (PIs). The aim of our experiments was to investigate the genotypes and the in vitro phenotypes of drug resistance of PNU-140690. We carried out drug susceptibility tests with peripheral blood mononuclear cells and a fixed amount of infectious virus (1,000 50% tissue culture infective doses) to determine the 50% inhibitory concentration (IC50) and IC90, PCR assays for the detection of drug resistance mutations in RNA in plasma, and direct sequencing of PCR products. Phenotypic resistance to PIs was invariably related to genotypic mutations. The substitutions among the amino acid residues of the protease included L10I, K20R, L24I, M36I, N37D, G48V, I54V, L63P, I64V, A71V, V77I, V82A, I84V, and L90M. Isolates from all of the patients had developed a maximal degree of resistance to indinavir, ritonavir, and nelfinavir (IC50s, >0.1 μM). We also compared these mutations with the amino acid changes previously described in association with in vivo tipranavir administration. The mutations included the following: I15V, E35D, N37D, R41K, D60E, and A71T. Infections with IIIB, 14aPre, and N70 were inhibited by an average drug IC90 of 0.18 ± 0.02 μM in multiple experiments. The average mean ± standard error of mean IC90 for the entire group of multidrug-resistant isolates derived from the mean values for two culture wells with p24 antigen supernatant appeared to be 0.619 ± 0.055 μM (range, 0.31 to 0.86 μM). Tipranavir retained a sustained antiviral activity against PI-MDR clinical isolates and might be useful in combination regimens with other antiretroviral agents for patients who have already failed other PI-containing therapies.

scholarly journals Resistance to Nucleoside Analog Reverse Transcriptase Inhibitors Mediated by Human Immunodeficiency Virus Type 1 p6 Protein

2001 ◽  
Vol 75 (20) ◽  
pp. 9644-9653 ◽  
Author(s):  
Solange Peters ◽  
Miguel Muñoz ◽  
Sabine Yerly ◽  
Victor Sanchez-Merino ◽  
Cecilio Lopez-Galindez ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Drug Resistance ◽  
Reverse Transcriptase ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Nucleoside Analog ◽  
Viral Protease ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Resistance of human immunodeficiency virus type 1 (HIV-1) to antiretroviral agents results from target gene mutation within thepol gene, which encodes the viral protease, reverse transcriptase (RT), and integrase. We speculated that mutations in genes other that the drug target could lead to drug resistance. For this purpose, the p1-p6 gag -p6 pol region of HIV-1, placed immediately upstream ofpol, was analyzed. This region has the potential to alter Pol through frameshift regulation (p1), through improved packaging of viral enzymes (p6Gag), or by changes in activation of the viral protease (p6Pol). Duplication of the proline-rich p6Gag PTAP motif, necessary for late viral cycle activities, was identified in plasma virus from 47 of 222 (21.2%) patients treated with nucleoside analog RT inhibitor (NRTI) antiretroviral therapy but was identified very rarely from drug-naı̈ve individuals. Molecular clones carrying a 3-amino-acid duplication, APPAPP (transframe duplication SPTSPT in p6Pol), displayed a delay in protein maturation; however, they packaged a 34% excess of RT and exhibited a marked competitive growth advantage in the presence of NRTIs. This phenotype is reminiscent of the inoculum effect described in bacteriology, where a larger input, or a greater infectivity of an organism with a wild-type antimicrobial target, leads to escape from drug pressure and a higher MIC in vitro. Though the mechanism by which the PTAP region participates in viral maturation is not known, duplication of this proline-rich motif could improve assembly and packaging at membrane locations, resulting in the observed phenotype of increased infectivity and drug resistance.

scholarly journals Unique Thermodynamic Response of Tipranavir to Human Immunodeficiency Virus Type 1 Protease Drug Resistance Mutations

2007 ◽  
Vol 81 (10) ◽  
pp. 5144-5154 ◽  
Author(s):  
S. Muzammil ◽  
A. A. Armstrong ◽  
L. W. Kang ◽  
A. Jakalian ◽  
P. R. Bonneau ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Drug Resistance ◽  
Protease Inhibitors ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Wild Type ◽  
High Potency ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Drug resistance is a major problem affecting the clinical efficacy of antiretroviral agents, including protease inhibitors, in the treatment of infection with human immunodeficiency virus type 1 (HIV-1)/AIDS. Consequently, the elucidation of the mechanisms by which HIV-1 protease inhibitors maintain antiviral activity in the presence of mutations is critical to the development of superior inhibitors. Tipranavir, a nonpeptidic HIV-1 protease inhibitor, has been recently approved for the treatment of HIV infection. Tipranavir inhibits wild-type protease with high potency (Ki = 19 pM) and demonstrates durable efficacy in the treatment of patients infected with HIV-1 strains containing multiple common mutations associated with resistance. The high potency of tipranavir results from a very large favorable entropy change (−TΔS = −14.6 kcal/mol) combined with a favorable, albeit small, enthalpy change (ΔH = −0.7 kcal/mol, 25°C). Characterization of tipranavir binding to wild-type protease, active site mutants I50V and V82F/I84V, the multidrug-resistant mutant L10I/L33I/M46I/I54V/L63I/V82A/I84V/L90M, and the tipranavir in vitro-selected mutant I13V/V32L/L33F/K45I/V82L/I84V was performed by isothermal titration calorimetry and crystallography. Thermodynamically, the good response of tipranavir arises from a unique behavior: it compensates for entropic losses by actual enthalpic gains or by sustaining minimal enthalpic losses when facing the mutants. The net result is a small loss in binding affinity. Structurally, tipranavir establishes a very strong hydrogen bond network with invariant regions of the protease, which is maintained with the mutants, including catalytic Asp25 and the backbone of Asp29, Asp30, Gly48 and Ile50. Moreover, tipranavir forms hydrogen bonds directly to Ile50, while all other inhibitors do so by being mediated by a water molecule.

scholarly journals Comparison of Human Immunodeficiency Virus Type 1 Pr55Gag and Pr160Gag-Pol Processing Intermediates That Accumulate in Primary and Transformed Cells Treated with Peptidic and Nonpeptidic Protease Inhibitors

2000 ◽  
Vol 44 (5) ◽  
pp. 1397-1403 ◽  
Author(s):  
R. Renae Speck ◽  
Charles Flexner ◽  
Chun-Juan Tian ◽  
Xiao-Fang Yu
Keyword(s):  
Human Immunodeficiency Virus ◽  
Protease Inhibitors ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Potent Inhibitor ◽  
Transformed Cells ◽  
Drug Concentrations ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) produces two polyproteins, Pr55Gag and Pr160Gag-Pol, that are cleaved into mature functional subunits by the virally encoded protease. Drugs that inhibit this protease are an important part of anti-HIV therapy. We studied the ordered accumulation of Gag and Gag-Pol processing intermediates by variably blocking the protease with HIV-1 protease inhibitors (PIs). Variable protease inhibition caused accumulation of a complex pattern of processing intermediates, which was the same after incubating HIV-1-infected cells with increasing concentrations of either one of the peptidomimetic inhibitors indinavir, saquinavir (SQV), ritonavir (RTV), nelfinavir, and SC-52151 or one of the nonpeptidomimetic inhibitors DMP450, DMP323, PNU-140135, and PNU-109112 for 3 days. The patterns of Gag and Gag-Pol processing intermediate accumulation were nearly identical when the following were compared: cell- versus virion-associated proteins, HIV-1-infected transformed cell lines versus primary human peripheral blood mononuclear cells (PBMCs) and HIV-1MN versus HIV-1IIIB virus strains. RTV was a more potent inhibitor of p24 production in PBMCs than SQV by approximately 7-fold, whereas SQV was a more potent inhibitor in transformed cells than RTV by approximately 30-fold. Although the antiretroviral potency of HIV-1 PIs may change as a function of cell type, the polyprotein intermediates that accumulate with increasing drug concentrations are the same. These results support sequential processing of Gag and Gag-Pol polyproteins by the HIV-1 protease and may have important implications for understanding common cross-resistance pathways.

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