Gp120 substitutions at positions associated with resistance to fostemsavir in treatment-naive HIV-1-positive individuals

2020 ◽  
Vol 75 (6) ◽  
pp. 1580-1587 ◽  
Author(s):  
Luciana Lepore ◽  
Claudia Fabrizio ◽  
Davide Fiore Bavaro ◽  
Eugenio Milano ◽  
Anna Volpe ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Position ◽  
High Rate ◽  
B Virus ◽  
Treatment Naïve ◽  
Constant Domains ◽  
Hiv Gp120 ◽  
Post Hoc ◽  
Hiv 1

Abstract Objectives Fostemsavir, a novel attachment inhibitor targeting the HIV-1 gp120, has demonstrated wide in vitro activity. However, the high rate of HIV gp120 substitutions could jeopardize its efficacy. We investigated envelope (env) substitutions at positions associated with resistance to fostemsavir in patients with a new HIV-1 diagnosis according to HIV subtype and tropism. Methods Gp120 sequences from 409 subjects were retrospectively analysed and the presence of the L116P, A204D, S375H/M/T, M426L, M434I and M475I mutations was evaluated. Other amino acid changes at the same positions were also recorded. The variability at each amino acid position was evaluated using Shannon entropy. Results The frequency of mutations was: S375T (13.2%); M426L (6.8%); M434I (2.9%); M475I (2.7%); S375H (1.0%)/M (0.8%) and L116P (0.31%). Statistically significant differences were found at positions 375 (R5/non-R5 strains and B/non-B subtypes) and 426 (B/non-B subtypes); post hoc analysis revealed that significance for position 375 was steered by S375T while for position 426 significance was governed by unusual substitutions, in particular M426R (B/non-B, P < 0.00001). The variability of env constant domains appeared to be more relevant in the non-B virus population. Conclusions In conclusion, gp120 substitutions were detected in different subtypes and in both R5 and non-R5 variants. Despite the great variability of gp120, the frequency of mutations was low overall and the predominant substitution was S375T, the role of which in reducing fostemsavir efficacy is less substantial.

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 Nature, Position, and Frequency of Mutations Made in a Single Cycle of HIV-1 Replication

2010 ◽  
Vol 84 (19) ◽  
pp. 9864-9878 ◽  
Author(s):  
Michael E. Abram ◽  
Andrea L. Ferris ◽  
Wei Shao ◽  
W. Gregory Alvord ◽  
Stephen H. Hughes
Keyword(s):  
Viral Replication ◽  
Mutation Rate ◽  
Hot Spots ◽  
High Rate ◽  
Published Data ◽  
Single Cycle ◽  
Efficient System ◽  
Hiv 1

ABSTRACT There is considerable HIV-1 variation in patients. The extent of the variation is due to the high rate of viral replication, the high viral load, and the errors made during viral replication. Mutations can arise from errors made either by host DNA-dependent RNA polymerase II or by HIV-1 reverse transcriptase (RT), but the relative contributions of these two enzymes to the mutation rate are unknown. In addition, mutations in RT can affect its fidelity, but the effect of mutations in RT on the nature of the mutations that arise in vivo is poorly understood. We have developed an efficient system, based on existing technology, to analyze the mutations that arise in an HIV-1 vector in a single cycle of replication. A lacZα reporter gene is used to identify viral DNAs that contain mutations which are analyzed by DNA sequencing. The forward mutation rate in this system is 1.4 × 10−5 mutations/bp/cycle, equivalent to the retroviral average. This rate is about 3-fold lower than previously reported for HIV-1 in vivo and is much lower than what has been reported for purified HIV-1 RT in vitro. Although the mutation rate was not affected by the orientation of lacZα, the sites favored for mutations (hot spots) in lacZα depended on which strand of lacZα was present in the viral RNA. The pattern of hot spots seen in lacZα in vivo did not match any of the published data obtained when purified RT was used to copy lacZα in vitro.

scholarly journals Hepatitis B Virus X-Associated Protein 2 Is a Subunit of the Unliganded Aryl Hydrocarbon Receptor Core Complex and Exhibits Transcriptional Enhancer Activity

1998 ◽  
Vol 18 (2) ◽  
pp. 978-988 ◽  
Author(s):  
Brian K. Meyer ◽  
Marilyn G. Pray-Grant ◽  
John P. Vanden Heuvel ◽  
Gary H. Perdew
Keyword(s):  
Amino Acid ◽  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Aryl Hydrocarbon Receptor ◽  
In Vitro Translation ◽  
Luciferase Reporter ◽  
Core Complex ◽  
Aryl Hydrocarbon ◽  
B Virus

ABSTRACT Prior to ligand activation, the unactivated aryl hydrocarbon receptor (AhR) exists in a heterotetrameric 9S core complex consisting of the AhR ligand-binding subunit, a dimer of hsp90, and an unknown subunit. Here we report the purification of an ∼38-kDa protein (p38) from COS-1 cell cytosol that is a member of this complex by coprecipitation with a FLAG-tagged AhR. Internal amino acid sequence information was obtained, and p38 was identified as the hepatitis B virus X-associated protein 2 (XAP2). The simian ortholog of XAP2 was cloned from a COS-1 cDNA library; it codes for a 330-amino-acid protein containing regions of homology to the immunophilins FKBP12 and FKBP52. A tetratricopeptide repeat (TPR) domain in the carboxy-terminal region of XAP2 was similar to the third and fourth TPR domains of human FKBP52 and the Saccharomyces cerevisiae transcriptional modulator SSN6, respectively. Polyclonal antibodies raised against XAP2 recognized p38 in the unliganded AhR complex in COS-1 and Hepa 1c1c7 cells. It was ubiquitously expressed in murine tissues at the protein and mRNA levels. It was not required for the assembly of an AhR-hsp90 complex in vitro. Additionally, XAP2 did not directly associate with hsp90 upon in vitro translation, but was present in a 9S form when cotranslated in vitro with murine AhR. XAP2 enhanced the ability of endogenous murine and human AhR complexes to activate a dioxin-responsive element–luciferase reporter twofold, following transient expression of XAP2 in Hepa 1c1c7 and HeLa cells.

scholarly journals A Naturally Occurring Rgg Variant in Serotype M3 Streptococcus pyogenes Does Not Activate speB Expression Due to Altered Specificity of DNA Binding

2009 ◽  
Vol 77 (12) ◽  
pp. 5411-5417 ◽  
Author(s):  
Kyle V. Kappeler ◽  
Srivishnupriya Anbalagan ◽  
Alexander V. Dmitriev ◽  
Emily J. McDowell ◽  
Melody N. Neely ◽  
...  
Keyword(s):  
Amino Acid ◽  
Streptococcus Pyogenes ◽  
Murine Model ◽  
Cysteine Protease ◽  
Phenotypic Diversity ◽  
Transcriptional Regulator ◽  
Amino Acid Position ◽  
Invasive Disease ◽  
Naturally Occurring

ABSTRACT The transcriptional regulator Rgg of Streptococcus pyogenes is essential for expression of the secreted cysteine protease SpeB. Although all isolates of S. pyogenes possess the speB gene, not all of them produce the protein in vitro. In a murine model of infection, the absence of SpeB production is associated with invasive disease. We speculated that naturally occurring mutations in rgg, which would also abrogate SpeB production, may be present in invasive isolates of S. pyogenes. Examination of the inferred Rgg sequences available in public databases revealed that the rgg gene in strain MGAS315 (a serotype M3 strain associated with invasive disease) encodes a proline at amino acid position 103 (Rgg103P); in contrast, all other strains encode a serine at this position (Rgg103S). A caseinolytic assay and Western blotting indicated that strain MGAS315 does not produce SpeB in vitro. Gene-swapping experiments showed that the rgg gene of MGAS315 is solely responsible for the lack of SpeB expression. In contrast to Rgg103S, Rgg103P does not bind to the speB promoter in gel shift assays, which correlates with a lack of speB expression. Despite its inability to activate speB expression, Rgg103P retains the ability to bind to DNA upstream of norA and to influence its expression. Overall, this study illustrates how variation at the rgg locus may contribute to the phenotypic diversity of S. pyogenes.

scholarly journals Impact of Human Immunodeficiency Virus Type 1 gp41 Amino Acid Substitutions Selected during Enfuvirtide Treatment on gp41 Binding and Antiviral Potency of Enfuvirtide In Vitro

2005 ◽  
Vol 79 (19) ◽  
pp. 12447-12454 ◽  
Author(s):  
M. Mink ◽  
S. M. Mosier ◽  
S. Janumpalli ◽  
D. Davison ◽  
L. Jin ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Amino Acid ◽  
Binding Affinity ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Fusion Inhibitor ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Enfuvirtide (ENF), a novel human immunodeficiency virus type 1 (HIV-1) fusion inhibitor, has potent antiviral activity against HIV-1 both in vitro and in vivo. Resistance to ENF observed after in vitro passaging was associated with changes in a three-amino-acid (aa) motif, GIV, at positions 36 to 38 of gp41. Patients with ongoing viral replication while receiving ENF during clinical trials acquired substitutions within gp41 aa 36 to 45 in the first heptad repeat (HR-1) of gp41 in both population-based plasma virus sequences and proviral DNA sequences from isolates showing reduced susceptibilities to ENF. To investigate their impact on ENF susceptibility, substitutions were introduced into a modified pNL4-3 strain by site-directed mutagenesis, and the susceptibilities of mutant viruses and patient-derived isolates to ENF were tested. In general, susceptibility decreases for single substitutions were lower than those for double substitutions, and the levels of ENF resistance seen for clinical isolates were higher than those observed for the site-directed mutant viruses. The mechanism of ENF resistance was explored for a subset of the substitutions by expressing them in the context of a maltose binding protein chimera containing a portion of the gp41 ectodomain and measuring their binding affinity to fluorescein-labeled ENF. Changes in binding affinity for the mutant gp41 fusion proteins correlated with the ENF susceptibilities of viruses containing the same substitutions. The combined results support the key role of gp41 aa 36 to 45 in the development of resistance to ENF and illustrate that additional envelope regions contribute to the ENF susceptibility of fusion inhibitor-naïve viruses and resistance to ENF.

scholarly journals In VitroCharacterization of GS-8374, a Novel Phosphonate-Containing Inhibitor of HIV-1 Protease with a Favorable Resistance Profile

2011 ◽  
Vol 55 (4) ◽  
pp. 1366-1376 ◽  
Author(s):  
Christian Callebaut ◽  
Kirsten Stray ◽  
Luong Tsai ◽  
Matt Williams ◽  
Zheng-Yu Yang ◽  
...  
Keyword(s):  
Synergistic Effects ◽  
Cell Types ◽  
Human Serum Protein ◽  
Treatment Naïve ◽  
Low Cytotoxicity ◽  
T Cell Lines ◽  
High Level ◽  
Hiv 1

ABSTRACTGS-8374 is a novel bis-tetrahydrofuran HIV-1 protease (PR) inhibitor (PI) with a unique diethylphosphonate moiety. It was selected from a series of analogs containing various di(alkyl)phosphonate substitutions connected via a linker to theparaposition of a P-1 phenyl ring. GS-8374 inhibits HIV-1 PR with high potency (Ki= 8.1 pM) and with no known effect on host proteases. Kinetic and thermodynamic analysis of GS-8374 binding to PR demonstrated an extremely slow off rate for the inhibitor and favorable contributions of both the enthalpic and entropic components to the total free binding energy. GS-8374 showed potent antiretroviral activity in T-cell lines, primary CD4+T cells (50% effective concentration [EC50] = 3.4 to 11.5 nM), and macrophages (EC50= 25.5 nM) and exhibited low cytotoxicity in multiple human cell types. The antiviral potency of GS-8374 was only moderately affected by human serum protein binding, and its combination with multiple approved antiretrovirals showed synergistic effects. When it was tested in a PhenoSense assay against a panel of 24 patient-derived viruses with high-level PI resistance, GS-8374 showed lower mean EC50s and lower fold resistance than any of the clinically approved PIs. Similar to other PIs,in vitrohepatic microsomal metabolism of GS-8374 was efficiently blocked by ritonavir, suggesting a potential for effective pharmacokinetic boostingin vivo. In summary, results from this broadin vitropharmacological profiling indicate that GS-8374 is a promising candidate to be further assessed as a new antiretroviral agent with potential for clinical efficacy in both treatment-naïve and -experienced patients.

scholarly journals HIV-1 Protease, Reverse Transcriptase, and Integrase Variation

2016 ◽  
Vol 90 (13) ◽  
pp. 6058-6070 ◽  
Author(s):  
Soo-Yon Rhee ◽  
Kris Sankaran ◽  
Vici Varghese ◽  
Mark A. Winters ◽  
Christopher B. Hurt ◽  
...  
Keyword(s):  
Quality Control ◽  
Amino Acid ◽  
Amino Acid Position ◽  
Antiretroviral Drugs ◽  
Resistance Testing ◽  
Genotypic Resistance ◽  
Naturally Occurring ◽  
Genotypic Resistance Testing ◽  
Hiv 1 ◽  
Amino Acid Variants

ABSTRACTHIV-1 protease (PR), reverse transcriptase (RT), and integrase (IN) variability presents a challenge to laboratories performing genotypic resistance testing. This challenge will grow with increased sequencing of samples enriched for proviral DNA such as dried blood spots and increased use of next-generation sequencing (NGS) to detect low-abundance HIV-1 variants. We analyzed PR and RT sequences from >100,000 individuals and IN sequences from >10,000 individuals to characterize variation at each amino acid position, identify mutations indicating APOBEC-mediated G-to-A editing, and identify mutations resulting from selective drug pressure. Forty-seven percent of PR, 37% of RT, and 34% of IN positions had one or more amino acid variants with a prevalence of ≥1%. Seventy percent of PR, 60% of RT, and 60% of IN positions had one or more variants with a prevalence of ≥0.1%. Overall 201 PR, 636 RT, and 346 IN variants had a prevalence of ≥0.1%. The median intersubtype prevalence ratios were 2.9-, 2.1-, and 1.9-fold for these PR, RT, and IN variants, respectively. Only 5.0% of PR, 3.7% of RT, and 2.0% of IN variants had a median intersubtype prevalence ratio of ≥10-fold. Variants at lower prevalences were more likely to differ biochemically and to be part of an electrophoretic mixture compared to high-prevalence variants. There were 209 mutations indicative of APOBEC-mediated G-to-A editing and 326 mutations nonpolymorphic treatment selected. Identification of viruses with a high number of APOBEC-associated mutations will facilitate the quality control of dried blood spot sequencing. Identifying sequences with a high proportion of rare mutations will facilitate the quality control of NGS.IMPORTANCEMost antiretroviral drugs target three HIV-1 proteins: PR, RT, and IN. These proteins are highly variable: many different amino acids can be present at the same position in viruses from different individuals. Some of the amino acid variants cause drug resistance and occur mainly in individuals receiving antiretroviral drugs. Some variants result from a human cellular defense mechanism called APOBEC-mediated hypermutation. Many variants result from naturally occurring mutation. Some variants may represent technical artifacts. We studied PR and RT sequences from >100,000 individuals and IN sequences from >10,000 individuals to quantify variation at each amino acid position in these three HIV-1 proteins. We performed analyses to determine which amino acid variants resulted from antiretroviral drug selection pressure, APOBEC-mediated editing, and naturally occurring variation. Our results provide information essential to clinical, research, and public health laboratories performing genotypic resistance testing by sequencing HIV-1 PR, RT, and IN.

Interleukin 21 augments the hepatitis B virus-specific CD8+ T-cell response in vitro in patients coinfected with HIV-1

AIDS ◽  
2012 ◽  
Vol 26 (17) ◽  
pp. 2145-2153 ◽  
Author(s):  
Guangxu Ren ◽  
Stefan Esser ◽  
Christoph Jochum ◽  
Joerg F. Schlaak ◽  
Guido Gerken ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
T Cell ◽  
Hepatitis B ◽  
Cell Response ◽  
Cd8 T Cell ◽  
T Cell Response ◽  
B Virus ◽  
Interleukin 21 ◽  
Hiv 1

scholarly journals Resistance to the Anti-Human Immunodeficiency Virus Type 1 Compound l-Chicoric Acid Results from a Single Mutation at Amino Acid 140 of Integrase

1998 ◽  
Vol 72 (10) ◽  
pp. 8420-8424 ◽  
Author(s):  
Peter J. King ◽  
W. Edward Robinson
Keyword(s):  
Human Immunodeficiency Virus ◽  
Amino Acid ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Single Mutation ◽  
Chicoric Acid ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT l-Chicoric acid is an inhibitor of human immunodeficiency virus type 1 (HIV-1) integrase in vitro and of HIV-1 replication in tissue culture. Following 3 months of selection in the presence of increasing concentrations of l-chicoric acid, HIV-1 was completely resistant to the compound. Introduction of the mutant integrase containing a single glycine-to-serine amino acid change at position 140 into the native, l-chicoric acid-sensitive virus demonstrated that this change was sufficient to confer resistance to l-chicoric acid. These results confirm through natural selection previous biochemical studies showing thatl-chicoric acid inhibits integrase and that the drug is likely to interact at residues near the catalytic triad in the integrase active site.

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