Characterization of twin-cysteine motif in the V2-loop region of gp120 in primate lentiviruses

Bowder, Dane; Thompson, Jay; Durst, Kate; Hollingsead, Haley; Hu, Duoyi; Wei, William; Xiang, Shi Hua

doi:10.1016/j.virol.2018.04.013

Cited by 5 publications

(5 citation statements)

References 43 publications

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“…After cells were cultured for 18 h, the medium was harvested, and the cells were lysed using a mild NP-40-based lysis buffer. The two fractions were treated, first with normal human serum (lot 6031174; Community Blood Bank) and protein A-Sepharose beads to clear background proteins and then with HIV-1 (subtype B)-positive patient sera (Community Blood Bank) and fresh beads to immunoprecipitate the radiolabeled Env proteins as described previously (41). The beads were mixed with SDS loading buffer, and then the protein was separated by SDS-PAGE.…”

Section: Methodsmentioning

confidence: 99%

“…The beads were mixed with SDS loading buffer, and then the protein was separated by SDS-PAGE. After vacuum fixation, protein expression was quantified using Quantity One software (Bio-Rad) (41). The association index, which indicates the ability of mutant gp120 to remain associated with the trimer complex on the cell membrane relative to WT gp120, was calculated using the following equation: association index ϭ (lysate gp120 mutant /supernatant gp120 mutant ) ϫ (supernatant gp120 wild-type /lysate gp120 wild-type ).…”

Section: Methodsmentioning

confidence: 99%

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The Polar Region of the HIV-1 Envelope Protein Determines Viral Fusion and Infectivity by Stabilizing the gp120-gp41 Association

Chen

et al. 2019

J Virol

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HIV-1 enters cells through binding between viral envelope glycoprotein (Env) and cellular receptors to initiate virus and cell fusion. HIV-1 Env precursor (gp160) is cleaved into two units noncovalently bound to form a trimer on virions, including a surface unit (gp120) and a transmembrane unit (gp41) responsible for virus binding and membrane fusion, respectively. The polar region (PR) at the N terminus of gp41 comprises 17 residues, including 7 polar amino acids. Previous studies suggested that the PR contributes to HIV-1 membrane fusion and infectivity; however, the precise role of the PR in Env-mediated viral entry and the underlying mechanisms remain unknown. Here, we show that the PR is critical for HIV-1 fusion and infectivity by stabilizing Env trimers. Through analyzing the PR sequences of 57,645 HIV-1 isolates, we performed targeted mutagenesis and functional studies of three highly conserved polar residues in the PR (S532P, T534A, and T536A) which have not been characterized previously. We found that single or combined mutations of these three residues abolished or significantly decreased HIV-1 infectivity without affecting viral production. These PR mutations abolished or significantly reduced HIV-1 fusion with target cells and also Env-mediated cell-cell fusion. Three PR mutations containing S532P substantially reduced gp120 and gp41 association, Env trimer stability, and increased gp120 shedding. Furthermore, S532A mutation significantly reduced HIV-1 infectivity and fusogenicity but not Env expression and cleavage. Our findings suggest that the PR of gp41, particularly the key residue S532, is structurally essential for maintaining HIV-1 Env trimer, viral fusogenicity, and infectivity. IMPORTANCE Although extensive studies of the transmembrane unit (gp41) of HIV-1 Env have led to a fusion inhibitor clinically used to block viral entry, the functions of different domains of gp41 in HIV-1 fusion and infectivity are not fully elucidated. The polar region (PR) of gp41 has been proposed to participate in HIV-1 membrane fusion in biochemical analyses, but its role in viral entry and infectivity remain unclear. In our effort to characterize three nucleotide mutations of an HIV-1 RNA element that partially overlaps the PR coding sequence, we identified a novel function of the PR that determines viral fusion and infectivity. We further demonstrated the structural and functional impact of six PR mutations on HIV-1 Env stability, viral fusion, and infectivity. Our findings reveal the previously unappreciated function of the PR and the underlying mechanisms, highlighting the important role of the PR in regulating HIV-1 fusion and infectivity.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

The Polar Region of the HIV-1 Envelope Protein Determines Viral Fusion and Infectivity by Stabilizing the gp120-gp41 Association

Chen

et al. 2019

J Virol

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“…Furthermore, V1 Ab responses were more frequent in SIV compared to HIV-1 immunizations. The presence of additional disulfide bonds in V1 and V2 of SIVmac is the likely cause for these observations (Figure 2) [70]. Differences in glycan content and composition (complex/high-mannose glycans) and the capacity of Env glycoproteins to stimulate the expression of genes involved in innate or adaptive immune responses in the context of different vectors and adjuvants may have contributed to the observed dissimilarities in immunogenicity between SIV and HIV-1 [38,69,71,72,73,74,75,76].…”

Section: The Multifaceted V1v2 Region and Its Epitope-specific Absmentioning

confidence: 99%

V2-Specific Antibodies in HIV-1 Vaccine Research and Natural Infection: Controllers or Surrogate Markers

Duerr

Gorny

2019

Vaccines

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Most human immunodeficiency virus (HIV) vaccine trials have lacked efficacy and empirical vaccine lead targets are scarce. Thus far, the only independent correlate of reduced risk of HIV-1 acquisition in humans is elevated levels of V2-specific antibodies identified in the modestly protective RV144 vaccine trial. Ten years after RV144, human and non-human primate vaccine studies have reassessed the potential contribution of V2-specific antibodies to vaccine efficacy. In addition, studies of natural HIV-1 infection in humans have provided insight into the development of V1V2-directed antibody responses and their impact on clinical parameters and disease progression. Functionally diverse anti-V2 monoclonal antibodies were isolated and their structurally distinct V2 epitope regions characterized. After RV144, a plethora of research studies were performed using different model systems, immunogens, protocols, and challenge viruses. These diverse studies failed to provide a clear picture regarding the contribution of V2 antibodies to vaccine efficacy. Here, we summarize the biological functions and clinical findings associated with V2-specific antibodies and discuss their impact on HIV vaccine research.

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“…They also demonstrated in silico that the twin-cysteines form a disulfide bond in the gp120 subunit, which interacts with the V1 loop to stabilize the envelope trimer (Bohl et al, 2013). In HIV-1, the V2 loop enforces trimer stabilization and co-receptor binding (Bowder et al, 2018). Analysis of plasma IgG anti-gp70V1V2 antibodies indicated an inverse correlation with risk for HIV-1 acquisition in a RV144 human vaccine efficacy trial (Powell et al, 2020).…”

Section: Variable Loopsmentioning

confidence: 98%

“…The second variable loop (V2) has extensive sequence variability between HIV-1 strains; nonetheless, its structural role as trimer stabilizer (Cimbro et al, 2014), functioning like a stapler, demands conservation in specific microdomain structural elements critical for functionality. The V2 loop is a conserved evolutionary element in all primate lentiviruses except for HIV-1, which carries variability in the loops sequence (Bowder et al, 2018). Experiments conducted by Bowder et al demonstrated that two cysteines in the V2 loop of SIVsm/HIV-2 lineage are critical for gp120 stability, and removal of the twin-cysteines decreases envelope trimer stability.…”

Section: Variable Loopsmentioning

confidence: 99%

gp120 Alters Its Conformation to Enhance Evasiveness and Infectivity

Ayariga¹,

Gildea²,

Ipimoroti³

et al. 2021

Preprint

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Infection by human immunodeficiency virus type I (HIV-1) requires virus particle binding to host cell-surface receptor CD4 via the viral envelope glycoprotein gp120. HIV-1 therapy and prevention efforts involve development of mimetic or recombinant gp120 vaccines or deployment of antiviral agents that target specific epitopes of gp120. The unliganded conformational state of gp120 is closed, whereas the CD4-bound state is open. However, in between, there exist dynamic conformational states, indicating intrinsically flexible region(s) of structural dynamics, imposing a structural challenge for developing drug or antibody targets. Known conformational states of gp120 were determined by X-ray crystallographic and cryo-electron microscopy, and neither method captures the population of gp120 species arising from conformational plasticity, motions, and transitions. gp120 plasticity brings up several important questions. How will differences in conformation affect receptor binding, antibody recognition, and neutralization? Which regions are crucial for gp120 structural plasticity? How could structural dynamics influence HIV-1 evasiveness against host immunity and drugs or vaccines, and facilitate the viral entry into its host? This review explores the structural constraints presented by conformational states of the glycoprotein to antibodies or drugs and how these conformational states provide structural avenues for the virus to escape neutralizing agents and evade host immunity.

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Characterization of twin-cysteine motif in the V2-loop region of gp120 in primate lentiviruses

Cited by 5 publications

References 43 publications

The Polar Region of the HIV-1 Envelope Protein Determines Viral Fusion and Infectivity by Stabilizing the gp120-gp41 Association

The Polar Region of the HIV-1 Envelope Protein Determines Viral Fusion and Infectivity by Stabilizing the gp120-gp41 Association

V2-Specific Antibodies in HIV-1 Vaccine Research and Natural Infection: Controllers or Surrogate Markers

gp120 Alters Its Conformation to Enhance Evasiveness and Infectivity

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