Mapping the Evolutionary Space of SARS-CoV-2 Variants to Anticipate Emergence of Subvariants Resistant to COVID-19 Therapeutics.

New sublineages of SARS-CoV-2 variants-of-concern (VOCs) continuously emerge with mutations in the spike glycoprotein. In most cases, the sublineage-defining mutations vary between the VOCs. It is unclear whether these differences reflect lineage-specific likelihoods for mutations at each spike position or the stochastic nature of their appearance. Here we show that SARS-CoV-2 lineages have distinct evolutionary spaces (a probabilistic definition of the sequence states that can be occupied by expanding virus subpopulations). This space can be accurately inferred from the patterns of amino acid variability at the whole-protein level. Robust networks of co-variable sites identify the highest-likelihood mutations in new VOC sublineages and predict remarkably well the emergence of subvariants with resistance mutations to COVID-19 therapeutics. Our studies reveal the contribution of low frequency variant patterns at heterologous sites across the protein to accurate prediction of the changes at each position of interest.

Commonly Elicited Antibodies against the Base of the HIV-1 Env Trimer Guide the Population-Level Evolution of a Structure-Regulating Region in gp41.

The antibody response against the HIV-1 envelope glycoproteins (Envs) guides evolution of this protein within each host. Whether antibodies with similar target specificities are elicited in different individuals and affect the population-level evolution of Env is poorly understood. To address this question, we analyzed properties of emerging variants in the gp41 fusion peptide-proximal region (FPPR) that exhibit distinct evolutionary patterns in HIV-1 clade B. For positions 534, 536, and 539 in the FPPR, alanine was the major emerging variant. However, 534A and 536A show a constant frequency in the population between 1979 and 2016, whereas 539A is gradually increasing. To understand the basis for these differences, we introduced alanine substitutions in the FPPR of primary HIV-1 strains and examined their functional and antigenic properties. Evolutionary patterns could not be explained by fusion competence or structural stability of the emerging variants. Instead, 534A and 536A exhibited modest but significant increases in sensitivity to antibodies against the membrane-proximal external region (MPER) and gp120-gp41 interface. These Envs were also more sensitive to poorly neutralizing sera from HIV-1-infected individuals than the clade ancestral form or 539A variant. Competition binding assays confirmed for all sera tested the presence of antibodies against the base of the Env trimer that compete with monoclonal antibodies targeting the MPER and gp120-gp41 interface. Our findings suggest that weakly neutralizing antibodies against the trimer base are commonly elicited; they do not exert catastrophic population size reduction effects on emerging variants but, instead, determine their set point frequencies in the population and historical patterns of change. IMPORTANCE Infection by HIV-1 elicits formation of antibodies that target the viral Env proteins and can inactivate the virus. The specific targets of these antibodies vary among infected individuals. It is unclear whether some target specificities are shared among the antibody responses of different individuals. We observed that antibodies against the base of the Env protein are commonly elicited during infection. The selective pressure applied by such antibodies is weak. As a result, they do not completely eliminate the sensitive forms of the virus from the population, but maintain their frequency at a low level that has not increased since the beginning of the AIDS pandemic. Interestingly, the changes in Env do not occur at the sites targeted by the antibodies, but at a distinct region of Env, the fusion peptide-proximal region, which regulates their exposure.

Phosphatidylserine receptors enhance SARS-CoV-2 infection.

Phosphatidylserine (PS) receptors enhance infection of many enveloped viruses through virion-associated PS binding that is termed apoptotic mimicry. Here we show that this broadly shared uptake mechanism is utilized by SARS-CoV-2 in cells that express low surface levels of ACE2. Expression of members of the TIM (TIM-1 and TIM-4) and TAM (AXL) families of PS receptors enhance SARS-CoV-2 binding to cells, facilitate internalization of fluorescently-labeled virions and increase ACE2-dependent infection of SARS-CoV-2; however, PS receptors alone did not mediate infection. We were unable to detect direct interactions of the PS receptor AXL with purified SARS-CoV-2 spike, contrary to a previous report. Instead, our studies indicate that the PS receptors interact with PS on the surface of SARS-CoV-2 virions. In support of this, we demonstrate that: 1) significant quantities of PS are located on the outer leaflet of SARS-CoV-2 virions, 2) PS liposomes, but not phosphatidylcholine liposomes, reduced entry of VSV/Spike pseudovirions and 3) an established mutant of TIM-1 which does not bind to PS is unable to facilitate entry of SARS-CoV-2. As AXL is an abundant PS receptor on a number of airway lines, we evaluated small molecule inhibitors of AXL signaling such as bemcentinib for their ability to inhibit SARS-CoV-2 infection. Bemcentinib robustly inhibited virus infection of Vero E6 cells as well as multiple human lung cell lines that expressed AXL. This inhibition correlated well with inhibitors that block endosomal acidification and cathepsin activity, consistent with AXL-mediated uptake of SARS-CoV-2 into the endosomal compartment. We extended our observations to the related betacoronavirus mouse hepatitis virus (MHV), showing that inhibition or ablation of AXL reduces MHV infection of murine cells. In total, our findings provide evidence that PS receptors facilitate infection of the pandemic coronavirus SARS-CoV-2 and suggest that inhibition of the PS receptor AXL has therapeutic potential against SARS-CoV-2.

The lipid membrane of HIV-1 stabilizes the viral envelope glycoproteins and modulates their sensitivity to antibody neutralization.

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 High Content of Fructose in Human Semen Competitively Inhibits Broad and Potent Antivirals That Target High-Mannose Glycans.

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.

Accurate predictions of population-level changes in sequence and structural properties of HIV-1 Env using a volatility-controlled diffusion model.

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.

Induction of a Tier-1-Like Phenotype in Diverse Tier-2 Isolates by Agents That Guide HIV-1 Env to Perturbation-Sensitive, Nonnative States.

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, Env-activating 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 coreceptor-binding 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 sensitivity by chemical agents that affect the structural stability of proteins can be applied to increase the potency of topical microbicides and vaccine-elicited antibodies.

Yellow Fever Virus, but Not Zika Virus or Dengue Virus, Inhibits T-Cell Receptor-Mediated T-Cell Function by an RNA-Based Mechanism.

The Flavivirus genus within the Flaviviridae family is comprised of many important human pathogens including yellow fever virus (YFV), dengue virus (DENV), and Zika virus (ZKV), all of which are global public health concerns. Although the related flaviviruses hepatitis C virus and human pegivirus (formerly named GBV-C) interfere with T-cell receptor (TCR) signaling by novel RNA and protein-based mechanisms, the effect of other flaviviruses on TCR signaling is unknown. Here, we studied the effect of YFV, DENV, and ZKV on TCR signaling. Both YFV and ZKV replicated in human T cells in vitro; however, only YFV inhibited TCR signaling. This effect was mediated at least in part by the YFV envelope (env) protein coding RNA. Deletion mutagenesis studies demonstrated that expression of a short, YFV env RNA motif (vsRNA) was required and sufficient to inhibit TCR signaling. Expression of this vsRNA and YFV infection of T cells reduced the expression of a Src-kinase regulatory phosphatase (PTPRE), while ZKV infection did not. YFV infection in mice resulted in impaired TCR signaling and PTPRE expression, with associated reduction in murine response to experimental ovalbumin vaccination. Together, these data suggest that viruses within the flavivirus genus inhibit TCR signaling in a species-dependent manner.

HIV-1 Adapts To Replicate in Cells Expressing Common Marmoset APOBEC3G and BST2.

UNLABELLED: Previous studies have shown that a major block to HIV-1 replication in common marmosets operates at the level of viral entry and that this block can be overcome by adaptation of the virus in tissue-cultured cells. However, our current studies indicate that HIV-1 encounters additional postentry blocks in common marmoset peripheral blood mononuclear cells. Here, we show that the common marmoset APOBEC3G (A3G) and BST2 proteins block HIV-1 in cell cultures. Using a directed-evolution method that takes advantage of the natural ability of HIV-1 to mutate during replication, we have been able to overcome these blocks in tissue-cultured cells. In the adapted viruses, specific changes were observed in gag, vif, env, and nef. The contribution of these changes to virus replication in the presence of the A3G and BST2 restriction factors was studied. We found that certain amino acid changes in Vif and Env that arise during adaptation to marmoset A3G and BST2 allow the virus to replicate in the presence of these restriction factors. The changes in Vif reduce expression levels and encapsidation of marmoset APOBEC3G, while the changes in Env increase viral fitness and discretely favor cell-to-cell transmission of the virus, allowing viral escape from these restriction factors. IMPORTANCE: HIV-1 can infect only humans and chimpanzees. The main reason for this narrow tropism is the presence in many species of dominant-acting factors, known as restriction factors, that block viral replication in a species-specific way. We have been exploring the blocks to HIV-1 in common marmosets, with the ultimate goal of developing a new animal model of HIV-1 infection in these monkeys. In this study, we observed that common marmoset APOBEC3G and BST2, two known restriction factors, are able to block HIV-1 in cell cultures. We have adapted HIV-1 to replicate in the presence of these restriction factors and have characterized the mechanisms of escape. These studies can help in the development of a novel animal model for in vivo infection of marmosets with HIV-1-like viruses.

The selection of low envelope glycoprotein reactivity to soluble CD4 and cold during simian-human immunodeficiency virus infection of rhesus macaques.

Envelope glycoprotein (Env) reactivity (ER) describes the propensity of human immunodeficiency virus type 1 (HIV-1) Env to change conformation from the metastable unliganded state in response to the binding of ligands (antibodies and soluble CD4 [sCD4]) or incubation in the cold. To investigate Env properties that favor in vivo persistence, we inoculated rhesus macaques with three closely related CCR5-tropic simian-human immunodeficiency viruses (SHIVs) that differ in ER to cold (ERcold) and ER to sCD4 (ERsCD4); these SHIVs were neutralized by antibodies equivalently and thus were similar in ERantibody. All three SHIVs achieved high levels of acute viremia in the monkeys without alteration of their Env sequences, indicating that neither ERcold nor ERsCD4 significantly influences the establishment of infection. Between 14 and 100 days following infection, viruses with high ERcold and ERsCD4 were counterselected. Remarkably, the virus variant with low ERcold and low ERsCD4 did not elicit a neutralizing antibody response against the infecting virus, despite the generation of high levels of anti-Env antibodies in the infected monkeys. All viruses that achieved persistent viremia escaped from any autologous neutralizing antibodies and exhibited low ERcold and low ERsCD4. One set of gp120 changes determined the decrease in ERcold and ERsCD4, and a different set of gp120 changes determined resistance to autologous neutralizing antibodies. Each set of changes contributed to a reduction in Env-mediated entry. During infection of monkeys, any Env replication fitness costs associated with decreases in ERcold and ERsCD4 may be offset by minimizing the elicitation of autologous neutralizing antibodies.

Loss of a conserved N-linked glycosylation site in the simian immunodeficiency virus envelope glycoprotein V2 region enhances macrophage tropism by increasing CD4-independent cell-to-cell transmission.

UNLABELLED: Human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) strains differ in their capacity to replicate in macrophages, but mechanisms underlying these differences are not fully understood. Here, we identify a highly conserved N-linked glycosylation site (N173 in SIV, corresponding to N160 in HIV) in the V2 region of the SIV envelope glycoprotein (Env) as a novel determinant of macrophage tropism and characterize mechanisms underlying this phenotype. Loss of the N173 glycosylation site in the non-macrophage-tropic SIVmac239 by introducing an N173Q mutation enhanced viral replication and multinucleated giant cell formation upon infection of rhesus macrophages, while the addition of N173 to SIVmac251 had the opposite effect. The removal of N173 in SIVmac239 enhanced CD4-independent cell-to-cell transmission to CCR5-expressing cells. SIVmac239 with N173Q mediated CD4-independent cell-cell fusion but could not infect CD4-negative cells in single-round infections. Thus, CD4-independent phenotypes were detected only in the context of cell-to-cell contact. Similar results were obtained in SIVmac251 with and without N173. N173 decreased the neutralization sensitivity of SIVmac251 but had no effect on the neutralization sensitivity of SIVmac239. The N173Q mutation had no effect on SIVmac239 binding to CD4 in Biacore assays, coimmunoprecipitation assays, and enzyme-linked immunosorbent assays (ELISAs). These findings suggest that the loss of the N173 N-linked glycosylation site increases SIVmac239 replication in macrophages by enhancing CD4-independent cell-to-cell virus transmission through CCR5-mediated fusion. This mechanism may facilitate the escape of macrophage-tropic viruses from neutralizing antibodies while promoting spreading infection by these viruses in vivo. IMPORTANCE: In this study, we identify a genetic determinant in the viral envelope (N173) that increases replication and spreading infection of SIV strains in macrophages by enhancing cell-to-cell virus transmission. This effect is explained by a novel mechanism involving increased cell-to-cell fusion in the absence of CD4, the primary receptor that normally mediates virus entry. The same genetic determinant also affects the sensitivity of these viruses to inhibition by neutralizing antibodies. Most macrophage-tropic HIV/SIV strains are known to be neutralization sensitive. Together, these findings suggest that this efficient mode of virus transmission may facilitate the escape of macrophage-tropic viruses from neutralizing antibodies while promoting spreading infection by these viruses to cells expressing little or no CD4 in vivo.

Proteolytic processing of the human immunodeficiency virus envelope glycoprotein precursor decreases conformational flexibility.

The mature envelope glycoprotein (Env) spike on the surface of human immunodeficiency virus type 1 (HIV-1) virions is derived by proteolytic cleavage of a trimeric gp160 glycoprotein precursor. Remarkably, proteolytic processing of the HIV-1 Env precursor results in changes in Env antigenicity that resemble those associated with glutaraldehyde fixation. Apparently, proteolytic processing of the HIV-1 Env precursor decreases conformational flexibility of the Env trimeric complex, differentially affecting the integrity/accessibility of epitopes for neutralizing and nonneutralizing antibodies.

Contribution of intrinsic reactivity of the HIV-1 envelope glycoproteins to CD4-independent infection and global inhibitor sensitivity.

Human immunodeficiency virus (HIV-1) enters cells following sequential activation of the high-potential-energy viral envelope glycoprotein trimer by target cell CD4 and coreceptor. HIV-1 variants differ in their requirements for CD4; viruses that can infect coreceptor-expressing cells that lack CD4 have been generated in the laboratory. These CD4-independent HIV-1 variants are sensitive to neutralization by multiple antibodies that recognize different envelope glycoprotein epitopes. The mechanisms underlying CD4 independence, global sensitivity to neutralization and the association between them are still unclear. By studying HIV-1 variants that differ in requirements for CD4, we investigated the contribution of CD4 binding to virus entry. CD4 engagement exposes the coreceptor-binding site and increases the "intrinsic reactivity" of the envelope glycoproteins; intrinsic reactivity describes the propensity of the envelope glycoproteins to negotiate transitions to lower-energy states upon stimulation. Coreceptor-binding site exposure and increased intrinsic reactivity promote formation/exposure of the HR1 coiled coil on the gp41 transmembrane glycoprotein and allow virus entry upon coreceptor binding. Intrinsic reactivity also dictates the global sensitivity of HIV-1 to perturbations such as exposure to cold and the binding of antibodies and small molecules. Accordingly, CD4 independence of HIV-1 was accompanied by increased susceptibility to inactivation by these factors. We investigated the role of intrinsic reactivity in determining the sensitivity of primary HIV-1 isolates to inhibition. Relative to the more common neutralization-resistant ("Tier 2-like") viruses, globally sensitive ("Tier 1") viruses exhibited increased intrinsic reactivity, i.e., were inactivated more efficiently by cold exposure or by a given level of antibody binding to the envelope glycoprotein trimer. Virus sensitivity to neutralization was dictated both by the efficiency of inhibitor/antibody binding to the envelope glycoprotein trimer and by envelope glycoprotein reactivity to the inhibitor/antibody binding event. Quantitative differences in intrinsic reactivity contribute to HIV-1 strain variability in global susceptibility to neutralization and explain the long-observed relationship between increased inhibitor sensitivity and decreased entry requirements for target cell CD4.

A classification algorithm based on dynamic ensemble selection to predict mutational patterns of the envelope protein in HIV-infected patients.

BACKGROUND: Therapeutics against the envelope (Env) proteins of human immunodeficiency virus type 1 (HIV-1) effectively reduce viral loads in patients. However, due to mutations, new therapy-resistant Env variants frequently emerge. The sites of mutations on Env that appear in each patient are considered random and unpredictable. Here we developed an algorithm to estimate for each patient the mutational state of each position based on the mutational state of adjacent positions on the three-dimensional structure of the protein. METHODS: We developed a dynamic ensemble selection algorithm designated k-best classifiers. It identifies the best classifiers within the neighborhood of a new observation and applies them to predict the variability state of each observation. To evaluate the algorithm, we applied amino acid sequences of Envs from 300 HIV-1-infected individuals (at least six sequences per patient). For each patient, amino acid variability values at all Env positions were mapped onto the three-dimensional structure of the protein. Then, the variability state of each position was estimated by the variability at adjacent positions of the protein. RESULTS: The proposed algorithm showed higher performance than the base learner and a panel of classification algorithms. The mutational state of positions in the high-mannose patch and CD4-binding site of Env, which are targeted by multiple therapeutics, was predicted well. Importantly, the algorithm outperformed other classification techniques for predicting the variability state at multi-position footprints of therapeutics on Env. CONCLUSIONS: The proposed algorithm applies a dynamic classifier-scoring approach that increases its performance relative to other classification methods. Better understanding of the spatiotemporal patterns of variability across Env may lead to new treatment strategies that are tailored to the unique mutational patterns of each patient. More generally, we propose the algorithm as a new high-performance dynamic ensemble selection technique.

Phenotypic and immunologic comparison of clade B transmitted/founder and chronic HIV-1 envelope glycoproteins.

Sexual transmission of human immunodeficiency virus type 1 (HIV-1) across mucosal barriers is responsible for the vast majority of new infections. This relatively inefficient process results in the transmission of a single transmitted/founder (T/F) virus, from a diverse viral swarm in the donor, in approximately 80% of cases. Here we compared the biological activities of 24 clade B T/F envelopes (Envs) with those from 17 chronic controls to determine whether the genetic bottleneck that occurs during transmission is linked to a particular Env phenotype. To maximize the likelihood of an intact mucosal barrier in the recipients and to enhance the sensitivity of detecting phenotypic differences, only T/F Envs from individuals infected with a single T/F variant were selected. Using pseudotyping to assess Env function in single-round infectivity assays, we compared coreceptor tropism, CCR5 utilization efficiencies, primary CD4(+) T cell subset tropism, dendritic cell trans-infections, fusion kinetics, and neutralization sensitivities. T/F and chronic Envs were phenotypically equivalent in most assays; however, T/F Envs were modestly more sensitive to CD4 binding site antibodies b12 and VRC01, as well as pooled human HIV Ig. This finding was independently validated with a panel of 14 additional chronic HIV-1 Env controls. Moreover, the enhanced neutralization sensitivity was associated with more efficient binding of b12 and VRC01 to T/F Env trimers. These data suggest that there are subtle but significant structural differences between T/F and chronic clade B Envs that may have implications for HIV-1 transmission and the design of effective vaccines.

Sensitive detection of SARS-CoV-2 spike protein using vertically-oriented silicon nanowire array-based biosensor.

The COVID-19 pandemic has caused tremendous damage to the world. In order to quickly and accurately diagnose the virus and contain the spread, there is a need for rapid, sensitive, accurate, and cost-effective SARS-CoV-2 biosensors. In this paper, we report on a novel biosensor based on angiotensin converting enzyme 2 (ACE-2)-conjugated vertically-oriented silicon nanowire (vSiNW) arrays that can detect the SARS-CoV-2 spike protein with high sensitivity and selectivity relative to negative controls. First, we demonstrate the efficacy of using ACE-2 receptor to detect the SARS-CoV-2 spike protein via a capture assay test, which confirms high specificity of ACE-2 against the mock protein, and high affinity between the spike and ACE-2. We then report on results for ACE-2-conjugated vSiNW arrays where the biosensor device architecture is based on a p-n junction transducer. We confirm via analytical modeling that the transduction mechanism of the biosensor involves induced surface charge depletion of the vSiNWs due to negative electrostatic surface potential induced by the spike protein after binding with ACE-2. This vSiNW surface charge modulation is measured via current-voltage characteristics of the functionalized biosensor. Calibrated concentration dependent electrical response of the vSiNW sensor confirms the limit-of-detection for virus spike concentration of 100 ng/ml (or 575 pM). The vSiNW sensor also exhibits highly specific response to the spike protein with respect to negative controls, offering a promising point-of-care detection method for SARS-CoV-2.

Mutability Patterns Across the Spike Glycoprotein Reveal the Diverging and Lineage-specific Evolutionary Space of SARS-CoV-2.

Mutations in the spike glycoprotein of SARS-CoV-2 allow the virus to probe the sequence space in search of higher-fitness states. New sublineages of SARS-CoV-2 variants-of-concern (VOCs) continuously emerge with such mutations. Interestingly, the sites of mutation in these sublineages vary between the VOCs. Whether such differences reflect the random nature of mutation appearance or distinct evolutionary spaces of spike in the VOCs is unclear. Here we show that each position of spike has a lineage-specific likelihood for mutations to appear and dominate descendent sublineages. This likelihood can be accurately estimated from the lineage-specific mutational profile of spike at a protein-wide level. The mutability environment of each position, including adjacent sites on the protein structure and neighboring sites on the network of comutability, accurately forecast changes in descendent sublineages. Mapping of imminent changes within the VOCs can contribute to the design of immunogens and therapeutics that address future forms of SARS-CoV-2.

Limited Variation between SARS-CoV-2-Infected Individuals in Domain Specificity and Relative Potency of the Antibody Response against the Spike Glycoprotein.

The spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is arranged as a trimer on the virus surface, composed of three S1 and three S2 subunits. Infected and vaccinated individuals generate antibodies against spike, which can neutralize the virus. Most antibodies target the receptor-binding domain (RBD) and N-terminal domain (NTD) of S1; however, antibodies against other regions of spike have also been isolated. The interhost variability in domain specificity and relative neutralization efficacy of the antibodies is still poorly characterized. To this end, we tested serum and plasma samples collected from 85 coronavirus disease 2019 (COVID-19) convalescent subjects. Samples were analyzed using seven immunoassays that employ different domains, subunits, and oligomeric forms of spike to capture the antibodies. Samples were also tested for their neutralization of pseudovirus containing SARS-CoV-2 spike and of replication-competent SARS-CoV-2. While the total amount of anti-spike antibodies produced varied among convalescent subjects, we observed an unexpectedly fixed ratio of RBD- to NTD-targeting antibodies. The relative potency of the response (defined as the measured neutralization efficacy relative to the total level of spike-targeting antibodies) also exhibited limited variation between subjects and was not associated with the overall amount of antispike antibodies produced. These studies suggest that host-to-host variation in the polyclonal response elicited against SARS-CoV-2 spike in early pandemic subjects is primarily limited to the quantity of antibodies generated rather than their domain specificity or relative neutralization potency. IMPORTANCE Infection by SARS-CoV-2 elicits antibodies against various domains of the spike protein, including the RBD and NTD of subunit S1 and against subunit S2. The antibody responses of different infected individuals exhibit different efficacies to inactivate (neutralize) the virus. Here, we show that the observed variation in the neutralizing activity of the antibody responses in COVID-19 convalescent subjects is caused by differences in the amounts of antibodies rather than their recognition properties or the potency of their antiviral activity. These findings suggest that COVID-19 vaccine strategies that focus on enhancing the overall level of the antibodies will likely elicit a more uniformly efficacious protective response.

Identification and characterization of persistent intracellular human immunodeficiency virus type 1 integrase strand transfer inhibitor activity.

Pharmacokinetic and pharmacodynamic considerations significantly impact infectious disease treatment options. One aspect of pharmacodynamics is the postantibiotic effect, classically defined as delayed bacterial growth after antibiotic removal. The same principle can apply to antiviral drugs. For example, significant delays in human immunodeficiency virus type 1 (HIV-1) replication can be observed after nucleoside/nucleotide reverse transcriptase inhibitor (N/NtRTI) removal from culture medium, because these prodrugs must be anabolized into active, phosphorylated forms once internalized into cells. A relatively new class of anti-HIV-1 drugs is the integrase strand transfer inhibitors (INSTIs), and the INSTIs raltegravir (RAL) and elvitegravir (EVG) were tested here alongside positive N/NtRTI controls tenofovir disoproxil fumarate (TDF) and azidothymidine (AZT), as well as the nonnucleoside reverse transcriptase inhibitor negative control nevirapine (NVP), to assess potential postantiviral effects. Transformed and primary CD4-positive cells pretreated with INSTIs significantly resisted subsequent challenge by HIV-1, revealing the following hierarchy of persistent intracellular drug strength: TDF > EVG ∼ AZT > RAL > NVP. A modified time-of-addition assay was moreover developed to assess residual drug activity levels. Approximately 0.8% of RAL and 2% of initial EVG and TDF 1-h pulse drug levels persisted during the acute phase of HIV-1 infection. EVG furthermore displayed significant virucidal activity. Although there is no reason to suspect obligate intracellular modification, this study nevertheless defines significant intracellular persistence of prototype INSTIs. Ongoing second-generation formulations should therefore consider the potential for significant postantiviral effects among this drug class. Combined intracellular persistence and virucidal activities suggest potential pre-exposure prophylaxis applications for EVG.

Soluble CD4 and CD4-mimetic compounds inhibit HIV-1 infection by induction of a short-lived activated state.

Binding to the CD4 receptor induces conformational changes in the human immunodeficiency virus (HIV-1) gp120 exterior envelope glycoprotein. These changes allow gp120 to bind the coreceptor, either CCR5 or CXCR4, and prime the gp41 transmembrane envelope glycoprotein to mediate virus-cell membrane fusion and virus entry. Soluble forms of CD4 (sCD4) and small-molecule CD4 mimics (here exemplified by JRC-II-191) also induce these conformational changes in the HIV-1 envelope glycoproteins, but typically inhibit HIV-1 entry into CD4-expressing cells. To investigate the mechanism of inhibition, we monitored at high temporal resolution inhibitor-induced changes in the conformation and functional competence of the HIV-1 envelope glycoproteins that immediately follow engagement of the soluble CD4 mimics. Both sCD4 and JRC-II-191 efficiently activated the envelope glycoproteins to mediate infection of cells lacking CD4, in a manner dependent on coreceptor affinity and density. This activated state, however, was transient and was followed by spontaneous and apparently irreversible changes of conformation and by loss of functional competence. The longevity of the activated intermediate depended on temperature and the particular HIV-1 strain, but was indistinguishable for sCD4 and JRC-II-191; by contrast, the activated intermediate induced by cell-surface CD4 was relatively long-lived. The inactivating effects of these activation-based inhibitors predominantly affected cell-free virus, whereas virus that was prebound to the target cell surface was mainly activated, infecting the cells even at high concentrations of the CD4 analogue. These results demonstrate the ability of soluble CD4 mimics to inactivate HIV-1 by prematurely triggering active but transient intermediate states of the envelope glycoproteins. This novel strategy for inhibition may be generally applicable to high-potential-energy viral entry machines that are normally activated by receptor binding.