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ABSTRACT:
PROVIDER: PRJNA1000967 | ENA |
REPOSITORIES: ENA
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Project description:HIV-1 Drug Resistance Genotyping of Reverse Transcriptase
| PRJNA743320 | ENA
Project description:The response of human immunodeficiency virus type 1 (HIV-1) quasispecies to antiretroviral therapy is influenced by the ensemble of mutants that composes the evolving population. Low-abundance subpopulations within HIV-1 quasispecies may determine the viral response to the administered drug combinations. However, routine sequencing assays available to clinical laboratories do not recognize HIV-1 minority variants representing less than 25% of the population. Although several alternative and more sensitive genotyping techniques have been developed, including next-generation sequencing (NGS) methods, they are usually very time consuming, expensive and require highly trained personnel, thus becoming unrealistic approaches in daily clinical practice. Here we describe the development and testing of a HIV-1 genotyping DNA microarray that detects and quantifies, in majority and minority viral subpopulations, relevant mutations and amino acid insertions in 42 codons of the pol gene associated with resistance and multidrug resistance to protease (PR) and reverse transcriptase (RT) inhibitors. A customized bioinformatics protocol has been implemented to analyze the microarray hybridization data by including a new normalization procedure and a stepwise filtering algorithm which resulted in the highly accurate (96.33%) detection of positive/negative signals. This microarray has been tested with 57 subtype B HIV-1 clinical samples extracted from multi-treated patients, showing an overall identification of 95.53% and 89.24% of the queried PR and RT codons, respectively, and enough sensitivity to detect minority subpopulations representing as low as 5-10% of the total quasispecies. Such a genotyping platform represents an efficient diagnostic and prognostic tool useful to personalize antiviral treatments in clinical practice.
2016-11-30 | GSE90621 | GEO
Project description:The goal of this study was to determine how an HIV quasispecies is maintained in the face of selection. We deep sequenced the HIV provirus from cell populations as well as single cells at different time points from in vitro evolution experiments and found that when a less fit and more fit infect the same cell, they share components (complmentation) and therefore allow the less fit to perpetuate. We reproduced a quasispecies to an HIV reverse transcriptase inhibitor. The drug resistant genotype never completely supplanted the drug sensitive genotype, which stabilized at about 20% of viral sequences. Single-cell sequencing showed that resistant genotype frequency plateaued when cells were co-infected with sensitive and resistant genotypes, suggesting a sharing of viral proteins in co-infected cells (complementation), masking genotypic differences. To test if complementation can confer phenotypic drug resistance, we co-transfected fluorescently labelled molecular clones of sensitive and resistant HIV and observed drug resistance in genotypically sensitive virus from co-transfected cells. Resistant virus preferentially co-infected cells with drug sensitive HIV, explaining initiation of co-infections. Modelling showed that a stable quasispecies could form at the experimental multiplicities of infection. Conclusions: Complementation can lead to a quasispecies in infection environments where multiple infections per cell are common
2020-05-13 | GSE150390 | GEO
Project description:Here we investigated whether a combination of cell-free infection and cell-to-cell spread confers a selective advantage in the evolution of resistance to an inhibitor relative to cell-to-cell spread alone due to the stronger selection pressure against drug sensitive virus. We propagated HIV infection using coculture of infected with uninfected cells in the face of the reverse transcriptase inhibitor efavirenz (EFV), and compared the effect on drug resistance evolution of including one cycle of cell-free infection. In the presence of a single cell-free infection step, we obtained earlier evolution of resistance to EFV. When we increased selective pressure by adding emtricitabine (FTC as a second drug, infection with the cell-free step evolved multidrug resistance and was able to replicate, while infection without a cell-free step failed to evolve multidrug resistance. In conclusion, our results suggest that, HIV cell-to-cell spread has a decreased capacity to rapidly evolve resistance to inhibitors, which is conferred by cell-free infection.
2020-09-05 | GSE157511 | GEO
Project description:Identifying resistance mutations in a drug target provides crucial information. Lentiviral transduction creates multiple types of mutations due to the error-prone nature of the HIV-1 reverse transcriptase (RT). We optimized and leveraged this property to identify drug resistance mutations, a technique we term LentiMutate. After validating this technique by identifying clinically relevant EGFR resistance mutations, we applied this technique to two additional anti-cancer drugs, imatinib and AMG 510. We find novel deletions in BCR-ABL1 that confer resistance to BCR-ABL1 inhibitors and point mutations in the AMG 510 binding pocket or oncogenic non-G12C mutations, in KRAS-G12C or wild-type KRAS, respectively, that confer resistance to AMG 510. LentiMutate may prove highly valuable to clinical and preclinical cancer drug development.
2021-07-28 | GSE180965 | GEO
Project description:Identifying resistance mutations in a drug target provides crucial information. Lentiviral transduction creates multiple types of mutations due to the error-prone nature of the HIV-1 reverse transcriptase (RT) and we show this property can be leveraged to identify mutations that confer resistance to targeted anti-cancer drugs, a technique we term “LentiMutate”. First, we improved LentiMutate by making the lentiviral RT more error-prone. Next, we applied this technique to two anti-cancer drugs, imatinib and AMG 510. We find novel deletions in BCR-ABL that confer resistance to BCR-ABL inhibitors and point mutations in the AMG 510 binding pocket or oncogenic non-G12C mutations, in KRAS-G12C or wild-type KRAS, respectively, that confer resistance to AMG 510. LentiMutate may prove highly valuable to clinical and preclinical cancer drug development
2021-07-25 | GSE164663 | GEO
Project description:Identifying resistance mutations in a drug target provides crucial information. Lentiviral transduction creates multiple types of mutations due to the error-prone nature of the HIV-1 reverse transcriptase (RT) and we show this property can be leveraged to identify mutations that confer resistance to targeted anti-cancer drugs, a technique we term “LentiMutate”. First, we improved LentiMutate by making the lentiviral RT more error-prone. Next, we applied this technique to two anti-cancer drugs, imatinib and AMG 510. We find novel deletions in BCR-ABL that confer resistance to BCR-ABL inhibitors and point mutations in the AMG 510 binding pocket or oncogenic non-G12C mutations, in KRAS-G12C or wild-type KRAS, respectively, that confer resistance to AMG 510. LentiMutate may prove highly valuable to clinical and preclinical cancer drug development
2021-07-25 | GSE164661 | GEO
Project description:Abacavir, a nucleoside reverse-transcriptase inhibitor used for the treatment of human immunodeficiency virus (HIV) infection, develops hypersensitivity in patients carrying HLA-B*57:01 allele through drug antigen presentation. We found that abacavir exposure induced HLA allotype-specific innate immune response in keratinocyte derived from HLA-B*57:01 transgenic mice. Therefore, the mechanism of the novel response was analyzed comprehensively.
2018-03-01 | GSE110394 | GEO
Project description:Nevirapine is a non-nucleoside reverse transcriptase inhibitor, a class of antiretroviral drug, used for the treatment of HIV-1 infection. Despite its wide use, nevirapine treatment has been associated with a significant incidence of different kind of hypersensitivity reactions (HSRs). We used microarrays to find significant genes that can relate to Nevirapine-persuaded hypersensitivity reactions in ‘acute’ patients compared to ‘recovered’ and/or ‘tolerant’ patients.
2015-11-30 | GSE42436 | GEO
Project description:We identified the alarmin S100A9 as a novel intracellular antiretroviral factor expressed in human monocyte-derived and skin-derived Langerhans cells (LC). The expression of S100A9 is significantly upregulated by the transforming growth factor beta in human monocyte-derived cells. We showed that S100A9 intracellular expression is decreased upon maturation and inversely correlated with an enhanced inversely correlates with the maturation level of LC and susceptibilityility to HIV-1 infection of LC. Furthermore, silencing of S100A9 in primary human LC relieves HIV-1 restriction while ectopic expression of S100A9 in various cell lines established an intrinsic resistance to both HIV-1 and MMLV infection by acting on reverse transcription. Mechanistically, the intracellular expression of S100A9 alters viral capsid uncoating and reverse transcription in a distal manner. Also, S100A9 demonstrates potent inhibitory effect against HIV-1 and MMLV reverse transcriptase (RTase) activity in-vitro in a divalent cation-dependent context. Our findings uncover an unexpected intracellular antiretroviral function of the human alarmin S100A9 and highlight a novel crosstalk betweenregulating antiretroviral immunity in Langerhans cells
2021-05-06 | GSE173939 | GEO