scholarly journals In Vivo Validation of a Bioinformatics Based Tool to Identify Reduced Replication Capacity in HIV-1

2010 ◽  
Vol 4 (1) ◽  
pp. 225-232
Author(s):  
Christina M.R Kitchen ◽  
Paul Krogstad ◽  
Scott G Kitchen
Keyword(s):  
Drug Resistance ◽  
In Silico ◽  
Viral Fitness ◽  
Biological Assessment ◽  
Relative Fitness ◽  
Replication Capacity ◽  
Resistance Mutations ◽  
Hiv 1

Although antiretroviral drug resistance is common in treated HIV infected individuals, it is not a consistent indicator of HIV morbidity and mortality. To the contrary, HIV resistance-associated mutations may lead to changes in viral fitness that are beneficial to infected individuals. Using a bioinformatics-based model to assess the effects of numerous drug resistance mutations, we determined that the D30N mutation in HIV-1 protease had the largest decrease in replication capacity among known protease resistance mutations. To test thisin silicoresult in anin vivoenvironment, we constructed several drug-resistant mutant HIV-1 strains and compared their relative fitness utilizing the SCID-hu mouse model. We found HIV-1 containing the D30N mutation had a significant defectin vivo, showing impaired replication kinetics and a decreased ability to deplete CD4+ thymocytes, compared to the wild-type or virus without the D30N mutation. In comparison, virus containing the M184V mutation in reverse transcriptase, which shows decreased replication capacityin vitro, did not have an effect on viral fitnessin vivo. Thus, in this study we have verified anin silicobioinformatics result with a biological assessment to identify a unique mutation in HIV-1 that has a significant fitness defectin vivo.

scholarly journals Impaired Replication Capacity of Acute/Early Viruses in Persons Who Become HIV Controllers

2010 ◽  
Vol 84 (15) ◽  
pp. 7581-7591 ◽  
Author(s):  
Toshiyuki Miura ◽  
Zabrina L. Brumme ◽  
Mark A. Brockman ◽  
Pamela Rosato ◽  
Jennifer Sela ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Viral Replication ◽  
Acute Infection ◽  
Viral Fitness ◽  
Replication Capacity ◽  
Transmitted Drug Resistance ◽  
Resistance Mutations ◽  
Drug Resistance Mutations ◽  
Hiv Controllers ◽  
Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) controllers maintain viremia at <2,000 RNA copies/ml without antiretroviral therapy. Viruses from controllers with chronic infection were shown to exhibit impaired replication capacities, in part associated with escape mutations from cytotoxic-T-lymphocyte (CTL) responses. In contrast, little is known about viruses during acute/early infection in individuals who subsequently become HIV controllers. Here, we examine the viral replication capacities, HLA types, and virus sequences from 18 HIV-1 controllers identified during primary infection. g ag-protease chimeric viruses constructed using the earliest postinfection samples displayed significantly lower replication capacities than isolates from persons who failed to control viremia (P = 0.0003). Protective HLA class I alleles were not enriched in these early HIV controllers, but viral sequencing revealed a significantly higher prevalence of drug resistance mutations associated with impaired viral fitness in controllers than in noncontrollers (6/15 [40.0%] versus 10/80 [12.5%], P = 0.018). Moreover, of two HLA-B57-positive (B57+) controllers identified, both harbored, at the earliest time point tested, signature escape mutations within Gag that likewise impair viral replication capacity. Only five controllers did not express “protective” alleles or harbor viruses with drug resistance mutations; intriguingly, two of them displayed typical B57 signature mutations (T242N), suggesting the acquisition of attenuated viruses from B57+ donors. These data indicate that acute/early stage viruses from persons who become controllers have evidence of reduced replication capacity during the initial stages of infection which is likely associated with transmitted or acquired CTL escape mutations or transmitted drug resistance mutations. These data suggest that viral dynamics during acute infection have a major impact on HIV disease outcome.

scholarly journals Relative Replicative Fitness of Human Immunodeficiency Virus Type 1 Mutants Resistant to Enfuvirtide (T-20)

2004 ◽  
Vol 78 (9) ◽  
pp. 4628-4637 ◽  
Author(s):  
Jing Lu ◽  
Prakash Sista ◽  
Françoise Giguel ◽  
Michael Greenberg ◽  
Daniel R. Kuritzkes
Keyword(s):  
Human Immunodeficiency Virus ◽  
Relative Order ◽  
Wild Type ◽  
Resistance Mutations ◽  
Recombinant Viruses ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Resistance to enfuvirtide (ENF; T-20), a fusion inhibitor of human immunodeficiency virus type 1 (HIV-1), is conferred by mutations in the first heptad repeat of the gp41 ectodomain. The replicative fitness of recombinant viruses carrying ENF resistance mutations was studied in growth competition assays. ENF resistance mutations, selected in vitro or in vivo, were introduced into the env gene of HIV-1NL4-3 by site-directed mutagenesis and expressed in HIV-1 recombinants carrying sequence tags in nef. The doubling time of ENF-resistant viruses was highly correlated with decreasing ENF susceptibility (R 2 = 0.859; P < 0.001). Initial fitness experiments focused on mutants identified by in vitro selection in the presence of ENF (L. T. Rimsky, D. C. Shugars, and T. J. Matthews, J. Virol. 72:986-993, 1998). In the absence of drug, these mutants displayed reduced fitness compared to wild-type virus with a relative order of fitness of wild type > I37T > V38 M > D36S/V38 M; this order was reversed in the presence of ENF. Likewise, recombinant viruses carrying ENF resistance mutations selected in vivo displayed reduced fitness in the absence of ENF with a relative order of wild type > N42T > V38A > N42T/N43K ≈ N42T/N43S > V38A/N42D ≈ V38A/N42T. Fitness and ENF susceptibility were inversely correlated (r = −0.988; P < 0.001). Similar results were obtained with recombinants expressing molecularly cloned full-length env genes obtained from patient-derived HIV-1 isolates before and after ENF treatment. Further studies are needed to determine whether the reduced fitness of ENF-resistant viruses alters their pathogenicity in vivo.

scholarly journals Elucidation of the Molecular Mechanism Driving Duplication of the HIV-1 PTAP Late Domain

2015 ◽  
Vol 90 (2) ◽  
pp. 768-779 ◽  
Author(s):  
Angelica N. Martins ◽  
Abdul A. Waheed ◽  
Sherimay D. Ablan ◽  
Wei Huang ◽  
Alicia Newton ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Antiretroviral Therapy ◽  
Protease Inhibitor ◽  
Protease Inhibitors ◽  
Biochemical Analysis ◽  
Replication Capacity ◽  
Resistance Mutations ◽  
Cellular Machinery ◽  
Ptap Motif ◽  
Hiv 1

ABSTRACTHIV-1 uses cellular machinery to bud from infected cells. This cellular machinery is comprised of several multiprotein complexes known as endosomal sorting complexes required for transport (ESCRTs). A conserved late domain motif, Pro-Thr-Ala-Pro (PTAP), located in the p6 region of Gag (p6Gag), plays a central role in ESCRT recruitment to the site of virus budding. Previous studies have demonstrated that PTAP duplications are selected in HIV-1-infected patients during antiretroviral therapy; however, the consequences of these duplications for HIV-1 biology and drug resistance are unclear. To address these questions, we constructed viruses carrying a patient-derived PTAP duplication with and without drug resistance mutations in the viral protease. We evaluated the effect of the PTAP duplication on viral release efficiency, viral infectivity, replication capacity, drug susceptibility, and Gag processing. In the presence of protease inhibitors, we observed that the PTAP duplication in p6Gagsignificantly increased the infectivity and replication capacity of the virus compared to those of viruses bearing only resistance mutations in protease. Our biochemical analysis showed that the PTAP duplication, in combination with mutations in protease, enhances processing between the nucleocapsid and p6 domains of Gag, resulting in more complete Gag cleavage in the presence of protease inhibitors. These results demonstrate that duplication of the PTAP motif in p6Gagconfers a selective advantage in viral replication by increasing Gag processing efficiency in the context of protease inhibitor treatment, thereby enhancing the drug resistance of the virus. These findings highlight the interconnected role of PTAP duplications and protease mutations in the development of resistance to antiretroviral therapy.IMPORTANCEResistance to current drug therapy limits treatment options in many HIV-1-infected patients. Duplications in a Pro-Thr-Ala-Pro (PTAP) motif in the p6 domain of Gag are frequently observed in viruses derived from patients on protease inhibitor (PI) therapy. However, the reason that these duplications arise and their consequences for virus replication remain to be established. In this study, we examined the effect of PTAP duplication on PI resistance in the context of wild-type protease or protease bearing PI resistance mutations. We observe that PTAP duplication markedly enhances resistance to a panel of PIs. Biochemical analysis reveals that the PTAP duplication reverses a Gag processing defect imposed by the PI resistance mutations in the context of PI treatment. The results provide a long-sought explanation for why PTAP duplications arise in PI-treated patients.

scholarly journals Mutations in HIV-1gagandpolCompensate for the Loss of Viral Fitness Caused by a Highly Mutated Protease

2012 ◽  
Vol 56 (8) ◽  
pp. 4320-4330 ◽  
Author(s):  
Milan Kožíšek ◽  
Sandra Henke ◽  
Klára Grantz Šašková ◽  
Graeme Brendon Jacobs ◽  
Anita Schuch ◽  
...  
Keyword(s):  
Highly Active Antiretroviral Therapy ◽  
Viral Fitness ◽  
Drug Pressure ◽  
Viral Loads ◽  
Highly Active ◽  
Subtype B ◽  
Hiv 1

ABSTRACTDuring the last few decades, the treatment of HIV-infected patients by highly active antiretroviral therapy, including protease inhibitors (PIs), has become standard. Here, we present results of analysis of a patient-derived, multiresistant HIV-1 CRF02_AG recombinant strain with a highly mutated protease (PR) coding sequence, where up to 19 coding mutations have accumulated in the PR. The results of biochemical analysisin vitroshowed that the patient-derived PR is highly resistant to most of the currently used PIs and that it also exhibits very poor catalytic activity. Determination of the crystal structure revealed prominent changes in the flap elbow region and S1/S1′ active site subsites. While viral loads in the patient were found to be high, the insertion of the patient-derived PR into a HIV-1 subtype B backbone resulted in reduction of infectivity by 3 orders of magnitude. Fitness compensation was not achieved by elevated polymerase (Pol) expression, but the introduction of patient-derivedgagandpolsequences in a CRF02_AG backbone rescued viral infectivity to near wild-type (wt) levels. The mutations that accumulated in the vicinity of the processing sites spanning the p2/NC, NC/p1, and p6pol/PR proteins lead to much more efficient hydrolysis of corresponding peptides by patient-derived PR in comparison to the wt enzyme. This indicates a very efficient coevolution of enzyme and substrate maintaining high viral loadsin vivounder constant drug pressure.

scholarly journals Improved HIV-1 drug resistance mutation prediction using quasispecies reconstruction supported analysis

2021 ◽  
Author(s):  
Jyoti Sutar ◽  
Shilpa Bhowmick ◽  
Varsha Padwal ◽  
Vidya Nagar ◽  
Priya Patil ◽  
...  
Keyword(s):  
Drug Resistance ◽  
High Throughput Sequencing ◽  
Resistance Mutation ◽  
Detection Sensitivity ◽  
Treatment As Prevention ◽  
Reconstruction Algorithms ◽  
Resistance Mutations ◽  
Proviral Dna ◽  
Hiv 1

Accurate and sensitive approaches to detect HIV-1 drug resistance mutations (DRMs) are indispensable for the paradigm of treatment as prevention. While HIV-1 proviral DNA allows sensitive high throughput sequencing (HTS)-based DRM detection, its applicability is limited by presence of defective genomes. This study demonstrates application of quasispecies reconstruction algorithms (QRAs) to improve DRM detection sensitivity from proviral DNA. A robust benchmarking of 5 QRAs was performed with 2 distinct experimental control-datasets including a stringent, novel control: DCPM, simulating in-vivo variant distribution (0.08%-86.5%). Selected QRA was further evaluated for its ability to differentiate DRMs from hypermutated sequences using an in-silico control. PredictHaplo outperformed all others in terms of precision and was selected for further analysis. Near full-genome HTS was performed on proviral DNA from 20 HIV-1C infected individuals, at different stages of ART, from Mumbai, India. DRM detection was performed through residue-wise variation analysis and implementation of QRAs. Both analyses were highly concordant for DRM frequencies >10% (spearman r=0.91, p<0.0001). Phylogenetic association in HTS datasets with shared transmission history could also be demonstrated by PredictHaplo. This study highlights utility of QRAs as an adjunct to traditional residue-wise variation-based DRM detection leading to optimal personalized ART as well as better disease management.

scholarly journals Influenza H3 and H1 hemagglutinins have different genetic barriers for resistance to broadly neutralizing stem antibodies

2019 ◽  
Author(s):  
Nicholas C. Wu ◽  
Andrew J. Thompson ◽  
Juhye M. Lee ◽  
Wen Su ◽  
Britni M. Arlian ◽  
...  
Keyword(s):  
Influenza Vaccine ◽  
Neutralizing Antibodies ◽  
Viral Fitness ◽  
Resistance Mutations ◽  
Genetic Barrier ◽  
Influenza Hemagglutinin ◽  
Genetic Barriers ◽  
Universal Influenza Vaccine

ABSTRACTIn the past decade, the discovery and characterization of broadly neutralizing antibodies (bnAbs) to the highly conserved stem region of influenza hemagglutinin (HA) have provided valuable insights for development of a universal influenza vaccine. However, the genetic barrier for resistance to stem bnAbs has not been thoroughly evaluated. Here, we performed a series of deep mutational scanning experiments to probe for resistance mutations. We found that the genetic barrier to resistance to stem bnAbs is generally very low for the H3 subtype but substantially higher for the H1 subtype. Several resistance mutations in H3 cannot be neutralized by stem bnAbs at the highest concentration tested, do not reduce in vitro viral fitness and in vivo pathogenicity, and are often present in circulating strains as minor variants. Thus, H3 HAs have a higher propensity than H1 HAs to escape major stem bnAbs and creates a potential challenge in the development of a bona fide universal influenza vaccine.ONE SENTENCE SUMMARYAcquisition of resistance by influenza virus to broadly neutralizing hemagglutinin stem antibodies varies tremendously depending on subtype.

scholarly journals Primary HIV-1 resistance: Persistence of transmitted drug resistance mutations

2012 ◽  
Vol 64 (4) ◽  
pp. 1301-1309 ◽  
Author(s):  
Valentina Nikolic ◽  
D. Salemovic ◽  
Dj. Jevtovic ◽  
I. Pesic-Pavlovic ◽  
S. Zerjav ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Antiretroviral Therapy ◽  
Viral Fitness ◽  
Transmitted Drug Resistance ◽  
Resistance Mutations ◽  
High Level ◽  
Hiv 1 ◽  
Level Resistance

Transmitted drug resistance (TDR) is one of the consequences of the high variability of HIV-1. The widespread use of antiretroviral therapy for the treatment of HIV-1 infection results in a large circulating pool of resistant virus variants. It is known that TDR mutations can persist for extended periods and may pose an important problem to the overall success of antiretroviral therapy. Factors that determine the duration of continuous persistence of resistance-associated mutations are the number and type of these mutations and their impact on viral fitness. Here we describe the follow-up of a case study of prolonged persistence of resistance-associated mutations, namely RT mutations Q151M, K65KR and Y181C conferring an intermediate-to-high level resistance to multiple NRTIs and NNRTIs that lasted for seven years. The infection was caused by subtype G virus.

Endophytic Fungi Penicillium species BCt Phytochemicals Inhibit Replication of Enzymes of Human Immuno-deficiency Virus 1 in in vitro and in silico Studies

2021 ◽  
Vol 14 (5) ◽  
pp. 5624-5633
Author(s):  
Govindappa M ◽  
Channabasava ◽  
Ritu Pawar ◽  
Chandrasekhar Srinivasa ◽  
Chandan Shivamallu ◽  
...  
Keyword(s):  
Amino Acids ◽  
Endophytic Fungi ◽  
In Silico ◽  
Methanol Extract ◽  
Penicillium Species ◽  
Ms Analysis ◽  
In Silico Studies ◽  
Hiv 1

The present investigation was aimed to know the coumarins in the methanol extract of endophytic fungi, Penicillium species BCt isolated from Calophyllum tomentosum bark tissues using qualitative and GC-MS analysis. The endophytic extract was evaluated for anti-HIV activity on three replicating enzymes in vitro and in silico. The methanol extract of Penicillium species confirmed the presence of coumarins in four qualitative methods and yielded four different types of coumarins in GC-MS. In GC-MS analysis, totally seven different phytochemicals were identified based on retention time and compared with available library data. The four coumarins are coumarin (2H-1-benzopyran-2-one), coumaric acid (3-benzofuran-carboxylic acid), hynecromone (coumarin 4), 4-hydroxy-9-(3-methyl-2-butyl) furo (3,2-g) chloronen-7-one) and other three are common phytochemicals. The HIV-1 RT (98) was strongly inhibited by the endophytic fungal extract compared to integrase (118) and protease (158) in vitro analysis. Highest inhibition of integrase was observed with coumarilic acid (-17.62) when attached to Glu-35, Asn-38, Ser-39 amino acids. The protease was inhibited strongly by hymecromone (-16.39) when attached to amino acids of Val-77, Glu-34, Pro-79, Gly-78. The inhibition of RT was observed with coumarilic acid by attaching to Ala-445, Arg-567, Asp-456, Glu-478, Ser-499, Asn-474 (-23.54) significantly. Based on above results, the endophytic fungal coumarins have the ability to inhibit the three replicating enzymes of HIV-1 significantly. The in-silico results are evidence for how coumarins inhibiting the HIV replicating proteins by binding at specific amino acids. The results will help to understand how and where phytochemicals bind to target proteins to inhibit their action and it may help to identification of drugs to treat HIV. To validate our results, the in vivo research is needed.   

scholarly journals Recapitulation in Saccharomyces cerevisiae of Cytochrome b Mutations Conferring Resistance to Atovaquone in Pneumocystis jiroveci

2003 ◽  
Vol 47 (9) ◽  
pp. 2725-2731 ◽  
Author(s):  
Philip Hill ◽  
Jacques Kessl ◽  
Nicholas Fisher ◽  
Steven Meshnick ◽  
Bernard L. Trumpower ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Drug Resistance ◽  
Efflux Pump ◽  
Pathogenic Fungus ◽  
Acquired Resistance ◽  
Resistance Mutations ◽  
Pneumocystis Jiroveci ◽  
Pump System

ABSTRACT Pneumocystis jiroveci (human-derived P. carinii) is an opportunistic pathogenic fungus which causes pneumonia and is life-threatening in immunocompromised individuals. Spontaneously acquired resistance to atovaquone, a hydroxynaphthoquinone that is used to treat P. jiroveci infections, was linked to mutations in the mitochondrially encoded cytochrome b gene. Because P. jiroveci cannot be easily cultivated, we have developed Saccharomyces cerevisiae as an alternative system to study atovaquone resistance mutations. In this work, we introduced seven mutations linked with atovaquone resistance in P. jiroveci into the S. cerevisiae cytochrome b gene. The effects of the mutations on the respiratory function and on the sensitivity to the inhibitor were then characterized. Six of the reported mutations lowered the sensitivity of the S. cerevisiae bc 1 complex to atovaquone, while one mutation had no effect on the drug resistance. These results were confirmed by monitoring the in vivo resistance of S. cerevisiae mutants which carried both the cytochrome b mutations and a deletion of the ABC transporter genes, allowing the drug to bypass the weakened efflux pump system. S. cerevisiae thus provides an easy-to-use system to characterize in vivo and in vitro cytochrome b mutations reported in pathogens and to assess their role in drug resistance.

scholarly journals In VitroResistance Profile of the Candidate HIV-1 Microbicide Drug Dapivirine

2011 ◽  
Vol 56 (2) ◽  
pp. 751-756 ◽  
Author(s):  
Susan M. Schader ◽  
Maureen Oliveira ◽  
Ruxandra-Ilinca Ibanescu ◽  
Daniela Moisi ◽  
Susan P. Colby-Germinario ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Reverse Transcriptase ◽  
Sexual Transmission ◽  
Transcriptase Inhibitor ◽  
Resistance Mutations ◽  
Drug Resistance Mutations ◽  
Development Of Resistance ◽  
Sexual Transmission Of Hiv ◽  
Hiv 1

ABSTRACTAntiretroviral-based microbicides may offer a means to reduce the sexual transmission of HIV-1. Suboptimal use of a microbicide may, however, lead to the development of drug resistance in users that are already, or become, infected with HIV-1. In such cases, the efficacy of treatments may be compromised since the same (or similar) antiretrovirals used in treatments are being developed as microbicides. To help predict which drug resistance mutations may develop in the context of suboptimal use, HIV-1 primary isolates of different subtypes and different baseline resistance profiles were used to infect primary cellsin vitroin the presence of increasing suboptimal concentrations of the two candidate microbicide antiretrovirals dapivirine (DAP) and tenofovir (TFV) alone or in combination. Infections were ongoing for 25 weeks, after which reverse transcriptase genotypes were determined and scrutinized for the presence of any clinically recognized reverse transcriptase drug resistance mutations. Results indicated that suboptimal concentrations of DAP alone facilitated the emergence of common nonnucleoside reverse transcriptase inhibitor resistance mutations, while suboptimal concentrations of DAP plus TFV gave rise to fewer mutations. Suboptimal concentrations of TFV alone did not frequently result in the development of resistance mutations. Sensitivity evaluations for stavudine (d4T), nevirapine (NVP), and lamivudine (3TC) revealed that the selection of resistance as a consequence of suboptimal concentrations of DAP may compromise the potential for NVP to be used in treatment, a finding of potential relevance in developing countries.

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