scholarly journals Rapid and Simple Phenotypic Assay for Drug Susceptibility of Human Immunodeficiency Virus Type 1 Using CCR5-Expressing HeLa/CD4+ Cell Clone 1-10 (MAGIC-5)

2001 ◽  
Vol 45 (2) ◽  
pp. 495-501 ◽  
Author(s):  
Atsuko Hachiya ◽  
Saori Aizawa-Matsuoka ◽  
Mari Tanaka ◽  
Yukiko Takahashi ◽  
Setsuko Ida ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Cell Clone ◽  
Drug Susceptibility ◽  
Phenotypic Resistance ◽  
Phenotypic Assay ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT We describe a rapid and simple novel phenotypic assay for drug susceptibility of human immunodeficiency virus type-1 (HIV-1) using a CCR5-expressing HeLa/CD4+ cell clone 1-10 (MAGIC-5). MAGIC-5 cells produced large amounts of HIV-1 in culture supernatants, which enabled us to perform the phenotypic resistance assay. Determination of HIV-1 susceptibility to various protease inhibitors (PI) and nucleoside reverse transcriptase inhibitors was completed within 15 days in T-cell-tropic (X4) and macrophage-tropic (R5) viruses using fresh plasma samples containing at least 104copies/ml. The nucleotide sequence of the envelope V3 region of HIV-1 in plasma was almost identical to that of the virus isolated by MAGIC-5 cells, suggesting a lack of selection bias in our assay. The assay variability was confined to within five-fold in all drugs examined. Accordingly, we used a 10-fold increase in the 50% inhibitory concentration as the cutoff value for viral resistance in the present assay. HIV-1 resistant to lamivudine, which was not detected by conventional genotypic assays, was isolated. In HIV-1 with PI-associated primary amino acid substitutions, our assay showed that drug resistance profiles correlated well with previously reported genotypic-assay data. Furthermore, our assay provided comprehensive results regarding PI resistance in the presence of multiple mutations. The novel assay successfully quantified the level of resistance of clinical HIV-1 isolates to a battery of anti-HIV drugs, indicating its clinical usefulness, particularly in patients who failed to respond to antiretroviral chemotherapy.

scholarly journals A recombinant retroviral system for rapid in vivo analysis of human immunodeficiency virus type 1 susceptibility to reverse transcriptase inhibitors.

1997 ◽  
Vol 41 (12) ◽  
pp. 2781-2785 ◽  
Author(s):  
C Shi ◽  
J W Mellors
Keyword(s):  
Human Immunodeficiency Virus ◽  
Reverse Transcriptase ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Drug Susceptibility ◽  
Phenotypic Resistance ◽  
Immunodeficiency Virus ◽  
Hiv 1

We have developed a new recombinant retroviral system in which a library of infectious molecular clones of human immunodeficiency virus type 1 (HIV-1) is constructed with reverse transcriptase (RT) genes derived from viral RNA sequences in plasma. HIV-1 RT is amplified from plasma HIV-1 RNA by nested RT-PCR and cloned into a RT-defective HIV-1 proviral vector (xxLAI-np), generating 10(3) to 10(4) recombinant proviral clones from each reaction. The bulk cloning products or individual molecular clones are transfected into MT-2 cells to generate infectious virus. The resultant viruses are assayed for drug susceptibility in CD4+ cell lines to determine either the dominant phenotype of the recombinant virus mixture or the phenotypes of the individual viral clones. DNA sequencing of the cloned RT genes can identify mutations associated with phenotypic resistance of clonal mixtures or individual clones. This method can be used to rapidly detect the in vivo emergence of HIV-1 quasispecies resistant to RT inhibitors.

scholarly journals The Impact of Individual Human Immunodeficiency Virus Type 1 Protease Mutations on Drug Susceptibility Is Highly Influenced by Complex Interactions with the Background Protease Sequence

2009 ◽  
Vol 83 (18) ◽  
pp. 9512-9520 ◽  
Author(s):  
H. Van Marck ◽  
I. Dierynck ◽  
G. Kraus ◽  
S. Hallenberger ◽  
T. Pattery ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Drug Susceptibility ◽  
Vast Number ◽  
Complex Interactions ◽  
Immunodeficiency Virus ◽  
The Impact ◽  
Hiv 1

ABSTRACT The requirement for multiple mutations for protease inhibitor (PI) resistance necessitates a better understanding of the molecular basis of resistance development. The novel bioinformatics resistance determination approach presented here elaborates on genetic profiles observed in clinical human immunodeficiency virus type 1 (HIV-1) isolates. Synthetic protease sequences were cloned in a wild-type HIV-1 background to generate a large number of close variants, covering 69 mutation clusters between multi-PI-resistant viruses and their corresponding genetically closely related, but PI-susceptible, counterparts. The vast number of mutants generated facilitates a profound and broad analysis of the influence of the background on the effect of individual PI resistance-associated mutations (PI-RAMs) on PI susceptibility. Within a set of viruses, all PI-RAMs that differed between susceptible and resistant viruses were varied while maintaining the background sequence from the resistant virus. The PI darunavir was used to evaluate PI susceptibility. Single sets allowed delineation of the impact of individual mutations on PI susceptibility, as well as the influence of PI-RAMs on one another. Comparing across sets, it could be inferred how the background influenced the interaction between two mutations, in some cases even changing antagonistic relationships into synergistic ones or vice versa. The approach elaborates on patient data and demonstrates how the specific mutational background greatly influences the impact of individual mutations on PI susceptibility in clinical patterns.

scholarly journals Genotypic and Phenotypic Resistance Patterns of Human Immunodeficiency Virus Type 1 Variants with Insertions or Deletions in the Reverse Transcriptase (RT): Multicenter Study of Patients Treated with RT Inhibitors

2001 ◽  
Vol 45 (6) ◽  
pp. 1836-1842 ◽  
Author(s):  
Bernard Masquelier ◽  
Esther Race ◽  
Catherine Tamalet ◽  
Diane Descamps ◽  
Jacques Izopet ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Reverse Transcriptase ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Multicenter Study ◽  
Phenotypic Resistance ◽  
Immunodeficiency Virus ◽  
Rt Inhibitors ◽  
Hiv 1

ABSTRACT Genomic rearrangements in the 5′ part of the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) have been involved in multidrug resistance to nucleoside RT inhibitors (NRTI). We carried out a retrospective, multicenter study to investigate the prevalence, variability, and phenotypic consequences of such rearrangements. Data concerning the HIV-1 RT genotype and the biological and clinical characteristics of NRTI-treated patients were collected from 10 virology laboratories. Sensitivities of the different HIV-1 variants to RT inhibitors were analyzed in a single-cycle recombinant virus assay. Fifty-two of 2,152 (2.4%) RT sequences had a rearrangement in the 5′ part of the RT, with an extensive molecular variation. The number of codons inserted between positions 68 and 69 ranged from 1 (3 samples) or 2 (41 samples) to 5 and 11 in one case each. In four cases, codon 67 was deleted. High levels of phenotypic resistance to zidovudine (AZT), lamivudine (3TC), stavudine (d4T), abacavir (ABC), and didanosine (ddI) were found in 95, 92, 72, 62, and 15% of the 40 samples analyzed, respectively. Resistance to AZT, d4T, and ABC could be found in the absence of the T215Y/F mutations. Resistance to 3TC could develop in the absence of specific mutations. Low-level resistance to ddI was noticed in 40% of the patients. The deletions of codon 67 seemed to have little effect on NRTI sensitivity. Most of the rearrangements were shown to contribute to cross-resistance to NRTI. The results regarding susceptibility to ddI raise the question of the interpretation of the phenotypic data concerning this drug.

scholarly journals A Rapid Non-Culture-Based Assay for Clinical Monitoring of Phenotypic Resistance of Human Immunodeficiency Virus Type 1 to Lamivudine (3TC)

1999 ◽  
Vol 43 (2) ◽  
pp. 264-270 ◽  
Author(s):  
J. Gerardo García Lerma ◽  
Raymond F. Schinazi ◽  
Amy S. Juodawlkis ◽  
Vincent Soriano ◽  
Yulin Lin ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Detection Threshold ◽  
Resistance Mutations ◽  
Phenotypic Resistance ◽  
Genotypic Analysis ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Monitoring for lamivudine (3TC) resistance is important both for the clinical management of human immunodeficiency virus type 1 (HIV-1)-infected patients treated with 3TC and for surveillance of transmission of 3TC-resistant HIV-1. We developed a novel non-culture-based assay for the rapid analysis of phenotypic resistance to 3TC of HIV-1 in plasma. The assay measures the susceptibility of HIV-1 reverse transcriptase (RT) activity to 3TC triphosphate (3TC-TP) in plasma. RT detection was done by the Amp-RT assay, an ultrasensitive PCR-based RT assay. Under our assay conditions, we found that 5 μM 3TC-TP inhibited RT activity from wild-type (WT), zidovudine-resistant, or nevirapine-resistant HIV-1 but not from HIV-1 carrying either the M184V mutation or multidrug (MD) resistance mutations (77L/116Y/151M or 62V/75I/77L/116Y/151M). Mixing experiments showed a detection threshold of 10% 3TC-resistant virus (M184V) in a background of WT HIV-1. To validate the assay for the detection of phenotypic resistance of HIV-1 to 3TC in plasma samples, HIV-1 RT in 30 plasma specimens collected from 15 patients before and during therapy with 3TC was tested for evidence of phenotypic resistance by the Amp-RT assay. The results were compared with those of genotypic analysis. The RT in 12 samples was found to be 3TC sensitive, while the RT in 18 samples had evidence of phenotypic resistance. All 12 samples with 3TC-sensitive RT had WT genotypes at codon 184 and were retrieved before treatment with 3TC. In contrast, all 18 specimens with 3TC-resistant RT were posttherapy samples. This assay provides a simple, rapid, and reliable method for the detection of phenotypic resistance of HIV-1 to 3TC in plasma.

scholarly journals Quantitation of Human Immunodeficiency Virus Type 1 Group O Load in Plasma by Measuring Reverse Transcriptase Activity

2000 ◽  
Vol 38 (1) ◽  
pp. 402-405
Author(s):  
J. Gerardo García Lerma ◽  
Vincent Soriano ◽  
Antonio Mas ◽  
Miguel E. Quiñones-Mateu ◽  
Eric J. Arts ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Reverse Transcriptase ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Reverse Transcriptase Activity ◽  
Plasma Viremia ◽  
Phenotypic Resistance ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT We have evaluated the use of an ultrasensitive reverse transcriptase (RT) activity assay to monitor plasma viremia in two human immunodeficiency virus type 1 (HIV-1) group O-infected patients treated with stavudine, lamivudine, and indinavir. After a initial decline in RT levels observed at 4 weeks of therapy, RT-based plasma viremia returned to baseline values at 28 or 44 weeks of treatment. The rebound in levels of RT activity was associated with the detection of phenotypic resistance to lamivudine and with the Met184Val mutation. Analysis of RT activity in plasma provides a sequence-independent means of monitoring virus loads in HIV-1 group O-infected patients.

scholarly journals Mutational Pathways, Resistance Profile, and Side Effects of Cyanovirin Relative to Human Immunodeficiency Virus Type 1 Strains with N-Glycan Deletions in Their gp120 Envelopes

2006 ◽  
Vol 80 (17) ◽  
pp. 8411-8421 ◽  
Author(s):  
Jan Balzarini ◽  
Kristel Van Laethem ◽  
Willy J. Peumans ◽  
Els J. M. Van Damme ◽  
Anders Bolmstedt ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Amino Acid ◽  
Side Effects ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Phenotypic Resistance ◽  
Immunodeficiency Virus ◽  
Virus Strains ◽  
Hiv 1

ABSTRACT Limited data are available on the genotypic and phenotypic resistance profile of the α-(1-2)mannose oligomer-specific prokaryotic lectin cyanovirin (CV-N). Therefore, a more systematic investigation was carried out to obtain a better view of the interaction between CV-N and human immunodeficiency virus type 1 (HIV-1) gp120. When HIV-1-infected CEM cell cultures were exposed to CV-N in a dose-escalating manner, a total of eight different amino acid mutations exclusively located at N-glycosylation sites in the envelope surface gp120 were observed. Six of the eight mutations resulted in the deletion of high-mannose type N-glycans (i.e., at amino acid positions 230, 332, 339, 386, 392, and 448). Two mutations (i.e., at position 136 and 160) deleted a complex type N-glycan in the variable V1/V2 domain of gp120. The level of phenotypic resistance of the mutated virus strains against CV-N generally correlated with the number of glycan deletions in gp120, although deletion of the glycans at N-230, N-392, and N-448 generally afforded a more pronounced CV-N resistance than other N-glycan deletions. However, the extent of the decrease of antiviral activity of CV-N against the mutated virus strains was markedly less pronounced than observed for α(1-3)- and α(1-6)-mannose-specific plant lectins Hippeastrum hybrid agglutinin (HHA) and Galanthus nivalis agglutinin (GNA), which points to the existence of a higher genetic barrier for CV-N. This is in agreement with a more consistent suppression of a wider variety of HIV-1 clades by CV-N than by HHA and GNA. Whereas the antiviral and in vitro antiproliferative activity of CV-N can be efficiently reversed by mannan, the pronounced mitogenic activity of CV-N on peripheral blood mononuclear cells was unaffected by mannan, indicating that some of the observed side effects of CV-N are unrelated to its carbohydrate specificity/activity.

A defective proviral DNA with a 2.6-kb deletion of human immunodeficiency virus type 1 (HIV-1) in a persistently HIV-1 infected cell clone

Virus Genes ◽  
1991 ◽  
Vol 5 (1) ◽  
pp. 81-88 ◽  
Author(s):  
Hideki Imai ◽  
Kumiko Maotani-Imai ◽  
Yeon-Sil Shin ◽  
Kazuyoshi Ikuta ◽  
Seishi Suehiro ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Infected Cell ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Cell Clone ◽  
Proviral Dna ◽  
Immunodeficiency Virus ◽  
Hiv 1

scholarly journals A Novel Human Immunodeficiency Virus Type 1 Reverse Transcriptase Mutational Pattern Confers Phenotypic Lamivudine Resistance in the Absence of Mutation 184V

2000 ◽  
Vol 44 (3) ◽  
pp. 568-573 ◽  
Author(s):  
Kurt Hertogs ◽  
Stuart Bloor ◽  
Veronique De Vroey ◽  
Christel van den Eynde ◽  
Pascale Dehertogh ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Reverse Transcriptase ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Site Directed Mutagenesis ◽  
Phenotypic Resistance ◽  
Mutational Pattern ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT We describe a new human immunodeficiency virus type 1 (HIV-1) mutational pattern associated with phenotypic resistance to lamivudine (3TC) in the absence of the characteristic replacement of methionine by valine at position 184 (M184V) of reverse transcriptase. Combined genotypic and phenotypic analyses of clinical isolates revealed the presence of moderate levels of phenotypic resistance (between 4- and 50-fold) to 3TC in a subset of isolates that did not harbor the M184V mutation. Mutational cluster analysis and comparison with the phenotypic data revealed a significant correlation between moderate phenotypic 3TC resistance and an increased incidence of replacement of glutamic acid by aspartic acid or alanine and of valine by isoleucine at residues 44 and 118 of reverse transcriptase, respectively. This occurred predominantly in those isolates harboring zidovudine resistance-associated mutations (41L, 215Y). The requirement of the combination of mutations 41L and 215Y with mutations 44D and 44A and/or 118I for phenotypic 3TC resistance was confirmed by site-directed mutagenesis experiments. These data support the assumption that HIV-1 may have access to several different genetic pathways to escape drug pressure or that the increase in the frequency of particular mutations may affect susceptibility to drugs that have never been part of a particular regimen.

Sign in / Sign up

Export Citation Format

Share Document