Étude du trafic intracellulaire de la protéine Gag du VIH-1 : rôle des facteurs de l'hôte

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal

Lineage-specific differences in the gp120 Inner Domain Layer 3 of Human and Simian Immunodeficiency Viruses

Binding of HIV-1 and SIV gp120 exterior envelope glycoprotein to CD4 triggers conformational changes in gp120 that promote its interaction with one of the chemokine receptors, usually CCR5, ultimately leading to gp41-mediated virus-cell membrane fusion and entry. We previously described that topological Layers (Layer 1, Layer 2 and Layer 3) in the gp120 inner domain contribute to gp120-trimer association in the unliganded state but also help secure CD4 binding. Relative to Layer 1 of HIV-1 gp120, the SIVmac239 gp120 Layer 1 plays a more prominent role in maintaining gp120-trimer association but is minimally involved in promoting CD4 binding, which could be explained by the existence of a well-conserved Tryptophan 375 (Trp 375) in HIV-2/SIVsmm. Here we investigated the role of SIV Layer 3 on viral entry, cell-to-cell fusion and CD4 binding. We observed that a network of interactions involving some residues of the β8-α5 region in SIVmac239 Layer 3 may contribute to CD4 binding by helping shape the nearby Phe 43 cavity which directly contacts CD4. In summary, our results suggest that SIV Layer 3 has a greater impact on CD4 binding than in HIV-1. This work defines lineage-specific differences in Layer 3 from HIV-1 and SIV

Lineage-specific differences in the gp120 Inner Domain Layer 3 of Human and Simian Immunodeficiency Viruses

Binding of HIV-1 and SIV gp120 exterior envelope glycoprotein to CD4 triggers conformational changes in gp120 that promote its interaction with one of the chemokine receptors, usually CCR5, ultimately leading to gp41-mediated virus-cell membrane fusion and entry. We previously described that topological Layers (Layer 1, Layer 2 and Layer 3) in the gp120 inner domain contribute to gp120-trimer association in the unliganded state but also help secure CD4 binding. Relative to Layer 1 of HIV-1 gp120, the SIVmac239 gp120 Layer 1 plays a more prominent role in maintaining gp120-trimer association but is minimally involved in promoting CD4 binding, which could be explained by the existence of a well-conserved Tryptophan 375 (Trp 375) in HIV-2/SIVsmm. Here we investigated the role of SIV Layer 3 on viral entry, cell-to-cell fusion and CD4 binding. We observed that a network of interactions involving some residues of the β8-α5 region in SIVmac239 Layer 3 may contribute to CD4 binding by helping shape the nearby Phe 43 cavity which directly contacts CD4. In summary, our results suggest that SIV Layer 3 has a greater impact on CD4 binding than in HIV-1. This work defines lineage-specific differences in Layer 3 from HIV-1 and SIV

HIV-1 Vpr-Mediated G2 Arrest Involves the DDB1-CUL4AVPRBP E3 Ubiquitin Ligase

Human immunodeficiency virus type 1 (HIV-1) viral protein R (Vpr) has been shown to cause G2 cell cycle arrest in human cells by inducing ATR-mediated inactivation of p34cdc2, but factors directly engaged in this process remain unknown. We used tandem affinity purification to isolate native Vpr complexes. We found that damaged DNA binding protein 1 (DDB1), viral protein R binding protein (VPRBP), and cullin 4A (CUL4A)—components of a CUL4A E3 ubiquitin ligase complex, DDB1-CUL4AVPRBP—were able to associate with Vpr. Depletion of VPRBP by small interfering RNA impaired Vpr-mediated induction of G2 arrest. Importantly, VPRBP knockdown alone did not affect normal cell cycle progression or activation of ATR checkpoints, suggesting that the involvement of VPRBP in G2 arrest was specific to Vpr. Moreover, leucine/isoleucine-rich domain Vpr mutants impaired in their ability to interact with VPRBP and DDB1 also produced strongly attenuated G2 arrest. In contrast, G2 arrest–defective C-terminal Vpr mutants were found to maintain their ability to associate with these proteins, suggesting that the interaction of Vpr with the DDB1-VPRBP complex is necessary but not sufficient to block cell cycle progression. Overall, these results point toward a model in which Vpr could act as a connector between the DDB1-CUL4AVPRBP E3 ubiquitin ligase complex and an unknown cellular factor whose proteolysis or modulation of activity through ubiquitination would activate ATR-mediated checkpoint signaling and induce G2 arrest

Contribution of the gp120 V3 loop to envelope glycoprotein trimer stability in primate immunodeficiency viruses

The V3 loop of the human immunodeficiency virus type 1 (HIV-1) gp120 exterior envelope glycoprotein (Env) becomes exposed after CD4 binding and contacts the coreceptor to mediate viral entry. Prior to CD4 engagement, a hydrophobic patch located at the tip of the V3 loop stabilizes the non-covalent association of gp120 with the Env trimer of HIV-1 subtype B strains. Here, we show that this conserved hydrophobic patch (amino acid residues 307, 309 and 317) contributes to gp120-trimer association in HIV-1 subtype C, HIV-2 and SIV. Changes that reduced the hydrophobicity of these V3 residues resulted in increased gp120 shedding and decreased Envmediated cell-cell fusion and virus entry in the different primate immunodeficiency viruses tested. Thus, the hydrophobic patch is an evolutionarily conserved element in the tip of the gp120 V3 loop that plays an essential role in maintaining the stability of the pre-triggered Env trimer in diverse primate immunodeficiency viruses

Paring Down HIV Env: Design and Crystal Structure of a Stabilized Inner Domain of HIV-1 gp120 Displaying a Major ADCC Target of the A32 Region

SummaryEvidence supports a role of antibody-dependent cellular cytotoxicity (ADCC) toward transitional epitopes in the first and second constant (C1-C2) regions of gp120 (A32-like epitopes) in preventing HIV-1 infection and in vaccine-induced protection. Here, we describe the first successful attempt at isolating the inner domain (ID) of gp120 as an independent molecule that encapsulates the A32-like region within a minimal structural unit of the HIV-1 Env. Through structure-based design, we developed ID2, which consists of the ID expressed independently of the outer domain and stabilized in the CD4-bound conformation by an inter-layer disulfide bond. ID2 expresses C1-C2 epitopes in the context of CD4-triggered full-length gp120 but without any known neutralizing epitope present. Thus, ID2 represents a novel probe for the analysis and/or selective induction of antibody responses to the A32 epitope region. We also present the crystal structure of ID2 complexed with mAb A32, which defines its epitope

A Highly-Conserved Residue of the HIV-1-gp120 Inner Domain is Important for ADCC Responses Mediated by Anti-Cluster A Antibodies

Previous studies have shown that sera from HIV-1-infected individuals contain antibodies able to mediate antibody-dependent cellular cytotoxicity (ADCC). These antibodies preferentially recognize envelope glycoprotein (Env) epitopes induced upon CD4 binding. Here, we show that a highly conserved tryptophan at position 69 of the gp120 inner domain is important for ADCC mediated by anti-cluster A antibodies and sera from HIV-1-infected individuals

SARS-CoV-2 Variants Increase Kinetic Stability of Open Spike Conformations as an Evolutionary Strategy

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) harbor mutations in the spike (S) glycoprotein that confer more efficient transmission and dampen the efficacy of COVID-19 vaccines and antibody therapies. S mediates virus entry and is the primary target for antibody responses, with structural studies of soluble S variants revealing an increased propensity toward conformations accessible to the human angiotensin-converting enzyme 2 (hACE2) receptor. However, real-time observations of conformational dynamics that govern the structural equilibriums of the S variants have been lacking. Here, we report single-molecule Förster resonance energy transfer (smFRET) studies of critical mutations observed in VOCs, including D614G and E484K, in the context of virus particles. Investigated variants predominately occupied more open hACE2-accessible conformations, agreeing with previous structures of soluble trimers. Additionally, these S variants exhibited slower transitions in hACE2-accessible/bound states. Our finding of increased S kinetic stability in the open conformation provides a new perspective on SARS-CoV-2 adaptation to the human population

The molecular basis of the neutralization breadth of the RBD-specific antibody CoV11

SARS-CoV-2, the virus behind the COVID-19 pandemic, has changed over time to the extent that the current virus is substantially different from what originally led to the pandemic in 2019–2020. Viral variants have modified the severity and transmissibility of the disease and continue do so. How much of this change is due to viral fitness versus a response to immune pressure is hard to define. One class of antibodies that continues to afford some level of protection from emerging variants are those that closely overlap the binding site for angiotensin-converting enzyme 2 (ACE2) on the receptor binding domain (RBD). Some members of this class that were identified early in the course of the pandemic arose from the VH 3-53 germline gene (IGHV3-53*01) and had short heavy chain complementarity-determining region 3s (CDR H3s). Here, we describe the molecular basis of the SARS-CoV-2 RBD recognition by the anti-RBD monoclonal antibody CoV11 isolated early in the COVID-19 pandemic and show how its unique mode of binding the RBD determines its neutralization breadth. CoV11 utilizes a heavy chain VH 3-53 and a light chain VK 3-20 germline sequence to bind to the RBD. Two of CoV11’s four heavy chain changes from the VH 3-53 germline sequence, ThrFWR H128 to Ile and SerCDR H131 to Arg, and some unique features in its CDR H3 increase its affinity to the RBD, while the four light chain changes from the VK 3-20 germline sequence sit outside of the RBD binding site. Antibodies of this type can retain significant affinity and neutralization potency against variants of concern (VOCs) that have diverged significantly from original virus lineage such as the prevalent omicron variant. We also discuss the mechanism by which VH 3-53 encoded antibodies recognize spike antigen and show how minimal changes to their sequence, their choice of light chain, and their mode of binding influence their affinity and impact their neutralization breadth