scholarly journals Physiological magnesium concentrations increase fidelity of diverse reverse transcriptases from HIV-1, HIV-2, and foamy virus, but not MuLV or AMV

2021 ◽  
Vol 102 (12) ◽  
Author(s):  
Ruofan Wang ◽  
Ashton T. Belew ◽  
Vasudevan Achuthan ◽  
Najib El Sayed ◽  
Jeffrey J. DeStefano
Keyword(s):  
Foamy Virus ◽  
Fold Increase ◽  
Reverse Transcriptases ◽  
Subtype B ◽  
Mutation Profile ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing ◽  
Hiv 1 ◽  
Current Report

Reverse transcriptases (RTs) are typically assayed using optimized Mg2+ concentrations (~5–10 mM) several-fold higher than physiological cellular free Mg2+ (~0.5 mM). Recent analyses demonstrated that HIV-1, but not Moloney murine leukaemia (MuLV) or avain myeloblastosis (AMV) virus RTs has higher fidelity in low Mg2+. In the current report, lacZα-based α-complementation assays were used to measure the fidelity of several RTs including HIV-1 (subtype B and A/E), several drug-resistant HIV-1 derivatives, HIV-2, and prototype foamy virus (PFV), all which showed higher fidelity using physiological Mg2+, while MuLV and AMV RTs demonstrated equivalent fidelity in low and high Mg2+. In 0.5 mM Mg2+, all RTs demonstrated approximately equal fidelity, except for PFV which showed higher fidelity. A Next Generation Sequencing (NGS) approach that used barcoding to determine mutation profiles was used to examine the types of mutations made by HIV-1 RT (type B) in low (0.5 mM) and high (6 mM) Mg2+ on a lacZα template. Unlike α-complementation assays which are dependent on LacZα activity, the NGS assay scores mutations at all positions and of every type. Consistent with α-complementation assays, a ~four-fold increase in mutations was observed in high Mg2+. These findings help explain why HIV-1 RT displays lower fidelity in vitro (with high Mg2+ concentrations) than other RTs (e.g. MuLV and AMV), yet cellular fidelity for these viruses is comparable. Establishing in vitro conditions that accurately represent RT’s activity in cells is pivotal to determining the contribution of RT and other factors to the mutation profile observed with HIV-1.

scholarly journals Physiological Magnesium Concentrations Increase Fidelity of Diverse Reverse Transcriptases from HIV-1, HIV-2, and Foamy Virus, but not MuLV or AMV

2021 ◽  
Author(s):  
Ruofan Wang ◽  
Ashton T. Belew ◽  
Vasudevan Achuthan ◽  
Najib M. El-Sayed ◽  
Jeffrey J DeStefano
Keyword(s):  
Leukemia Virus ◽  
Foamy Virus ◽  
Fold Increase ◽  
Common Mutation ◽  
Avian Myeloblastosis Virus ◽  
Reverse Transcriptases ◽  
Subtype B ◽  
Substitution Mutations ◽  
Hiv 1

Reverse transcriptases (RTs) are typically assayed in vitro using optimized Mg2+ concentrations (~ 5-10 mM) that are several-fold higher than physiological cellular free Mg2+ (~ 0.5 mM). Analysis of fidelity using lacZα-based α-complementation assays showed that tested HIV RTs, including HIV-1 from subtype B (HXB2-derived), HIV-2, subtype A/E, and several drug-resistant HXB2 derivatives all showed significantly higher fidelity using physiological Mg2+. This also occurred with prototype foamy virus (PFV) RT. In contrast, Moloney murine leukemia virus (MuLV) and avian myeloblastosis virus (AMV) RTs demonstrated equivalent fidelity in both low and high Mg2+. In 0.5 mM Mg2+, all RTs demonstrated ≈ equal fidelity, except for PFV RT which showed higher fidelity. A Next Generation Sequencing (NGS) approach that used barcoding to accurately determine mutation rates and profiles was used to examine the types of mutations made by HIV-1 (subtype B, wild type) in low (0.5 mM) and high (6 mM) Mg2+ with DNA or RNA that coded for lacZα. Unlike the α-complementation assay, which is dependent on LacZα activity, the NGS assay scores mutations at all positions and of every type. A ~ 4-fold increase in substitution mutations was observed in high Mg2+. The general trend was an exacerbation in high Mg2+ of more common mutation in low Mg2+, rather than the creation of new mutation hotspots. These findings help explain why HIV RT displays lower fidelity in vitro (with high Mg2+ concentrations) than other RTs (e.g., MuLV and AMV), yet cellular fidelity for these viruses is comparable.

scholarly journals Use of Coreceptors Other Than CCR5 by Non-Syncytium-Inducing Adult and Pediatric Isolates of Human Immunodeficiency Virus Type 1 Is Rare In Vitro

1998 ◽  
Vol 72 (11) ◽  
pp. 9337-9344 ◽  
Author(s):  
Yi-jun Zhang ◽  
Tatjana Dragic ◽  
Yunzhen Cao ◽  
Leondios Kostrikis ◽  
Douglas S. Kwon ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Viral Entry ◽  
Infected Infant ◽  
Subtype B ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT We have tested a panel of pediatric and adult human immunodeficiency virus type 1 (HIV-1) primary isolates for the ability to employ the following proteins as coreceptors during viral entry: CCR1, CCR2b, CCR3, CCR4, CCR5, CCR8, CXCR4, Bonzo, BOB, GPR1, V28, US28, and APJ. Most non-syncytium-inducing isolates could utilize only CCR5. All syncytium-inducing viruses used CXCR4, some also employed V28, and one (DH123) used CCR8 and APJ as well. A longitudinal series of HIV-1 subtype B isolates from an infected infant and its mother utilized Bonzo efficiently, as well as CCR5. The maternal isolates, which were syncytium inducing, also used CXCR4, CCR8, V28, and APJ.

scholarly journals Neutralizing antibodies induced by first-generation gp41-stabilized HIV-1 envelope trimers and nanoparticles

2020 ◽  
Author(s):  
Sonu Kumar ◽  
Xiaohe Lin ◽  
Timothy Ngo ◽  
Benjamin Shapero ◽  
Cindy Sou ◽  
...  
Keyword(s):  
Cell Sorting ◽  
Neutralizing Antibodies ◽  
First Generation ◽  
Heptad Repeat ◽  
X Ray ◽  
X Ray Crystallography ◽  
Clade C ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing ◽  
Hiv 1

ABSTRACTAntigen-specific B-cell sorting and next-generation sequencing (NGS) were combined to isolate HIV-1 neutralizing antibodies (NAbs) from mice and rabbits immunized with BG505 trimers and nanoparticles. Three mouse NAbs potently neutralize BG505.T332N and recognize a glycan epitope centered at the C3/V4 region, as revealed by electron microscopy (EM), x-ray crystallography, and epitope mapping. Three potent NAbs were sorted from rabbit B cells that target glycan holes on the BG505 envelope glycoprotein (Env) and account for a significant portion of autologous NAb response. We then determined a 3.4Å-resolution crystal structure for the clade C transmitted/founder Du172.17 Env with a redesigned heptad repeat 1 (HR1) bend. This clade C Env, as a soluble trimer and attached to a ferritin nanoparticle, along with a clade A Q482-d12 Env trimer, elicited distinct NAb responses in rabbits. Our study demonstrates that nanoparticles presenting gp41-stabilized trimers can induce potent NAb responses in mice and rabbits with Env-dependent breadth.TEASERMouse and rabbit NAbs elicited by gp41-stabilized trimers and nanoparticles neutralize autologous HIV-1 by targeting different epitopes

scholarly journals Molecular Dynamics Approaches Estimate the Binding Energy of HIV-1 Integrase Inhibitors and Correlate withIn VitroActivity

2011 ◽  
Vol 56 (1) ◽  
pp. 411-419 ◽  
Author(s):  
Barry C. Johnson ◽  
Mathieu Métifiot ◽  
Yves Pommier ◽  
Stephen H. Hughes
Keyword(s):  
Molecular Dynamics ◽  
Binding Energy ◽  
Foamy Virus ◽  
Complex Model ◽  
Integrase Inhibitors ◽  
Training Set ◽  
Viral Dna ◽  
Starting Point ◽  
Hiv 1

ABSTRACTThe design of novel integrase (IN) inhibitors has been aided by recent crystal structures revealing the binding mode of these compounds with a full-length prototype foamy virus (PFV) IN and synthetic viral DNA ends. Earlier docking studies relied on incomplete structures and did not include the contribution of the viral DNA to inhibitor binding. Using the structure of PFV IN as the starting point, we generated a model of the corresponding HIV-1 complex and developed a molecular dynamics (MD)-based approach that correlates with thein vitroactivities of novel compounds. Four well-characterized compounds (raltegravir, elvitegravir, MK-0536, and dolutegravir) were used as a training set, and the data for theirin vitroactivity against the Y143R, N155H, and G140S/Q148H mutants were used in addition to the wild-type (WT) IN data. Three additional compounds were docked into the IN-DNA complex model and subjected to MD simulations. All three gave interaction potentials within 1 standard deviation of values estimated from the training set, and the most active compound was identified. Additional MD analysis of the raltegravir- and dolutegravir-bound complexes gave internal and interaction energy values that closely match the experimental binding energy of a compound related to raltegravir that has similar activity. These approaches can be used to gain a deeper understanding of the interactions of the inhibitors with the HIV-1 intasome and to identify promising scaffolds for novel integrase inhibitors, in particular, compounds that retain activity against a range of drug-resistant mutants, making it possible to streamline synthesis and testing.

scholarly journals Antiviral Activity of Bictegravir and Cabotegravir against Integrase Inhibitor-Resistant SIVmac239 and HIV-1

2017 ◽  
Vol 61 (12) ◽  
Author(s):  
Said A. Hassounah ◽  
Ahmad Alikhani ◽  
Maureen Oliveira ◽  
Simrat Bharaj ◽  
Ruxandra-Ilinca Ibanescu ◽  
...  
Keyword(s):  
Treatment Strategies ◽  
Fold Increase ◽  
Integrase Inhibitor ◽  
Antiretroviral Drugs ◽  
Strand Transfer ◽  
Single Cycle ◽  
Genetic Barrier ◽  
Hiv 1

ABSTRACT Animal models are essential to study novel antiretroviral drugs, resistance-associated mutations (RAMs), and treatment strategies. Bictegravir (BIC) is a novel potent integrase strand transfer inhibitor (INSTI) that has shown promising results against HIV-1 infection in vitro and in vivo and against clinical isolates with resistance against INSTIs. BIC has a higher genetic barrier to the development of resistance than two clinically approved INSTIs, termed raltegravir and elvitegravir. Another clinically approved INSTI, dolutegravir (DTG) also possesses a high genetic barrier to resistance, while a fourth compound, termed cabotegravir (CAB), is currently in late phases of clinical development. Here we report the susceptibilities of simian immunodeficiency virus (SIV) and HIV-1 integrase (IN) mutants containing various RAMs to BIC, CAB, and DTG. BIC potently inhibited SIV and HIV-1 in single cycle infection with 50% effective concentrations (EC50s) in the low nM range. In single cycle SIV infections, none of the E92Q, T97A, Y143R, or N155H substitutions had a significant effect on susceptibility to BIC (≤4-fold increase in EC50), whereas G118R and R263K conferred ∼14-fold and ∼6-fold increases in EC50, respectively. In both single and multiple rounds of HIV-1 infections, BIC remained active against the Y143R, N155H, R263K, R263K/M50I, and R263K/E138K mutants (≤4-fold increase in EC50). In multiple rounds of infection, the G140S/Q148H combination of substitutions decreased HIV-1 susceptibility to BIC 4.8-fold compared to 16.8- and 7.4-fold for CAB and DTG, respectively. BIC possesses an excellent resistance profile in regard to HIV and SIV and could be useful in nonhuman primate models of HIV infection.

scholarly journals Identification and Characterization of BMS-955176, a Second-Generation HIV-1 Maturation Inhibitor with Improved Potency, Antiviral Spectrum, and Gag Polymorphic Coverage

2016 ◽  
Vol 60 (7) ◽  
pp. 3956-3969 ◽  
Author(s):  
Beata Nowicka-Sans ◽  
Tricia Protack ◽  
Zeyu Lin ◽  
Zhufang Li ◽  
Sharon Zhang ◽  
...  
Keyword(s):  
Human Serum ◽  
Second Generation ◽  
Mononuclear Cells ◽  
Integrase Inhibitor ◽  
Wild Type Virus ◽  
Peripheral Blood Mononuclear ◽  
Subtype B ◽  
Maturation Inhibitor ◽  
Hiv 1

ABSTRACTBMS-955176 is a second-generation human immunodeficiency virus type 1 (HIV-1) maturation inhibitor (MI). A first-generation MI, bevirimat, showed clinical efficacy in early-phase studies, but ∼50% of subjects had viruses with reduced susceptibility associated with naturally occurring polymorphisms in Gag near the site of MI action. MI potency was optimized using a panel of engineered reporter viruses containing site-directed polymorphic changes in Gag that reduce susceptibility to bevirimat (including V362I, V370A/M/Δ, and T371A/Δ), leading incrementally to the identification of BMS-955176. BMS-955176 exhibits potent activity (50% effective concentration [EC50], 3.9 ± 3.4 nM [mean ± standard deviation]) toward a library (n= 87) ofgag/prrecombinant viruses representing 96.5% of subtype B polymorphic Gag diversity near the CA/SP1 cleavage site. BMS-955176 exhibited a median EC50of 21 nM toward a library of subtype B clinical isolates assayed in peripheral blood mononuclear cells (PBMCs). Potent activity was maintained against a panel of reverse transcriptase, protease, and integrase inhibitor-resistant viruses, with EC50s similar to those for the wild-type virus. A 5.4-fold reduction in EC50occurred in the presence of 40% human serum plus 27 mg/ml of human serum albumin (HSA), which corresponded well to anin vitromeasurement of 86% human serum binding. Time-of-addition and pseudotype reporter virus studies confirm a mechanism of action for the compound that occurs late in the virus replication cycle. BMS-955176 inhibits HIV-1 protease cleavage at the CA/SP1 junction within Gag in virus-like particles (VLPs) and in HIV-1-infected cells, and it binds reversibly and with high affinity to assembled Gag in purified HIV-1 VLPs. Finally,in vitrocombination studies showed no antagonistic interactions with representative antiretrovirals (ARVs) of other mechanistic classes. In conclusion, BMS-955176 is a second-generation MI with potentin vitroanti-HIV-1 activity and a greatly improved preclinical profile compared to that of bevirimat.

scholarly journals Nucleotide specificity of HIV-1 reverse transcriptases with amino acid substitutions affecting Ala-114

2005 ◽  
Vol 387 (1) ◽  
pp. 221-229 ◽  
Author(s):  
Clara E. CASES-GONZÁLEZ ◽  
Luis MENÉNDEZ-ARIAS
Keyword(s):  
Catalytic Efficiency ◽  
Fold Increase ◽  
Polymerase Activity ◽  
Site Directed Mutagenesis ◽  
Van Der Waals Interactions ◽  
Reverse Transcriptases ◽  
Nucleotide Incorporation ◽  
Steady State Kinetic ◽  
Nucleotide Specificity ◽  
Hiv 1

Ala-114, together with Asp-113, Tyr-115 and Gln-151, form the pocket that accommodates the 3′-OH of the incoming dNTP in the HIV-1 RT (reverse transcriptase). Four mutant RTs having serine, glycine, threonine or valine instead of Ala-114 were obtained by site-directed mutagenesis. While mutants A114S and A114G retained significant DNA polymerase activity, A114T and A114V showed very low catalytic efficiency in nucleotide incorporation assays, due to their high apparent Km values for dNTP. Discrimination between AZTTP (3′-azido-3′-deoxythymidine triphosphate) and dTTP was not significantly affected by mutations A114S and A114G in assays carried out with heteropolymeric template/primers. However, both mutants showed decreased susceptibility to AZTTP when poly(rA)/(dT)16 was used as substrate. Steady-state kinetic analysis of the incorporation of ddNTPs compared with dNTPs showed that substituting glycine for Ala-114 produced a 5–6-fold increase in the RT's ability to discriminate against ddNTPs (including the physiologically relevant metabolites of zalcitabine and didanosine), a result that was confirmed in primer-extension assays. In contrast, A114S and A114V showed wild-type ddNTP/dNTP discrimination efficiencies. Discrimination against ribonucleotides was not affected by mutations at position 114. Misinsertion and mispair extension fidelity assays as well as determinations of G→A mutation frequencies using a lacZ complementation assay showed that, unlike Tyr-115 or Gln-151 mutants, the fidelity of HIV-1 RT was not largely affected by substitutions of Ala-114. The role of the side-chain of Ala-114 in ddNTP/dNTP discrimination appears to be determined by its participation in van der Waals interactions with the ribose moiety of the incoming nucleotide.

scholarly journals Enrichment of Food With Tannin Extracts Promotes Healthy Changes in the Human Gut Microbiota

2021 ◽  
Vol 12 ◽  
Author(s):  
Silvia Molino ◽  
Alberto Lerma-Aguilera ◽  
Nuria Jiménez-Hernández ◽  
María José Gosalbes ◽  
José Ángel Rufián-Henares ◽  
...  
Keyword(s):  
Gut Microbiota ◽  
Short Chain Fatty Acids ◽  
Human Gut ◽  
Human Gut Microbiota ◽  
Wood Extracts ◽  
Potential Benefits ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing ◽  
Booster Effect

Food and food bioactive components are major drivers of modulation of the human gut microbiota. Tannin extracts consist of a mix of bioactive compounds, which are already exploited in the food industry for their chemical and sensorial properties. The aim of our study was to explore the viability of associations between tannin wood extracts of different origin and food as gut microbiota modulators. 16S rRNA amplicon next-generation sequencing (NGS) was used to test the effects on the gut microbiota of tannin extracts from quebracho, chestnut, and tara associated with commercial food products with different composition in macronutrients. The different tannin-enriched and non-enriched foods were submitted to in vitro digestion and fermentation by the gut microbiota of healthy subjects. The profile of the short chain fatty acids (SCFAs) produced by the microbiota was also investigated. The presence of tannin extracts in food promoted an increase of the relative abundance of the genus Akkermansia, recognized as a marker of a healthy gut, and of various members of the Lachnospiraceae and Ruminococcaceae families, involved in SCFA production. The enrichment of foods with tannin extracts had a booster effect on the production of SCFAs, without altering the profile given by the foods alone. These preliminary results suggest a positive modulation of the gut microbiota with potential benefits for human health through the enrichment of foods with tannin extracts.

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