Catalytic Efficiency and Phenotype of HIV-1 Proteases Encoding Single Critical Resistance Substitutions

Cabana, Marta; Fernàndez, Guerau; Parera, Mariona; Clotet, Bonaventura; Martı́nez, Miguel Ángel

doi:10.1006/viro.2002.1520

Cited by 15 publications

(24 citation statements)

References 48 publications

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“…As we have previously demonstrated, and as also shown in Fig. 5, the cI repressor cleavage is directly proportional to the protease catalytic efficiency (5,20,36,37,46,47). The enzymatic activities of the different master and variant proteases of the three quasispecies analyzed in this study were related to the activity of the HXB2 protease (100%) (Fig.…”

Section: Resultssupporting

confidence: 74%

“…The HIV-1 protease gene was amplified by PCR from proviral PBMC DNA. PCR amplification and phage lambda cloning were performed as previously described (5,36). Briefly, for the first PCR, protease oligonucleotides 5Јprot1 (5Ј-AGGCTAATTTTTTAGGGA AGATCTGGCCTTCC-3Ј [HXB2 residues 2078 to 2108]) and 3Јprot1 (5Ј-GC AAATACTGGAGTATTGTATGGATTTTCAGG-3Ј [HXB2 residues 2703 to 2734]) were used.…”

Section: Methodsmentioning

confidence: 99%

“…The catalytic efficiencies of the different HIV-1 proteases were calculated using a bacteriophage lambda-based genetic screening, as previously described (5,20,36,37,45,46,55). Briefly, Escherichia coli JM109 cells containing plasmid p2X-cI-HIV were transformed with plasmid pcI-HIV-cro.…”

Section: Methodsmentioning

confidence: 99%

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Fitness Landscape of Human Immunodeficiency Virus Type 1 Protease Quasispecies

Fernàndez

Clotet

Martı́nez

2007

J Virol

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Here we show, at a high resolution (1%), the human immunodeficiency virus type 1 (HIV-1) protease gene quasispecies landscape from three infected naïve individuals. A huge range of genetic configurations was found (67%, 71%, and 80% of the nucleotide clones from the three individuals, respectively, were different), and these configurations created a dense net that linked different parts of the viral population. Similarly, a vast diversity of different protease activities was also found. Importantly, 65% of the analyzed enzymes had detectable protease activity, and 11% of the minority individual variants showed similar or better fitness than the master (most abundant) enzyme, suggesting that the viral complexity in this genomic region does not exclusively depend on the enzyme's catalytic efficiency. Several high-fitness minority variants had only one substitution compared to the master sequence, supporting the possibility that the rugged HIV-1 protease quasispecies fitness landscape may be formed by a continuous network that can be traversed by single mutational steps without passing through defective or less-adapted proteins.

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Section: Resultssupporting

confidence: 74%

Section: Methodsmentioning

confidence: 99%

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Fitness Landscape of Human Immunodeficiency Virus Type 1 Protease Quasispecies

Fernàndez

Clotet

Martı́nez

2007

J Virol

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“…The enzymatic activity of the different identified single mutant proteases (33,47, and 32, for quasispecies A, B, and C, respectively) was determined by using a bateriophage based genetic screen. 26,27,[41][42][43][44][45] First, we evaluated the enzymatic activities of single-variant proteases by engineering in the cI repressor the HCV polyprotein NS5A/NS5B cleavage site. 27 The enzymatic activities were related to the activity of the HCV subgenomic replicon I389/NS3-3 NS3/4 protease (100%).…”

Section: Resultsmentioning

confidence: 99%

Genetic and catalytic efficiency structure of an HCV protease quasispecies

Franco¹,

Parera²,

Aparicio³

et al. 2007

Hepatology

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The HCV nonstructural protein (NS)3/4A serine protease is not only involved in viral polyprotein processing but also efficiently blocks the retinoic-acid-inducible gen I and Toll-like receptor 3 signaling pathways and contributes to virus persistence by enabling HCV to escape the interferon antiviral response. Therefore, the NS3/4A protease has emerged as an ideal target for the control of the disease and the development of new anti-HCV agents. Here, we analyzed, at a high resolution (approximately 100 individual clones), the HCV NS3 protease gene quasispecies from three infected individuals. Nucleotide heterogeneity of 49%, 84%, and 91% were identified, respectively, which created a dense net that linked different parts of the viral population. Minority variants having mutations involved in the acquisition of resistance to current NS3/4A protease inhibitors (PIs) were also found. A vast diversity of different catalytic efficiencies could be distinguished. Importantly, 67% of the analyzed enzymes displayed a detectable protease activity. Moreover, 35% of the minority individual variants showed similar or better catalytic efficiency than the master (most abundant) enzyme. Nevertheless, and in contrast to minority variants, master enzymes always displayed a high catalytic efficiency when different viral polyprotein cleavage sites were tested. Finally, genetic and catalytic efficiency differences were observed when the 3 quasispecies were compared, suggesting that different selective forces were acting in different infected individuals. Conclusion: The rugged HCV protease quasispecies landscape should be able to react to environmental changes that may threaten its survival. (HEPATOLOGY 2007;45:899-910.)

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“…Inferred levels of resistance* to protease (PI) and to nucleoside reverse transcriptase (NRTI) inhibitors**. appear to be necessary for the development of a highly PIresistant virus (8,22). Accordingly, among PI, we detected seven samples (36.8%) resistant to APV and one sample (5.3%) resistant to NFV, while all NRTI had at least one sample classified as resistant.…”

Section: Discussionmentioning

confidence: 99%

HIV-1 subtypes and mutations associated to antiretroviral drug resistance in human isolates from Central Brazil

Cerqueira¹,

Ramalho²,

Oliveira³

et al. 2004

Braz. J. Microbiol.

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The detection of polymorphisms associated to HIV-1 drug-resistance and genetic subtypes is important for the control and treatment of HIV-1 disease. Drug pressure selects resistant variants that carry mutations in the viral reverse transcriptase (RT) and protease (PR) genes. For a contribution to the public health authorities in planning the availability of therapeutic treatment, we therefore described the genetic variability, the prevalence of mutations associated to drug resistance and the antiretroviral resistance profile in HIV-1 isolates from infected individuals in Central Brazil. Nineteen HIV-1 RNA samples from a Public Health Laboratory of the Federal District were reversely transcribed and cDNAs were amplified by nested PCR. One fragment of 297 bp coding the entire protease gene, and another of 647 bp, corresponding to the partial RT gene (codons 19-234), were obtained. Automated sequencing and BLAST analysis revealed the presence of 17 B and 2 F1 HIV-1 subtypes. The amino acid sequences were analyzed for the presence of resistance-associated mutations. A total of 6 PR mutations, 2 major and 4 accessory, and 8 RT mutations related to drug resistance were found. Our data suggest a high prevalence of HIV-1 B subtype in the studied population of Federal District as well as the presence of genetically-resistant strains in individuals failing treatment.

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Catalytic Efficiency and Phenotype of HIV-1 Proteases Encoding Single Critical Resistance Substitutions

Cited by 15 publications

References 48 publications

Fitness Landscape of Human Immunodeficiency Virus Type 1 Protease Quasispecies

Fitness Landscape of Human Immunodeficiency Virus Type 1 Protease Quasispecies

Genetic and catalytic efficiency structure of an HCV protease quasispecies

HIV-1 subtypes and mutations associated to antiretroviral drug resistance in human isolates from Central Brazil

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