Functional Anatomy of the Trimer Apex Reveals Key Hydrophobic Constraints That Maintain the HIV-1 Envelope Spike in a Closed State.

The human immunodeficiency virus type 1 (HIV-1) envelope trimer maintains a closed, metastable configuration to protect vulnerable epitopes from neutralizing antibodies. Here, we identify key hydrophobic constraints at the trimer apex that function as global stabilizers of the HIV-1 envelope spike configuration. Mutation of individual residues within four hydrophobic clusters that fasten together the V1V2, V3, and C4 domains at the apex of gp120 dramatically increases HIV-1 sensitivity to weak and restricted neutralizing antibodies targeting epitopes that are largely concealed in the prefusion Env spike, consistent with the adoption of a partially open trimer configuration. Conversely, the same mutations decrease the sensitivity to broad and potent neutralizing antibodies that preferentially recognize the closed trimer. Sera from chronically HIV-infected patients neutralize open mutants with enhanced potency, compared to the wild-type virus, suggesting that a large fraction of host-generated antibodies target concealed epitopes. The identification of structural constraints that maintain the HIV-1 envelope in an antibody-protected state may inform the design of a protective vaccine.IMPORTANCE Elucidating the structure and function of the HIV-1 envelope proteins is critical for the design of an effective vaccine. Despite the availability of many high-resolution structures, key functional correlates in the envelope trimer remain undefined. We utilized a combination of structural analysis, in silico energy calculation, mutagenesis, and neutralization profiling to dissect the functional anatomy of the trimer apex, which acts as a global regulator of the HIV-1 spike conformation. We identify four hydrophobic clusters that stabilize the spike in a tightly closed configuration and, thereby, play a critical role in protecting it from the reach of neutralizing antibodies.

Structural Constraints at the Trimer Apex Stabilize the HIV-1 Envelope in a Closed, Antibody-Protected Conformation.

The human immunodeficiency virus type 1 (HIV-1) envelope (Env) trimer evades antibody recognition by adopting a closed prefusion conformation. Here, we show that two conserved tyrosines (Y173, Y177) within the second variable (V2) loop of the gp120 Env glycoprotein are key regulators of the closed, antibody-protected state of the trimer by establishing intramolecular interaction with the base of the third variable (V3) loop. Mutation of Y177 and/or Y173 to phenylalanine or alanine dramatically altered the susceptibility of diverse HIV-1 strains to neutralization, increasing sensitivity to weakly and nonneutralizing antibodies directed against diverse Env regions, consistent with the adoption of an open trimer configuration. Conversely, potent broadly neutralizing antibodies (bNAbs) against different supersites of HIV-1 vulnerability exhibited reduced potency against V2 loop tyrosine mutants, consistent with their preferential targeting of the closed trimer. Mutation of V3 loop residues predicted to interact with the V2 loop tyrosines yielded a similar neutralization phenotype. Sera from chronically HIV-1-infected patients contained very high titers of antibodies capable of neutralizing V2 loop tyrosine mutants but not wild-type viruses, indicating that the bulk of antibodies produced in infected hosts are unable to penetrate the protective shield of the closed trimer. These results identify the tyrosine-mediated V2-V3 loop complex at the trimer apex as a key structural constraint that facilitates HIV-1 evasion from the bulk of host antibodies.IMPORTANCE The extraordinary ability of human immunodeficiency virus type 1 (HIV-1) to evade host immunity represents a major obstacle to the development of a protective vaccine. Thus, elucidating the mechanisms whereby HIV-1 protects its external envelope (Env), which is the sole target of virus-neutralizing antibodies, is an essential step toward vaccine design. We identified a key structural element that maintains the HIV-1 Env trimer in a closed, antibody-resistant conformation. A major role is played by two conserved tyrosines at the apex of the Env spike, whose mutation causes a global opening of the trimer structure, exposing multiple concealed targets for neutralizing antibodies. We also found that HIV-infected individuals produce very large amounts of antibodies that neutralize the open Env form; however, the bulk of these antibodies are unable to penetrate the tight defensive shield of the native virus. This work may help to devise new strategies to overcome the viral defensive mechanisms and facilitate the development of an effective HIV-1 vaccine.