scholarly journals Genetic Variation and Susceptibilities to Protease Inhibitors among Subtype B and F Isolates in Brazil

1999 ◽  
Vol 43 (2) ◽  
pp. 253-258 ◽  
Author(s):  
Amilcar Tanuri ◽  
Ana C. P. Vicente ◽  
Koko Otsuki ◽  
Carlos A. Ramos ◽  
Orlando C. Ferreira ◽  
...  
Keyword(s):  
Genetic Variation ◽  
Amino Acid ◽  
Protease Inhibitors ◽  
Consensus Sequence ◽  
Antiretroviral Drugs ◽  
Protease Gene ◽  
Subtype B ◽  
Hiv 1 ◽  
The U.S

ABSTRACT The genetic variation of the human immunodeficiency virus type 1 (HIV-1) protease gene (prt) permits the classification of HIV-1 strains into five distinct protease subtypes, which follow thegag subtyping patterns. The susceptibilities of non-B-subtype strains to protease inhibitors (PIs) and other antiretroviral drugs remain largely unknown. Subtype F is the main non-B strain contributing to the Brazilian epidemic, accounting for 15 to 20% of these infections. In this work, we report the findings on 81 isolates from PI-naive Brazilian patients collected between 1993 and 1997. In addition, the relevant PI resistance mutations and their phenotypes were determined in vitro for 15 of these patients (B = 9 and F = 6). Among these, the subtype F samples evidenced high sensitivities in vitro to ritonavir and indinavir, with MICs at which 50 and 90% of the isolates are inhibited similar to those of both the Brazilian and the U.S. subtype B isolates. Analysis of the 81 Brazilianprt sequences demonstrated that the subtype F consensus sequence differs from the U.S. and Brazilian subtype B consensus in eight positions (I15V, E35D, M36I, R41K, R57K, Q61N, L63P, and L89M). The frequency of critical PI resistance substitutions (amino acid changes D30N, V82A/F/T, I84V, N88D, and L90M) among Brazilian isolates is very low (mean, 2.5%), and the associated secondary substitutions (amino acid positions 10L, 20K, 36M, 46M, 48G, 54I, 63P, 71A, and 77A) are infrequent. These observations document the relative rarity of resistance to PIs in the treatment of patients infected with HIV-1 subtype F in South America.

scholarly journals Genetic Diversity of Protease and Reverse Transcriptase Sequences in Non-Subtype-B Human Immunodeficiency Virus Type 1 Strains: Evidence of Many Minor Drug Resistance Mutations in Treatment-Naive Patients

2000 ◽  
Vol 38 (11) ◽  
pp. 3919-3925 ◽  
Author(s):  
Laurence Vergne ◽  
Martine Peeters ◽  
Eitel Mpoudi-Ngole ◽  
Anke Bourgeois ◽  
Florian Liegeois ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Reverse Transcriptase ◽  
Antiretroviral Drugs ◽  
Protease Gene ◽  
Resistance Mutations ◽  
Subtype B ◽  
Immunodeficiency Virus ◽  
The Impact ◽  
Hiv 1

Most human immunodeficiency virus (HIV) drug susceptibility studies have involved subtype B strains. Little information on the impact of viral diversity on natural susceptibility to antiretroviral drugs has been reported. However, the prevalence of non-subtype-B (non-B) HIV type 1 (HIV-1) strains continues to increase in industrialized countries, and antiretroviral treatments have recently become available in certain developing countries where non-B subtypes predominate. We sequenced the protease and reverse transcriptase (RT) genes of 142 HIV-1 isolates from antiretroviral-naive patients: 4 belonged to group O and 138 belonged to group M (9 subtype A, 13 subtype B, 2 subtype C, 5 subtype D, 2 subtype F1, 9 subtype F2, 4 subtype G, 5 subtype J, 2 subtype K, 3 subtype CRF01-AE, 67 subtype CRF02-AG, and 17 unclassified isolates). No major mutations associated with resistance to nucleoside reverse transcriptase inhibitors (NRTIs) or protease inhibitors were detected. Major mutations linked to resistance to non-NRTI agents were detected in all group O isolates (A98G and Y181C) and in one subtype J virus (V108I). In contrast, many accessory mutations were found, especially in the protease gene. Only 5.6% of the 142 strains, all belonging to subtype B or D, had no mutations in the protease gene. Sixty percent had one mutation, 22.5% had two mutations, 9.8% had three mutations, and 2.1% (all group O strains) had four mutations. In order of decreasing frequency, the following mutations were identified in the protease gene: M36I (86.6%), L10I/V (26%), L63P (12.6%), K20M/R (11.2%), V77I (5.6%), A71V (2.8%), L33F (0.7%), and M46I (0.7%). R211K, an accessory mutation associated with NRTI resistance, was also observed in 43.6% of the samples. Phenotypic and clinical studies are now required to determine whether multidrug-resistant viruses emerge more rapidly during antiretroviral therapy when minor resistance-conferring mutations are present before treatment initiation.

scholarly journals Construction of a Human Immunodeficiency Virus Type 1 (HIV-1) Library Containing Random Combinations of Amino Acid Substitutions in the HIV-1 Protease due to Resistance by Protease Inhibitors

2002 ◽  
Vol 76 (6) ◽  
pp. 3031-3037 ◽  
Author(s):  
Keisuke Yusa ◽  
Wei Song ◽  
Matthias Bartelmann ◽  
Shinji Harada
Keyword(s):  
Human Immunodeficiency Virus ◽  
Amino Acid ◽  
Protease Inhibitors ◽  
Human Immunodeficiency Virus Type ◽  
Amino Acid Substitutions ◽  
In Vitro System ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) heterogeneity contributes to the emergence of drug-resistant virus, escape from host defense systems, and/or conversion of the cellular tropism. To establish an in vitro system to address a heterogeneous virus population, we constructed a library of HIV-1 molecular clones containing a set of random combinations of zero to 11 amino acid substitutions associated with resistance to protease inhibitors by the HIV-1 protease. The complexity (2.1 × 105) of the HIV-1 library pNG-PRL was large enough to cover all of the possible combinations of zero to 11 amino acid substitutions (a total of 4,096 substitutions possible). The T-cell line MT-2 was infected with the HIV-1 library, and resistant viruses were selected after treatment by the protease inhibitor ritonavir (0.03 to 0.30 μM). The viruses that contained three to eight amino acid substitutions could be selected within 2 weeks. These results demonstrate that this HIV-1 library could serve as an alternative in vitro system to analyze the emergence of drug resistance and to evaluate the antiviral activity of novel compounds against multidrug-resistant viruses.

Impact of frequent natural polymorphisms at the protease gene on the in vitro susceptibility to protease inhibitors in HIV-1 non-B subtypes

2004 ◽  
Vol 31 (3) ◽  
pp. 215-220 ◽  
Author(s):  
Africa Holguı́n ◽  
Ellen Paxinos ◽  
Kurt Hertogs ◽  
Chad Womac ◽  
Vincent Soriano
Keyword(s):  
Protease Inhibitors ◽  
Protease Gene ◽  
In Vitro Susceptibility ◽  
Hiv 1

scholarly journals Highly prevalent Russian HIV-1 V3-loop sequence variants are susceptible to maraviroc

2021 ◽  
Vol 29 ◽  
pp. 204020662110251
Author(s):  
ME Lewis ◽  
B Jubb ◽  
P Simpson ◽  
A Lopatukhin ◽  
D Kireev ◽  
...  
Keyword(s):  
Amino Acid ◽  
Virologic Failure ◽  
Natural Resistance ◽  
V3 Loop ◽  
Amino Acid Residues ◽  
Subtype B ◽  
Loop Sequence ◽  
Hiv 1 ◽  
Amino Acid Variants

Introduction Maraviroc inhibits CCR5-tropic HIV-1 across different subtypes in vitro and has demonstrated efficacy in clinical trials. V3-loop amino acid variants observed in individual maraviroc-resistant viruses have not been found to be predictive of reduced susceptibility. Sequence-database searches have demonstrated that approximately 7.3% of viruses naturally encode these variants, raising concerns regarding potential pre-existing resistance. A study from Russia reported that combinations of these same amino acids are present in the V3 loops of the Russian variant subtype A (IDU-A, now A6) with a much greater prevalence (range: 74.4%–92.3%) depending on the combination. However, these studies and database searches did not include phenotypic evaluation. Methods Sixteen Russian HIV-1 isolates (including sub-subtype A6 viruses) were assessed for V3 loop sequence and phenotypic susceptibility to maraviroc. Results All 12 of the A6 viruses and 2/4 subtype B isolates encoded V3-loop variants that have previously been identified in individual virus isolates with reduced susceptibility to maraviroc. However, despite the prevalence of these V3-loop amino acid variants among the tested viruses, phenotypic sensitivity to maraviroc was observed in all instances. Similarly, reduced susceptibility to maraviroc was not found in virus from participants who experienced virologic failure in a clinical study of maraviroc in Russia (A4001101, [NCT01275625]). Discussion Altogether, these data confirm that the presence of individual or combinations of V3-loop amino acid residues in sub-subtype A6 viruses alone does not predict natural resistance to maraviroc and that V3-loop genotype analysis of R5 virus prior to treatment is not helpful in predicting clinical outcome.

scholarly journals Effect of Natural Polymorphisms in the HIV-1 CRF02_AG Protease on Protease Inhibitor Hypersusceptibility

2012 ◽  
Vol 56 (5) ◽  
pp. 2719-2725 ◽  
Author(s):  
André F. A. Santos ◽  
Denis M. Tebit ◽  
Matthew S. Lalonde ◽  
Ana B. Abecasis ◽  
Annette Ratcliff ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Treatment Outcome ◽  
Protease Inhibitor ◽  
Protease Inhibitors ◽  
Antiretroviral Drugs ◽  
Response To Treatment ◽  
Resistance Mutations ◽  
Subtype B ◽  
Direct Head ◽  
Hiv 1

ABSTRACTHypersusceptibility (HS) to inhibition by different antiretroviral drugs (ARVs) among diverse HIV-infected individuals may be a misnomer because clinical response to treatment is evaluated in relation to subtype B infections while drug susceptibility of the infecting virus, regardless of subtype, is compared to a subtype B HIV-1 laboratory strain (NL4-3 or IIIB). Mounting evidence suggests that HS to different ARVs may result in better treatment outcome just as drug resistance leads to treatment failure. We have identified key amino acid polymorphisms in the protease coding region of a non-B HIV-1 subtype linked to protease inhibitor HS, namely, 17E and 64M in CRF02_AG. These HS-linked polymorphisms were introduced in the BD6-15 CRF02_AG molecular clone and tested for inhibition using a panel of protease inhibitors. In general, suspected HS-linked polymorphisms did increase susceptibility to specific protease inhibitors such as amprenavir and atazanavir, but the combination of the 17E/64M polymorphisms showed greater HS. These two mutations were found at low frequencies but linked in a sequence database of over 700 protease sequences of CRF02_AG. In direct head-to-head virus competitions, CRF02_AG harboring the 17E/64M polymorphisms also had higher replicative fitness than did the 17E or the 64M polymorphism in the CFR02_AG clone. These findings suggest that subtype-specific, linked polymorphisms can result in hypersusceptibility to ARVs. Considering the potential benefit of HS to treatment outcome, screening for potential HS-linked polymorphisms as well as preexisting drug resistance mutations in treatment-naïve patients may guide the choice of ARVs for the best treatment outcome.

scholarly journals Specificity of the HIV-1 Protease on Substrates Representing the Cleavage Site in the Proximal Zinc-Finger of HIV-1 Nucleocapsid Protein

Viruses ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 1092
Author(s):  
János András Mótyán ◽  
Márió Miczi ◽  
Stephen Oroszlan ◽  
József Tőzsér
Keyword(s):  
Amino Acid ◽  
Zinc Finger ◽  
Cleavage Site ◽  
Potential Role ◽  
Binding Mode ◽  
Interaction Energies ◽  
Vitro Assay ◽  
Hiv 1

To explore the sequence context-dependent nature of the human immunodeficiency virus type 1 (HIV-1) protease’s specificity and to provide a rationale for viral mutagenesis to study the potential role of the nucleocapsid (NC) processing in HIV-1 replication, synthetic oligopeptide substrates representing the wild-type and modified versions of the proximal cleavage site of HIV-1 NC were assayed as substrates of the HIV-1 protease (PR). The S1′ substrate binding site of HIV-1 PR was studied by an in vitro assay using KIVKCF↓NCGK decapeptides having amino acid substitutions of N17 residue of the cleavage site of the first zinc-finger domain, and in silico calculations were also performed to investigate amino acid preferences of S1′ site. Second site substitutions have also been designed to produce “revertant” substrates and convert a non-hydrolysable sequence (having glycine in place of N17) to a substrate. The specificity constants obtained for peptides containing non-charged P1′ substitutions correlated well with the residue volume, while the correlation with the calculated interaction energies showed the importance of hydrophobicity: interaction energies with polar residues were related to substantially lower specificity constants. Cleavable “revertants” showed one residue shift of cleavage position due to an alternative productive binding mode, and surprisingly, a double cleavage of a substrate was also observed. The results revealed the importance of alternative binding possibilities of substrates into the HIV-1 PR. The introduction of the “revertant” mutations into infectious virus clones may provide further insights into the potential role of NC processing in the early phase of the viral life-cycle.

scholarly journals Multiple Mutations in the Human Immunodeficiency Virus Protease Gene Are Responsible for Decreased Susceptibility to Protease Inhibitors

1995 ◽  
Vol 6 (2) ◽  
pp. 80-88 ◽  
Author(s):  
R. W. King ◽  
S. Garber ◽  
D. L. Winslow ◽  
C. Reid ◽  
L. T. Bacheler ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Protease Inhibitors ◽  
Clinical Isolate ◽  
Protease Gene ◽  
Human Immunodeficiency Virus Protease ◽  
Fold Reduction ◽  
Antiretroviral Drug ◽  
Immunodeficiency Virus ◽  
Hiv 1

The protease (PR) of the human immunodeficiency virus (HIV) is essential for replication of the virus, and accordingly has become an attractive target for the development of an antiretroviral drug. We have previously reported that passage of HIV-1 in the presence of increasing concentrations of the C-2 symmetrical, linear diol P9941 resulted in the isolation of virus with a valine-to-alanine change at position 82 (V82A) of the PR, and reduced sensitivity to certain PR inhibitors. In this study, we passaged four different variants of HIV-1 in increasing concentrations of XM323, and isolated variants with reduced sensitivity to inhibitors of PR. Twenty-three passages of HIV-1 (RF) in the presence of XM323 resulted in a variant that exhibited an approximately 100-fold reduction in susceptibility to XM323 and that contained V82F and I84V changes. When two other viruses, HIV-1 (RF41D2) and HIV-1(RF41E4), previously derived from HIV-1 (RF) by passage in the presence of P9941, were passaged in the presence of XM323, variants with V82A/L97V and M46L/V82A/L97V changes, respectively, were obtained. The M46L/V82A/L97V variant showed a 6-fold reduction in sensitivity to XM323, whereas the susceptibility of the V82A/L97V mutant remained unchanged. Seventeen passages of a clinical isolate of HIV-1, HIV-1 (Pat.E), in the presence of XM323 produced a V82F/L97V mutant with an approximately 9-fold reduction in sensitivity to XM323.

Advancing RNA Virus Discovery and Biology with Whole Genome Sequencing

2021 ◽  
Author(s):  
◽  
Mariah Taylor ◽  
Keyword(s):  
Genetic Variation ◽  
Amino Acid ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Rna Virus ◽  
Animal Health ◽  
Functional Domains ◽  
Whole Genome ◽  
Coding Region

Two RNA virus families that pose a threat to human and animal health are Hantaviridae and Coronaviridae. These RNA viruses which originate in wildlife continue and will continue to cause disease, and hence, it is critical that scientific research define the mechanisms as to how these viruses spillover and adapt to new hosts to become endemic. One gap in our ability to define these mechanisms is the lack of whole genome sequences for many of these viruses. To address this specific gap, I developed a versatile amplicon-based whole-genome sequencing (WGS) approach to identify viral genomes of hantaviruses and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) within reservoir and spillover hosts. In my research studies, I used the amplicon-based WGS approach to define the genetic plasticity of viral RNA within pathogenic and nonpathogenic hantavirus species. The standing genetic variation of Andes orthohantavirus and Prospect Hill orthohantavirus was mapped out and amino acid changes occurring outside of functional domains were identified within the nucleocapsid and glycoprotein. I observed several amino acid changes in functional domains of the RNA-dependent RNA polymerase, as well as single nucleotide polymorphisms (SNPs) within the 3’ non-coding region (NCR) of the S-segment. To identify whether virus adaptation would occur within the S- and L-segments we attempted to adapt hantaviruses in vitro in a spillover host model through passaging experiments. In early passages we identified few mutations in the M-segment with the majority being identified in the S-segment 3’ NCR and the L-segment. This work suggests that hantavirus adaptation occurs in the S- and L-segments although the effect of these mutants on pathology is yet to be determined. While sequencing laboratory isolates is easily accomplished, sequencing low concentrations of virus within the reservoir is a formidable task. I further translated our amplicon-based WGS approach into a pan-oligonucleotide amplicon-based WGS approach to sequence hantavirus vRNA and mRNA from reservoir and spillover hosts in Ukraine. This approach successfully identified a novel Puumala orthohantavirus (PUUV) strain in Ukraine and using Bayesian phylogenetics we found this strain to be associated with the PUUV Latvian lineage. Early during the SARS-CoV-2 pandemic, I applied the knowledge gained in the hantavirus WGS efforts to sequencing of SARS-CoV-2 from nasopharyngeal swabs collected in April 2020. The genetic diversity of 45 SARS-CoV-2 isolates was evaluated with the methods I developed. We identified D614G, a notable mutation known for increasing transmission, in over 90% of our isolates. Two major lineages distinguish SARS-CoV-2 variants worldwide, lineages A and B. While most of our isolates were found within B lineage, we also identified one isolate within lineage A. We performed in vitro work which confirmed A lineage isolates as having poor replication in the trachea as compared to the nasal cavity. Five of these isolates presented a unique array of mutations which were assessed in the keratin 18 human angiotensin-converting enzyme 2 (K18-hACE2) mouse model for its response immunologically and pathogenically. We identified a distinction of pathogenesis between the A and B lineages with emphysema being common amongst A lineage isolates. Additionally, we discovered a small cohort of likely SNPs that defined the late induction of eosinophils during infection. In summary, this work will further define the dynamics of genetic variation and plasticity within virus populations that cause disease outbreaks and will allow a deeper understanding of the virus-host relationship.

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