Insights into the activity of maturation inhibitor PF-46396 on HIV-1 clade C

Ghimire, Dibya; Timilsina, Uddhav; Srivastava, Tryambak; Gaur, Ritu

doi:10.1038/srep43711

Cited by 8 publications

(11 citation statements)

References 45 publications

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“…The second mutant K3016 CA:I201V was identified in the presence of BVM analog CV-8613 and conferred partial resistance and compound dependence on the HIV-1 subtype C virus consistent with our previous study using another MI, PF-46396 [ 37 ]. In contrast, the HIV-1 subtype B CA:I1201V mutant conferred resistance but not compound dependence against MIs: GSK3532795 and PF-46396 [ 36 , 39 ].…”

Section: Discussionsupporting

confidence: 89%

“…In contrast, the HIV-1 subtype B CA:I1201V mutant conferred resistance but not compound dependence against MIs: GSK3532795 and PF-46396 [ 36 , 39 ]. This phenotype of the HIV-1 subtype C CA:I201V mutant reflected a unique characteristic of this virus [ 37 ]. It is interesting to note that mutation at this residue did not impair virus assembly, but reduced infectivity and replicative fitness which was markedly improved on addition of BVM analog CV-8613 (Fig.…”

Section: Discussionmentioning

confidence: 99%

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A single G10T polymorphism in HIV-1 subtype C Gag-SP1 regulates sensitivity to maturation inhibitors

Ghimire

Timilsina

et al. 2021

Retrovirology

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Background Maturation inhibitors (MIs) potently block HIV-1 maturation by inhibiting the cleavage of the capsid protein and spacer peptide 1 (CA-SP1). Bevirimat (BVM), a highly efficacious first-in-class MI against HIV-1 subtype B isolates, elicited sub-optimal efficacy in clinical trials due to polymorphisms in the CA-SP1 region of the Gag protein (SP1:V7A). HIV-1 subtype C inherently contains this polymorphism thus conferring BVM resistance, however it displayed sensitivity to second generation BVM analogs. Results In this study, we have assessed the efficacy of three novel second-generation MIs (BVM analogs: CV-8611, CV-8612, CV-8613) against HIV-1 subtype B and C isolates. The BVM analogs were potent inhibitors of both HIV-1 subtype B (NL4-3) and subtype C (K3016) viruses. Serial passaging of the subtype C, K3016 virus strain in the presence of BVM analogs led to identification of two mutant viruses—Gag SP1:A1V and CA:I201V. While the SP1:A1V mutant was resistant to the MIs, the CA:I120V mutant displayed partial resistance and a MI-dependent phenotype. Further analysis of the activity of the BVM analogs against two additional HIV-1 subtype C strains, IndieC1 and ZM247 revealed that they had reduced sensitivity as compared to K3016. Sequence analysis of the three viruses identified two polymorphisms at SP1 residues 9 and 10 (K3016: N9, G10; IndieC1/ZM247: S9, T10). The N9S and S9N mutants had no change in MI-sensitivity. On the other hand, replacing glycine at residue 10 with threonine in K3016 reduced its MI sensitivity whereas introducing glycine at SP1 10 in place of threonine in IndieC1 and ZM247 significantly enhanced their MI sensitivity. Thus, the specific glycine residue 10 of SP1 in the HIV-1 subtype C viruses determined sensitivity towards BVM analogs. Conclusions We have identified an association of a specific glycine at position 10 of Gag-SP1 with an MI susceptible phenotype of HIV-1 subtype C viruses. Our findings have highlighted that HIV-1 subtype C viruses, which were inherently resistant to BVM, may also be similarly predisposed to exhibit a significant degree of resistance to second-generation BVM analogs. Our work has strongly suggested that genetic differences between HIV-1 subtypes may produce variable MI sensitivity that needs to be considered in the development of novel, potent, broadly-active MIs. Graphic abstract

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Section: Discussionsupporting

confidence: 89%

Section: Discussionmentioning

confidence: 99%

A single G10T polymorphism in HIV-1 subtype C Gag-SP1 regulates sensitivity to maturation inhibitors

Ghimire

Timilsina

et al. 2021

Retrovirology

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“…Replication fitness of these infectious viruses was subsequently tested in a multiple-cycle infectivity assay in the HUT-R5 cell line (93)(94)(95). Infected cells were split every 3rd day, and virus replication was quantitated by measuring the HIV-1 p24 antigen released in culture supernatants at different time points, post-infection.…”

Section: Hiv-1 Env Glycosylation Conformation and Viral Infectivitymentioning

confidence: 99%

“…MCs were produced in HEK-293T cells as described above for pseudoviral production, by transfection of pLAI-JRFL, pLAI-⌬G13 gp160, or pLAI-⌬G15F gp160 plasmid using PEI. HUT-R5 cells were infected with these MCs to analyze spreading multiple-cycle HIV-1 infection as described previously (93)(94)(95). HUT-R5 cells refer to the human T-cell line HUT-78, which is stably transduced with CCR5 co-receptor.…”

Section: Generation Of Full-length Hiv-1 Molecular Clones and Multiplmentioning

confidence: 99%

Glycosylation of the core of the HIV-1 envelope subunit protein gp120 is not required for native trimer formation or viral infectivity

Rathore

Saha

Kesavardhana

et al. 2017

Journal of Biological Chemistry

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The gp120 subunit of the HIV-1 envelope (Env) protein is heavily glycosylated at ∼25 glycosylation sites, of which ∼7-8 are located in the V1/V2 and V3 variable loops and the others in the remaining core gp120 region. Glycans partially shield Env from recognition by the host immune system and also are believed to be indispensable for proper folding of gp120 and for viral infectivity. Previous attempts to alter glycosylation sites in Env typically involved mutating the glycosylated asparagine residues to structurally similar glutamines or alanines. Here, we confirmed that such mutations at multiple glycosylation sites greatly diminish viral infectivity and result in significantly reduced binding to both neutralizing and non-neutralizing antibodies. Therefore, using an alternative approach, we combined evolutionary information with structure-guided design and yeast surface display to produce properly cleaved HIV-1 Env variants that lack all 15 core gp120 glycans, yet retain conformational integrity and multiple-cycle viral infectivity and bind to several broadly neutralizing antibodies (bNAbs), including trimer-specific antibodies and a germline-reverted version of the bNAb VRC01. Our observations demonstrate that core gp120 glycans are not essential for folding, and hence their likely primary role is enabling immune evasion. We also show that our glycan removal approach is not strain restricted. Glycan-deficient Env derivatives can be used as priming immunogens because they should engage and activate a more divergent set of germlines than fully glycosylated Env. In conclusion, these results clarify the role of core gp120 glycosylation and illustrate a general method for designing glycan-free folded protein derivatives.

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“…To map epitopes in polyclonal sera, multi-cycle infectivity assays (56) Table S3). The PCR products have an average read length of 420 bp, with an overlap of ~63 bp between successive fragments.…”

Section: Multi-cycle Infectivity Of Labeled Excyslib With Polyclonal mentioning

confidence: 99%

A facile method of mapping HIV-1 neutralizing epitopes using chemically masked cysteines and deep sequencing

Datta

Chowdhury

Manjunath

et al. 2020

Preprint

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13Identification of specific epitopes targeted by neutralizing antibodies is essential to advance 14 epitope-based vaccine design strategies. We report a methodology for rapid epitope mapping 15 of neutralizing antibodies against HIV-1 Env at single residue resolution, using viral 16 neutralization assays and deep sequencing. We extended our methodology to map multiple 17 specificities of epitopes targeted in polyclonal sera, elicited in immunized animals as well as 18 in HIV-We recently described a methodology to decipher epitopes of monoclonal antibody panels 35 using yeast surface display, Cys labeling and deep sequencing 1, 2 . Here we built on this to map 36 epitopes of neutralizing monoclonal antibodies and polyclonal sera against the HIV-1 virus. 37 The design of an effective vaccine against HIV-1 has met with limited success so far, because 38 of the inability of immunogens to elicit broadly neutralizing antibodies (bNAbs) targeted to the 39 trimeric envelope (Env) glycoprotein, the sole viral antigen on the surface of the virus. Most 40 bNAbs isolated from infected individuals are directed to one of the major sites of vulnerability 41 on Env: V1/V2 loop apex, V3 loop, CD4-binding site, centre of the gp120 silent face, gp120-42 gp41 subunit interface, gp41 fusion peptide and membrane proximal external region (MPER) 43 of gp41. 44 We developed a facile methodology for mapping neutralizing HIV-1 epitopes at single residue 45 resolution. To this end, a library of single-site Cys mutations were engineered at selected 46 solvent-exposed sites in the Env glycoprotein on the surface of the virus. Cys mutants were 47 covalently labeled using a Cys reactive reagent (Maleimide-PEG2-Biotin) that masks epitope 48 residues, followed by infection of the labeled mutant viruses in mammalian cells in the 49 presence of bNAbs. Subsequently, deep sequencing of the env gene from bNAb-resistant 50 viruses was used to delineate epitopes ( Figure 1). We focused our epitope mapping efforts to 51 the Env ectodomain of the clade B, CCR5-tropic primary HIV-1 isolate JRFL, which has been 52 extensively characterized both biochemically and structurally, and is used as a model primary 53 virus. 54A total of 81 solvent-exposed residues (Table S1) were selected from the X-ray crystal structure 55 of the pre-fusion HIV-1 Env ectodomain using a combined criterion of >30% solvent 56 accessibility and sidechain-sidechain centroid distance >8 Å ( Figure S1). This led to the 57 identification of a set of solvent-exposed residues which collectively exhibit uniform surface 58 coverage of the Env trimer ( Figure S2). These Env residues were mutated to Cys in the pLAI-59 JRFL molecular clone, pooled in equimolar quantities to create a plasmid DNA library, and 60 transfected in HEK293T cells to produce the Exposed Cys Library, a library of single-site Cys 61 mutant viruses. Wt Env lacks free Cys residues. In single-cycle and multi-cycle infectivity 62 assays, the Exposed Cys Library retained significant viral infectivity ( Figure S3) and ...

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Insights into the activity of maturation inhibitor PF-46396 on HIV-1 clade C

Cited by 8 publications

References 45 publications

A single G10T polymorphism in HIV-1 subtype C Gag-SP1 regulates sensitivity to maturation inhibitors

A single G10T polymorphism in HIV-1 subtype C Gag-SP1 regulates sensitivity to maturation inhibitors

Glycosylation of the core of the HIV-1 envelope subunit protein gp120 is not required for native trimer formation or viral infectivity

A facile method of mapping HIV-1 neutralizing epitopes using chemically masked cysteines and deep sequencing

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