The envelope glycoproteins (Envs) of HIV-1 are embedded in the cholesterol-rich lipid membrane of the virus. Chemical depletion of cholesterol from HIV-1 particles inactivates their infectivity. We observed that diverse HIV-1 strains exhibit a range of sensitivities to such treatment. Differences in sensitivity to cholesterol depletion could not be explained by variation in Env components known to interact with cholesterol, including the cholesterol-recognition motif and cytoplasmic tail of gp41. Using antibody-binding assays, measurements of virus infectivity, and analyses of lipid membrane order, we found that depletion of cholesterol from HIV-1 particles decreases the conformational stability of Env. It enhances exposure of partially cryptic epitopes on the trimer and increases sensitivity to structure-perturbing treatments such as antibodies and cold denaturation. Substitutions in the cholesterol-interacting motif of gp41 induced similar effects as depletion of cholesterol. Surface-acting agents, which are incorporated into the virus lipid membrane, caused similar effects as disruption of the Env-cholesterol interaction. Furthermore, substitutions in gp120 that increased structural stability of Env (i.e. induced a “closed” conformation of the trimer) increased virus resistance to cholesterol depletion and to the surface-acting agents. Collectively, these results indicate a critical contribution of the viral membrane to the stability of the Env trimer and to neutralization resistance against antibodies. Our findings suggest that the potency of poorly neutralizing antibodies, which are commonly elicited in vaccinated individuals, may be markedly enhanced by altering the lipid composition of the viral membrane.

The envelope glycoproteins (Envs) on the surfaces of HIV-1 particles are targeted by host antibodies. Primary HIV-1 isolates demonstrate different global sensitivities to antibody neutralization; tier-1 isolates are sensitive, whereas tier-2 isolates are more resistant. Single-site mutations in Env can convert tier-2 into tier-1-like viruses. We hypothesized that such global change in neutralization sensitivity results from weakening of intramolecular interactions that maintain Env integrity. Three strategies commonly applied to perturb protein structure were tested for their effects on global neutralization sensitivity: exposure to low temperature, Envactivating ligands, and a chaotropic agent. A large panel of diverse tier-2 isolates from clades B and C was analyzed. Incubation at 0°C, which globally weakens hydrophobic interactions, causes gradual and reversible exposure of the coreceptorbinding site. In the cold-induced state, Envs progress at isolate-specific rates to unstable forms that are sensitive to antibody neutralization and then gradually lose function. Agents that mimic the effects of CD4 (CD4Ms) also induce reversible structural changes to states that exhibit isolate-specific stabilities. The chaotropic agent urea (at low concentrations) does not affect the structure or function of native Env. However, urea efficiently perturbs metastable states induced by cold and CD4Ms and increases their sensitivity to antibody neutralization and their inactivation rates Therefore, chemical and physical agents can guide Env from the stable native state to perturbation-sensitive forms and modulate their stability to bestow tier-1-like properties on primary tier-2 strains. These concepts can be applied to enhance the potency of vaccine-elicited antibodies and microbicides at mucosal sites of HIV-1 transmission.IMPORTANCE An effective vaccine to prevent transmission of HIV-1 is a primary goal of the scientific and health care communities. Vaccine-elicited antibodies target the viral envelope glycoproteins (Envs) and can potentially inhibit infection. However, the potency of such antibodies is generally low. Single-site mutations in Env can enhance the global sensitivity of HIV-1 to neutralization by antibodies. We found that such a hypersensitivity phenotype can also be induced by agents that destabilize protein structure. Exposure to 0°C or low concentrations of Env-activating ligands gradually guides Env to metastable forms that expose cryptic epitopes and that are highly sensitive to neutralization. Low concentrations of the chaotropic agent urea do not affect native Env but destabilize perturbed states induced by cold or CD4Ms and increase their neutralization. The concept of enhancing antibody sen-

The envelope glycoproteins (Envs) of HIV-1 continuously evolve in the host by random mutations and recombination events. The resulting diversity of Env variants circulating in the population and their continuing diversification process limit the efficacy of AIDS vaccines. We examined the historic changes in Env sequence and structural features (measured by integrity of epitopes on the Env trimer) in a geographically defined population in the United States. As expected, many Env features were relatively conserved during the 1980s. From this state, some features diversified whereas others remained conserved across the years. We sought to identify “clues” to predict the observed historic diversification patterns. Comparison of viruses that cocirculate in patients at any given time revealed that each feature of Env (sequence or structural) exists at a defined level of variance. The in-host variance of each feature is highly conserved among individuals but can vary between different HIV-1 clades. We designate this property “volatility” and apply it to model evolution of features as a linear diffusion process that progresses with increasing genetic distance. Volatilities of different features are highly correlated with their divergence in longitudinally monitored patients. Volatilities of features also correlate highly with their population-level diversification. Using volatility indices measured from a small number of patient samples, we accurately predict the population diversity that developed for each feature over the course of 30 years. Amino acid variants that evolved at key antigenic sites are also predicted well. Therefore, small “fluctuations” in feature values measured in isolated patient samples accurately describe their potential for population-level diversification. These tools will likely contribute to the design of population-targeted AIDS vaccines by effectively capturing the diversity of currently circulating strains and addressing properties of variants expected to appear in the future.

Semen is the primary transmission vehicle for various pathogenic viruses. Initial steps of transmission, including cell attachment and entry, likely occur in the presence of semen. However, the unstable nature of human seminal plasma and its toxic effects on cells in culture limit the ability to study in vitro virus infection and inhibition in this medium. We found that whole semen significantly reduces the potency of antibodies and microbicides that target glycans on the envelope glycoproteins (Envs) of HIV-1. The extraordinarily high concentration of the monosaccharide fructose in semen contributes significantly to the effect by competitively inhibiting the binding of ligands to α1,2-linked mannose residues on Env. Infection and inhibition in whole human seminal plasma are accurately mimicked by a stable synthetic simulant of seminal fluid that we formulated. Our findings indicate that, in addition to the protein content of biological secretions, their small-solute composition impacts the potency of antiviral microbicides and mucosal antibodies. IMPORTANCE Biological secretions allow viruses to spread between individuals. Each type of secretion has a unique composition of proteins, salts, and sugars, which can affect the infectivity potential of the virus and inhibition of this process. Here, we describe HIV-1 infection and inhibition in whole human seminal plasma and a synthetic simulant that we formulated. We discovered that the sugar fructose in semen decreases the activity of a broad and potent class of antiviral agents that target mannose sugars on the envelope protein of HIV-1. This effect of semen fructose likely reduces the efficacy of such inhibitors to prevent the sexual transmission of HIV-1. Our findings suggest that the preclinical evaluation of microbicides and vaccine-elicited antibodies will be improved by their in vitro assessment in synthetic formulations that simulate the effects of semen on HIV-1 infection and inhibition.

HIV-1 group M was transmitted to humans nearly one century ago. The virus has since evolved to form distinct clades, which spread to different regions of the world. The envelope glycoproteins (Envs) of HIV-1 have rapidly diversified in all infected populations. We examined whether key antigenic sites of Env and signatures of vaccine efficacy are evolving toward similar or distinct structural forms in different populations worldwide. Patterns of amino acid variants that emerged at each position of Env were compared between diverse HIV-1 clades and isolates from different geographic regions. Interestingly, at each Env position, the amino acid in the clade ancestral or regional-founder virus was replaced by a unique frequency distribution (FD) of amino acids. FDs are highly conserved in populations from different regions worldwide and in paraphyletic and monophyletic subclade groups. Remarkably, founder effects of Env mutations at the clade and regional levels have gradually decreased during the pandemic by evolution of each site toward the unique combination of variants. Therefore, HIV-1 Env is evolving at a population level toward well-defined “target” states; these states are not specific amino acids but rather specific distributions of amino acid frequencies. Our findings reveal the powerful nature of the forces that guide evolution of Env and their conservation across different populations. Such forces have caused a gradual decrease in the interpopulation diversity of Env despite an increasing intrapopulation diversity. IMPORTANCE The Env protein of HIV-1 is the primary target in AIDS vaccine design. Frequent mutations in the virus increase the number of Env forms in each population, limiting the efficacy of AIDS vaccines. Comparison of newly emerging forms in different populations showed that each position of Env is evolving toward a specific combination of amino acids. Similar changes are occurring in different HIV-1 subtypes and geographic regions toward the same position-specific combinations of amino acids, often from distinct ancestral sequences. The predictable nature of HIV-1 Env evolution, as shown here, provides a new framework for designing vaccines that are tailored to the unique combination of variants expected to emerge in each virus subtype and geographic region.