scholarly journals HIV-1 Protease Evolvability Is Affected by Synonymous Nucleotide Recoding

2018 ◽  
Vol 92 (16) ◽  
Author(s):  
Maria Nevot ◽  
Ana Jordan-Paiz ◽  
Glòria Martrus ◽  
Cristina Andrés ◽  
Damir García-Cehic ◽  
...  
Keyword(s):  
Sequence Space ◽  
Large Scale ◽  
Mononuclear Cells ◽  
Selective Pressure ◽  
Population Diversity ◽  
Wild Type ◽  
Evolutionary Trajectory ◽  
Synonymous Mutations ◽  
Mutant Spectrum ◽  
Hiv 1

ABSTRACTOne unexplored aspect of HIV-1 genetic architecture is how codon choice influences population diversity and evolvability. Here we compared the levels of development of HIV-1 resistance to protease inhibitors (PIs) between wild-type (WT) virus and a synthetic virus (MAX) carrying a codon-pair-reengineered protease sequence including 38 (13%) synonymous mutations. The WT and MAX viruses showed indistinguishable replication in MT-4 cells or peripheral blood mononuclear cells (PBMCs). Both viruses were subjected to serial passages in MT-4 cells, with selective pressure from the PIs atazanavir (ATV) and darunavir (DRV). After 32 successive passages, both the WT and MAX viruses developed phenotypic resistance to PIs (50% inhibitory concentrations [IC50s] of 14.6 ± 5.3 and 21.2 ± 9 nM, respectively, for ATV and 5.9 ± 1.0 and 9.3 ± 1.9, respectively, for DRV). Ultradeep sequence clonal analysis revealed that both viruses harbored previously described mutations conferring resistance to ATV and DRV. However, the WT and MAX virus proteases showed different resistance variant repertoires, with the G16E and V77I substitutions observed only in the WT and the L33F, S37P, G48L, Q58E/K, and L89I substitutions detected only in the MAX virus. Remarkably, the G48L and L89I substitutions are rarely foundin vivoin PI-treated patients. The MAX virus showed significantly higher nucleotide and amino acid diversity of the propagated viruses with and without PIs (P< 0.0001), suggesting a higher selective pressure for change in this recoded virus. Our results indicate that the HIV-1 protease position in sequence space delineates the evolution of its mutant spectrum. Nevertheless, the investigated synonymously recoded variant showed mutational robustness and evolvability similar to those of the WT virus.IMPORTANCELarge-scale synonymous recoding of virus genomes is a new tool for exploring various aspects of virus biology. Synonymous virus genome recoding can be used to investigate how a virus's position in sequence space defines its mutant spectrum, evolutionary trajectory, and pathogenesis. In this study, we evaluated how synonymous recoding of the human immunodeficiency virus type 1 (HIV-1) protease affects the development of protease inhibitor (PI) resistance. HIV-1 protease is a main target of current antiretroviral therapies. Our present results demonstrate that the wild-type (WT) virus and a virus with recoded protease exhibited different patterns of resistance mutations after PI treatment. Nevertheless, the developed PI resistance phenotypes were indistinguishable between the recoded virus and the WT virus, suggesting that the HIV-1 strain with synonymously recoded protease and the WT virus are equally robust and evolvable.

scholarly journals HIV-1 Protease Evolvability is Affected by Synonymous Nucleotide Recoding

2018 ◽  
Author(s):  
Maria Nevot ◽  
Ana Jordan-Paiz ◽  
Glòria Martrus ◽  
Cristina Andrés ◽  
Damir García-Cehic ◽  
...  
Keyword(s):  
Sequence Space ◽  
Large Scale ◽  
Selective Pressure ◽  
Population Diversity ◽  
Wild Type ◽  
Resistance Mutations ◽  
Mutational Robustness ◽  
Evolutionary Trajectory ◽  
Synonymous Mutations ◽  
Hiv 1

ABSTRACTOne unexplored aspect of HIV-1 genetic architecture is how codon choice influences population diversity and evolvability. Here we compared the development of HIV-1 resistance to protease inhibitors (PIs) between wild-type (WT) virus and a synthetic virus (MAX) carrying a codon-pair re-engineered protease sequence including 38 (13%) synonymous mutations. WT and MAX viruses showed indistinguishable replication in MT-4 cells or PBMCs. Both viruses were subjected to serial passages in MT-4 cells with selective pressure from the PIs atazanavir (ATV) and darunavir (DRV). After 32 successive passages, both the WT and MAX viruses developed phenotypic resistance to PIs (IC5014.6 ± 5.3 and 21.2 ± 9 nM for ATV, and 5. 9 ± 1.0 and 9.3 ± 1.9 for DRV, respectively). Ultra-deep sequence clonal analysis revealed that both viruses harbored previously described resistance mutations to ATV and DRV. However, the WT and MAX virus proteases showed different resistance variant repertoires, with the G16E and V77I substitutions observed only in WT, and the L33F, S37P, G48L, Q58E/K, and L89I substitutions detected only in MAX. Remarkably, G48L and L89I are rarely foundin vivoin PI-treated patients. The MAX virus showed significantly higher nucleotide and amino acid diversity of the propagated viruses with and without PIs (P< 0.0001), suggesting higher selective pressure for change in this recoded virus. Our results indicate that HIV-1 protease position in sequence space delineates the evolution of its mutant spectra. Nevertheless, the investigated synonymously recoded variant showed mutational robustness and evolvability similar to the WT virus.IMPORTANCELarge-scale synonymous recoding of virus genomes is a new tool for exploring various aspects of virus biology. Synonymous virus genome recoding can be used to investigate how a virus’s position in sequence space defines its mutant spectrum, evolutionary trajectory, and pathogenesis. In this study, we evaluated how synonymous recoding of the human immunodeficiency virus type 1 (HIV-1) protease impacts the development of protease inhibitor (PI) resistance. HIV-1 protease is a main target of current antiretroviral therapies. Our present results demonstrate that the wild-type (WT) virus and the virus with the recoded protease exhibited different patterns of resistance mutations after PI treatment. Nevertheless, the developed PI resistance phenotype was indistinguishable between the recoded virus and the WT virus, suggesting that the synonymously recoded protease HIV-1 and the WT protease virus were equally robust and evolvable.

scholarly journals The HIV-1 Central Polypurine Tract Functions as a Second Line of Defense against APOBEC3G/F

2010 ◽  
Vol 84 (22) ◽  
pp. 11981-11993 ◽  
Author(s):  
Chunling Hu ◽  
Dyana T. Saenz ◽  
Hind J. Fadel ◽  
William Walker ◽  
Mary Peretz ◽  
...  
Keyword(s):  
Mononuclear Cells ◽  
Human Peripheral Blood ◽  
Target Cells ◽  
Second Line ◽  
Wild Type ◽  
Single Cycle ◽  
Polypurine Tract ◽  
Producer Cell ◽  
Central Polypurine Tract ◽  
Hiv 1

ABSTRACT HIV-1 and certain other retroviruses initiate plus-strand synthesis in the center of the genome as well as at the standard retroviral 3′ polypurine tract. This peculiarity of reverse transcription results in a central DNA “flap” structure that has been of controversial functional significance. We mutated both HIV-1 flap-generating elements, the central polypurine tract (cPPT) and the central termination sequence (CTS). To avoid an ambiguity of previous studies, we did so without affecting integrase coding. DNA flap formation was disrupted but single-cycle infection was unaffected in all target cells tested, regardless of cell cycle status. Spreading HIV-1 infection was also normal in most T cell lines, and flap mutant viruses replicated equivalently to the wild type in nondividing cells, including macrophages. However, spreading infection of flap mutant HIV-1 was impaired in non-vif-permissive cells (HuT78, H9, and primary human peripheral blood mononuclear cells [PBMCs]), suggesting APOBEC3G (A3G) restriction. Single-cycle infections confirmed that vif-intact flap mutant HIV-1 is restricted by producer cell A3G/F. Combining the Δvif and cPPT-CTS mutations increased A3G restriction synergistically. Moreover, RNA interference knockdown of A3G in HuT78 cells released the block to flap mutant HIV-1 replication. Flap mutant HIV-1 also accrued markedly increased A3G-mediated G→A hypermutation compared to that of wild-type HIV-1 (a full log10 in the 0.36 kb downstream of the mutant cPPT). We suggest that the triple-stranded DNA structure, the flap, is not the consequential outcome. The salient functional feature is central plus-strand initiation, which functions as a second line of defense against single-stranded DNA editing by A3 proteins that survive producer cell degradation by Vif.

scholarly journals Antiviral Activities and Cellular Toxicities of Modified 2′,3′-Dideoxy-2′,3′-Didehydrocytidine Analogues

2002 ◽  
Vol 46 (12) ◽  
pp. 3854-3860 ◽  
Author(s):  
Lieven J. Stuyver ◽  
Stefania Lostia ◽  
Marjorie Adams ◽  
Judy S. Mathew ◽  
Balakrishna S. Pai ◽  
...  
Keyword(s):  
Antiviral Activity ◽  
Mononuclear Cells ◽  
Human Peripheral Blood ◽  
Strain Range ◽  
Nucleoside Analogs ◽  
Wild Type ◽  
Peripheral Blood Mononuclear ◽  
Diarrhea Virus ◽  
Antiviral Activities ◽  
Hiv 1

ABSTRACT The antiviral efficacies and cytotoxicities of 2′,3′- and 4′-substituted 2′,3′-didehydro-2′,3′-dideoxycytidine analogs were evaluated. All compounds were tested (i) against a wild-type human immunodeficiency virus type 1 (HIV-1) isolate (strain xxBRU) and lamivudine-resistant HIV-1 isolates, (ii) for their abilities to inhibit hepatitis B virus (HBV) production in the inducible HepAD38 cell line, and (iii) for their abilities to inhibit bovine viral diarrhea virus (BVDV) production in acutely infected Madin-Darby bovine kidney cells. Some compounds demonstrated potent antiviral activities against the wild-type HIV-1 strain (range of 90% effective concentrations [EC90s], 0.14 to 5.2 μM), but marked increases in EC90s were noted when the compounds were tested against the lamivudine-resistant HIV-1 strain (range of EC90s, 53 to >100 μM). The β-l-enantiomers of both classes of compounds were more potent than the corresponding β-d-enantiomers. None of the compounds showed antiviral activity in the assay that determined their abilities to inhibit BVDV, while two compounds inhibited HBV production in HepAD38 cells (EC90, 0.25 μM). The compounds were essentially noncytotoxic in human peripheral blood mononuclear cells and HepG2 cells. No effect on mitochondrial DNA levels was observed after a 7-day incubation with the nucleoside analogs at 10 μM. These studies demonstrate that (i) modification of the sugar ring of cytosine nucleoside analogs with a 4′-thia instead of an oxygen results in compounds with the ability to potently inhibit wild-type HIV-1 but with reduced potency against lamivudine-resistant virus and (ii) the antiviral activity of β-d-2′,3′-didehydro-2′,3′-dideoxy-5-fluorocytidine against wild-type HIV-1 (EC90, 0.08 μM) and lamivudine-resistant HIV-1 (EC90 = 0.15 μM) is markedly reduced by introduction of a 3′-fluorine in the sugar (EC90s of compound 2a, 37.5 and 494 μM, respectively).

scholarly journals The Dimer Initiation Sequence Stem-Loop of Human Immunodeficiency Virus Type 1 Is Dispensable for Viral Replication in Peripheral Blood Mononuclear Cells

2003 ◽  
Vol 77 (15) ◽  
pp. 8329-8335 ◽  
Author(s):  
M. K. Hill ◽  
M. Shehu-Xhilaga ◽  
S. M. Campbell ◽  
P. Poumbourios ◽  
S. M. Crowe ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Mononuclear Cells ◽  
Wild Type ◽  
Peripheral Blood Mononuclear ◽  
Stem Loop ◽  
Immunodeficiency Virus ◽  
Blood Mononuclear Cells ◽  
T Cell Lines ◽  
Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) contains two copies of genomic RNA that are noncovalently linked via a palindrome sequence within the dimer initiation site (DIS) stem-loop. In contrast to the current paradigm that the DIS stem or stem-loop is critical for HIV-1 infectivity, which arose from studies using T-cell lines, we demonstrate here that HIV-1 mutants with deletions in the DIS stem-loop are replication competent in peripheral blood mononuclear cells (PBMCs). The DIS mutants contained either the wild-type (5′GCGCGC3′) or an arbitrary (5′ACGCGT3′) palindrome sequence in place of the 39-nucleotide DIS stem-loop (NLCGCGCG and NLACGCGT). These DIS mutants were replication defective in SupT1 cells, concurring with the current model in which DIS mutants are replication defective in T-cell lines. All of the HIV-1 DIS mutants were replication competent in PBMCs over a 40-day infection period and had retained their respective DIS mutations at 40 days postinfection. Although the stability of the virion RNA dimer was not affected by our DIS mutations, the RNA dimers exhibited a diffuse migration profile when compared to the wild type. No defect in protein processing of the Gag and GagProPol precursor proteins was found in the DIS mutants. Our data provide direct evidence that the DIS stem-loop is dispensable for viral replication in PBMCs and that the requirement of the DIS stem-loop in HIV-1 replication is cell type dependent.

scholarly journals Detection of Latent HIV-1 Infection and Drug Resistant Mutation Testing in Nepal: HIV-1 env V3 DNA Sequence and RT Gene (M184V) Mutation

2016 ◽  
Vol 4 (1) ◽  
pp. 18-25
Author(s):  
Rupendra Shrestha ◽  
Sundar Khadka ◽  
Susbin Raj Wagle ◽  
Alisha Sapkota
Keyword(s):  
Dna Sequence ◽  
Large Scale ◽  
Mutation Screening ◽  
Resistance Mutation ◽  
Hiv Infections ◽  
Wild Type ◽  
Proviral Dna ◽  
Latent Hiv ◽  
Hiv 1 ◽  
M184v Mutation

HIV-1 resistance to antiretroviral therapy (ART) is a crucial issue, despite various effective drugs are available for the treatment. Although the viral RNA is suppressed below the detection limit (<50 copies/ml) with the use of potent antiviral drugs, the mutation can be archived in the cellular reservoir as proviral DNA. The detection of proviral DNA and mutation screening in HIV 1 RNA for genotypic resistance is the sole basis for monitoring the effectiveness of ART. Our study aim to access the extent of latent HIV infections by detecting env V3 DNA and also testing of M184V (meth184val; ATG - GTG substitution at 184th codon) specific mutations in HIV-1 RT gene to monitor the effectiveness of ART. The HIV-1 env V3 DNA sequence was amplified using multiple upstream and downstream primes to show the latent HIV infections, whereas polymerase chain reaction- restriction fragment digestion assay (PCR-RFDA) was used for testing M184V mutation in HIV-1 RT gene. In the study, out of 15 HIV infected patient blood samples, 12 shows amplification of env V3 DNA, confirming the latent HIV infections while 3 were negative for env V3 DNA. HIV-1 RT gene tested for M184V mutation in all 15 samples showed wild type after analysis using PCR-RFDA. After digestion with CviAII, three bands were observed in wild type whereas in mutant only two bands. Although the study shows negative for the M184V resistance mutation, screening of various panels of drug resistance mutations should be performed in recently infected HIV-1 patients for planning the effective ART strategy. The data is not enough to compare the overall scenario of the Nepal thus warrant urgency for large scale study with standard genotypic tools.

scholarly journals Impact of Synonymous Genome Recoding on the HIV Life Cycle

2021 ◽  
Vol 12 ◽  
Author(s):  
Ana Jordan-Paiz ◽  
Sandra Franco ◽  
Miguel Angel Martínez
Keyword(s):  
Life Cycle ◽  
Large Scale ◽  
Viral Gene Expression ◽  
Virus Genome ◽  
Infection Process ◽  
Replication Capacity ◽  
Viral Gene ◽  
Mutational Robustness ◽  
Synonymous Mutations ◽  
Hiv 1

Synonymous mutations within protein coding regions introduce changes in DNA or messenger (m) RNA, without mutating the encoded proteins. Synonymous recoding of virus genomes has facilitated the identification of previously unknown virus biological features. Moreover, large-scale synonymous recoding of the genome of human immunodeficiency virus type 1 (HIV-1) has elucidated new antiviral mechanisms within the innate immune response, and has improved our knowledge of new functional virus genome structures, the relevance of codon usage for the temporal regulation of viral gene expression, and HIV-1 mutational robustness and adaptability. Continuous improvements in our understanding of the impacts of synonymous substitutions on virus phenotype – coupled with the decreased cost of chemically synthesizing DNA and improved methods for assembling DNA fragments – have enhanced our ability to identify potential HIV-1 and host factors and other aspects involved in the infection process. In this review, we address how silent mutagenesis impacts HIV-1 phenotype and replication capacity. We also discuss the general potential of synonymous recoding of the HIV-1 genome to elucidate unknown aspects of the virus life cycle, and to identify new therapeutic targets.

scholarly journals Comparison of G-to-A Mutation Frequencies Induced by APOBEC3 Proteins in H9 Cells and Peripheral Blood Mononuclear Cells in the Context of Impaired Processivities of Drug-Resistant Human Immunodeficiency Virus Type 1 Reverse Transcriptase Variants

2008 ◽  
Vol 82 (13) ◽  
pp. 6536-6545 ◽  
Author(s):  
Stefanie Andrea Knoepfel ◽  
Nadine Christina Salisch ◽  
Peter Michael Huelsmann ◽  
Pia Rauch ◽  
Hauke Walter ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Mononuclear Cells ◽  
Wild Type ◽  
Peripheral Blood Mononuclear ◽  
Virus Variant ◽  
Immunodeficiency Virus ◽  
Blood Mononuclear Cells ◽  
Apobec3 Proteins ◽  
Hiv 1

ABSTRACT APOBEC3 proteins can inhibit human immunodeficiency virus type 1 (HIV-1) replication by inducing G-to-A mutations in newly synthesized viral DNA. However, HIV-1 is able to overcome the antiretroviral activity of some of those enzymes by the viral protein Vif. We investigated the impact of different processivities of HIV-1 reverse transcriptases (RT) on the frequencies of G-to-A mutations introduced by APOBEC3 proteins. Wild-type RT or the M184V, M184I, and K65R+M184V RT variants, which are increasingly impaired in their processivities, were used in the context of a vif-deficient molecular HIV-1 clone to infect H9 cells and peripheral blood mononuclear cells (PBMCs). After two rounds of infection, a part of the HIV-1 env gene was amplified, cloned, and sequenced. The M184V mutation led to G-to-A mutation frequencies that were similar to those of the wild-type RT in H9 cells and PBMCs. The frequencies of G-to-A mutations were increased after infection with the M184I virus variant. This effect was augmented when using the K65R+M184V virus variant (P < 0.001). Overall, the G-to-A mutation frequencies were lower in PBMCs than in H9 cells. Remarkably, 38% ± 18% (mean ± standard deviation) of the env clones derived from PBMCs did not harbor any G-to-A mutation. This was rarely observed in H9 cells (3% ± 3%). Our data imply that the frequency of G-to-A mutations induced by APOBEC3 proteins can be influenced by the processivities of HIV-1 RT variants. The high number of nonmutated clones derived from PBMCs leads to several hypotheses, including that additional antiretroviral mechanisms of APOBEC3 proteins other than their deamination activity might be involved in the inhibition of vif-deficient viruses.

scholarly journals Development of Hexadecyloxypropyl Tenofovir (CMX157) for Treatment of Infection Caused by Wild-Type and Nucleoside/Nucleotide-Resistant HIV

2010 ◽  
Vol 54 (7) ◽  
pp. 2901-2909 ◽  
Author(s):  
E. Randall Lanier ◽  
Roger G. Ptak ◽  
Bernhard M. Lampert ◽  
Laurie Keilholz ◽  
Tracy Hartman ◽  
...  
Keyword(s):  
Mononuclear Cells ◽  
Human Peripheral Blood ◽  
Systemic Exposure ◽  
Target Cells ◽  
Drug Resistant ◽  
Wild Type ◽  
Peripheral Blood Mononuclear ◽  
Nucleotide Analog ◽  
Antiretroviral Drug ◽  
Hiv 1

ABSTRACT CMX157 is a lipid (1-0-hexadecyloxypropyl) conjugate of the acyclic nucleotide analog tenofovir (TFV) with activity against both wild-type and antiretroviral drug-resistant HIV strains, including multidrug nucleoside/nucleotide analog-resistant viruses. CMX157 was consistently >300-fold more active than tenofovir against multiple viruses in several different cell systems. CMX157 was active against all major subtypes of HIV-1 and HIV-2 in fresh human peripheral blood mononuclear cells (PBMCs) and against all HIV-1 strains evaluated in monocyte-derived macrophages, with 50% effective concentrations (EC50s) ranging between 0.20 and 7.2 nM. The lower CMX157 EC50s can be attributed to better cellular uptake of CMX157, resulting in higher intracellular levels of the active antiviral anabolite, TFV-diphosphate (TFV-PP), inside target cells. CMX157 produced >30-fold higher levels of TFV-PP in human PBMCs exposed to physiologically relevant concentrations of the compounds than did TFV. Unlike conventional prodrugs, including TFV disoproxil fumarate (Viread), CMX157 remains intact in plasma, facilitating uptake by target cells and decreasing relative systemic exposure to TFV. There was no detectable antagonism with CMX157 in combination with any marketed antiretroviral drug, and it possessed an excellent in vitro cytotoxicity profile. CMX157 is a promising clinical candidate to treat wild-type and antiretroviral drug-resistant HIV, including strains that fail to respond to all currently available nucleoside/nucleotide reverse transcriptase inhibitors.

scholarly journals Mechanisms Involved in the Selection of HIV-1 Reverse Transcriptase Thumb Subdomain Polymorphisms Associated with Nucleoside Analogue Therapy Failure

2010 ◽  
Vol 54 (11) ◽  
pp. 4799-4811 ◽  
Author(s):  
Gilberto Betancor ◽  
Maria C. Puertas ◽  
María Nevot ◽  
César Garriga ◽  
Miguel A. Martínez ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Nucleoside Analogue ◽  
Mononuclear Cells ◽  
Rnase H ◽  
Replication Capacity ◽  
Wild Type ◽  
Recombinant Enzymes ◽  
Dna Complexes ◽  
Dna Template ◽  
Hiv 1

ABSTRACT Previous studies showed an increased prevalence of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) thumb subdomain polymorphisms Pro272, Arg277, and Thr286 in patients failing therapy with nucleoside analogue combinations. Interestingly, wild-type HIV-1BH10 RT contains Pro272, Arg277, and Thr286. Here, we demonstrate that in the presence of zidovudine, HIV-1BH10 RT mutations P272A/R277K/T286A produce a significant reduction of the viral replication capacity in peripheral blood mononuclear cells in both the absence and presence of M41L/T215Y. In studies carried out with recombinant enzymes, we show that RT thumb subdomain mutations decrease primer-unblocking activity on RNA/DNA complexes, but not on DNA/DNA template-primers. These effects were observed with primers terminated with thymidine analogues (i.e., zidovudine and stavudine) and carbovir (the relevant derivative of abacavir) and were more pronounced when mutations were introduced in the wild-type HIV-1BH10 RT sequence context. RT thumb subdomain mutations increased by 2-fold the apparent dissociation equilibrium constant (Kd ) for RNA/DNA without affecting the Kd for DNA/DNA substrates. RNase H assays carried out with RNA/DNA complexes did not reveal an increase in the reaction rate or in secondary cleavage events that could account for the decreased excision activity. The interaction of Arg277 with the phosphate backbone of the RNA template in HIV-1 RT bound to RNA/DNA and the location of Thr286 close to the RNA strand are consistent with thumb polymorphisms playing a role in decreasing nucleoside RT inhibitor excision activity on RNA/DNA template-primers by affecting interactions with the template-primer duplex without involvement of the RNase H activity of the enzyme.

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