Molecular mechanism of HIV-1 resistance to sifuvirtide, a clinical trial–approved membrane fusion inhibitor

Yu, Danwei; Ding, Xiaohui; Liu, Zixuan; Wu, Xiyuan; Zhu, Yuanmei; Wei, Huanmian; Chong, Huihui; Cui, Sheng; He, Yuxian

doi:10.1074/jbc.ra118.003538

Cited by 22 publications

(27 citation statements)

References 57 publications

(51 reference statements)

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“…Several N/CHR mutations also enhanced entry into cells bearing macaque receptors by a second transmitted Env strain, indicating that their effects were not strain specific. While N/CHR mutations have previously been identified that heighten fusogenic activity [45,46] and increase infectivity of cells expressing low levels of CD4 and CCR5 receptors [40,47], none have been shown to enhance entry of cells bearing macaque receptors. Many of the N/CHR mutations we identified also enhanced entry into cells bearing human CD4 and CCR5 receptors, but the magnitude of this effect was smaller than during infection of cells bearing macaque receptors.…”

Section: Discussionmentioning

confidence: 99%

Identification of HIV-1 Envelope Mutations that Enhance Entry Using Macaque CD4 and CCR5

Roop

Cassidy

Dingens

et al. 2020

Viruses

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Although Rhesus macaques are an important animal model for HIV-1 vaccine development research, most transmitted HIV-1 strains replicate poorly in macaque cells. A major genetic determinant of this species-specific restriction is a non-synonymous mutation in macaque CD4 that results in reduced HIV-1 Envelope (Env)-mediated viral entry compared to human CD4. Recent research efforts employing either laboratory evolution or structure-guided design strategies have uncovered several mutations in Env's gp120 subunit that enhance binding of macaque CD4 by transmitted/founder HIV-1 viruses. In order to identify additional Env mutations that promote infection of macaque cells, we utilized deep mutational scanning to screen thousands of Env point mutants for those that enhance HIV-1 entry via macaque receptors. We identified many uncharacterized amino acid mutations in the N-terminal heptad repeat (NHR) and C-terminal heptad repeat (CHR) regions of gp41 that increased entry into cells bearing macaque receptors up to 9-fold. Many of these mutations also modestly increased infection of cells bearing human CD4 and CCR5 (up to 1.5-fold). NHR/CHR mutations identified by deep mutational scanning that enhanced entry also increased sensitivity to neutralizing antibodies targeting the MPER epitope, and to inactivation by cold-incubation, suggesting that they promote sampling of an intermediate trimer conformation between closed and receptor bound states. Identification of this set of mutations can inform future macaque model studies, and also further our understanding of the relationship between Env structure and function.Viruses 2020, 12, 241 2 of 20 in macaque cells [7], most studies employing the SHIV/macaque model have used HIV-1 Envs that were lab adapted to enhance macaque infection, and/or that were derived from HIV-1 variants isolated from the chronic stages of infection [7]. These evolved and chronic-stage Envs are highly pathogenic and transmissible in macaques [8]. While these characteristics enabled robust infection of macaques, evolved and chronic-stage Envs display altered structural conformations and increased susceptibility to neutralizing antibodies compared to Envs from circulating HIV-1 strains [8,9]. Use of these Envs in SHIVs therefore compromises the relevance of the SHIV/macaque model for informing HIV-1 infection in humans [7].In 2012, it was discovered that a single amino acid mutation in macaque CD4 was responsible for the poor ability of HIV-1 to utilize macaque receptors for entry [3]. In vitro evolution experiments also revealed that single amino acid substitutions at sites 204 or 312 in Env's gp120 subunit allow HIV-1 to more effectively use macaque CD4 as an entry receptor [4]. These mutations disrupted quaternary contacts between Env trimer subunits, and similar to Envs encoded by highly pathogenic SHIVs, increased sensitivity to neutralizing antibodies [10].Identification of these mutants prompted further efforts to identify additional Env mutations that would enhance usage of macaque CD4. In 2016, L...

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Section: Discussionmentioning

confidence: 99%

Identification of HIV-1 Envelope Mutations that Enhance Entry Using Macaque CD4 and CCR5

Roop

Cassidy

Dingens

et al. 2020

Viruses

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“…N/CHR mutations enhanced entry into cells bearing macaque receptors by a second transmitted Env strain, indicating that their effects were not strain specific, but they did not confer a CD4 independent phenotype. While N/CHR mutations have previously been identified that heighten fusogenic activity (39, 40) and increase infectivity of cells expressing low levels of CD4 and CCR5 receptors (34, 41), none have been shown to enhance entry of cells bearing macaque receptors. Many of the N/CHR mutations we identified also enhanced entry into cells bearing human CD4 and CCR5 receptors, but the magnitude of this effect was smaller than during infection of cells bearing macaque receptors.…”

Section: Discussionmentioning

confidence: 99%

Identification of HIV-1 Envelope Mutations that Enhance Entry Using Macaque CD4 and CCR5

Roop

Cassidy

Dingens

et al. 2019

Preprint

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8Although Rhesus macaques are an important animal model for HIV-1 vaccine development 9research, most transmitted HIV-1 strains replicate poorly in macaque cells. A major genetic 10 determinant of this species-specific restriction is a non-synonymous mutation in macaque CD4 11 that results in reduced HIV-1 Envelope (Env)-mediated viral entry compared to human CD4. 12 Recent research efforts employing either laboratory evolution or structure-guided design 13 strategies have uncovered several mutations in Env's gp120 subunit that enhance binding of 14 macaque CD4 by transmitted/founder HIV-1 viruses. In order to identify additional Env 15 mutations that promote infection of macaque cells, we utilized deep mutational scanning to 16 screen thousands of Env point mutants for those that enhance HIV-1 entry via macaque 17 receptors. We identified many uncharacterized amino acid mutations in the N-terminal heptad 18 repeat (NHR) and C-terminal heptad repeat (CHR) regions of gp41 that increased entry into cells 19 bearing macaque receptors by up to 38-fold. Many of these mutations also modestly increased 20 infection of cells bearing human CD4 and CCR5 (up to 13-fold). NHR/CHR mutations identified 21 by deep mutational scanning that enhanced entry also increased sensitivity to neutralizing 22 antibodies targeting the MPER epitope, and to inactivation by cold-incubation, suggesting that 23 they promote sampling of an intermediate trimer conformation between closed and receptor 24 bound states. Identification of this set of mutations can inform future macaque model studies, 25 and also further our understanding of the relationship between Env structure and function. 263 Importance 27Although Rhesus macaques are the favored non-human primate animal model used in HIV-1 28 research, most circulating HIV-1 strains poorly infect macaque cells. Studies using macaques to 29 model HIV-1 infection often use evolved, or mutant HIV-1 variants that are able to utilize 30 macaque CD4, but these HIV-1 variants poorly model infection by circulating strains. In this 31 work, we sought to identity HIV-1 mutations that would allow entry into macaque cells, but 32 that would maintain critical characteristics of circulating HIV-1 strains. We employed a powerful 33 experimental method to simultaneously assess the effects of thousands of individual HIV-1 34 mutations on infection of cells bearing macaque receptors. We identified many previously 35 uncharacterized mutations that enhance infection of circulating HIV-1 strains into cells bearing 36 macaque receptors by up to 38-fold. Identification of these mutations may be of use in future 37 macaque model studies. 38Env, used to model HIV-1 infection. As SHIVs encoding Envs from circulating HIV-1 strains 51 replicate poorly in macaque cells (7), most studies employing the SHIV/macaque model have 52 used HIV-1 Envs that were lab adapted to enhance macaque infection, and/or that were 53 derived from HIV-1 variants isolated from the chronic stages of infection (7). These evolved and 54 chronic s...

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“…By adding the M-T hook structure to the PBD sequence, we previously generated several highly potent short-peptide fusion inhibitors, including MTSC22EK, HP23, and 2P23, which mainly target the deep pocket sites rather than the T20 resistance sites [20][21][22]. As noted, the resistance profiles of both the C34 derivatives and M-T hook-based short peptides were previously characterized by the in vitro selection of HIV-1 variants that were resistant to the inhibitors, and interestingly, the secondary N126K mutation was universally found to accompany with the diverse primary resistance mutations [23][24][25][26][27][28][29][30]. In the CHR sequence, Asn126 locates immediately at the downstream of the PBD sequence and it is a highly conserved residue among all the HIV-1, HIV-2, and even simian immunodeficiency virus (SIV) isolates (www.hiv.lanl.gov).…”

Section: Introductionmentioning

confidence: 94%

“…The expression and processing profile of HIV-1 gp160 were determined by a capture enzyme-linked immunosorbent assay (ELISA) as described [25]. Briefly, the wells of an ELISA plate were coated with a sheep anti-gp120 antibody (D7324) at 10 µg/mL and blocked by 3% bovine serum albumin (BSA).…”

Section: Capture Elisamentioning

confidence: 99%

“…CD spectroscopy was performed to determine the α-helicity and thermostability of the peptide complexes as described previously [25]. Briefly, the NHR peptide N36 was incubated with an equal molar concentration of the CHR peptide C34 or its N126K mutant at 37 • C for 30 min in phosphate-buffered saline (PBS, pH 7.2).…”

Section: Circular Dichroism (Cd) Spectroscopymentioning

confidence: 99%

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Structural and Functional Characterization of the Secondary Mutation N126K Selected by Various HIV-1 Fusion Inhibitors

Sai

Ding

et al. 2020

Viruses

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Peptides derived from the C-terminal heptad repeat (CHR) region of HIV-1 gp41 is potent viral membrane fusion inhibitors, such as the first clinically approved peptide drug T20 and a group of newly-designed peptides. The resistance profiles of various HIV-1 fusion inhibitors were previously characterized, and the secondary mutation N126K in the gp41 CHR was routinely identified during the in vitro and in vivo selections. In this study, the functional and structural relevance of the N126K mutation has been characterized from multiple angles. First, we show that a single N126K mutation across several HIV-1 isolates conferred mild to moderate cross-resistances. Second, the N126K mutation exerted different effects on Env-mediated HIV-1 entry and cell-cell fusion. Third, the N126K mutation did not interfere with the expression and processing of viral Env glycoproteins, but it disrupted the Asn126-based glycosylation site in gp41. Fourth, the N126K mutation was verified to enhance the thermal stability of 6-HB conformation. Fifth, we determined the crystal structure of a 6-HB bearing the N126K mutation, which revealed the interhelical and intrahelical interactions underlying the increased thermostability. Therefore, our data provide new information to understand the mechanism of HIV-1 gp41-mediated cell fusion and its resistance mode to viral fusion inhibitors.

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Molecular mechanism of HIV-1 resistance to sifuvirtide, a clinical trial–approved membrane fusion inhibitor

Cited by 22 publications

References 57 publications

Identification of HIV-1 Envelope Mutations that Enhance Entry Using Macaque CD4 and CCR5

Identification of HIV-1 Envelope Mutations that Enhance Entry Using Macaque CD4 and CCR5

Identification of HIV-1 Envelope Mutations that Enhance Entry Using Macaque CD4 and CCR5

Structural and Functional Characterization of the Secondary Mutation N126K Selected by Various HIV-1 Fusion Inhibitors

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