Biology subjects

Anang, S.

Publications and source records attributed to Anang, S..

10 recordsLinked to original sources

bioRxiv2025

Characterization of full-length and cytoplasmic tail-truncated envelope glycoproteins incorporated into human immunodeficiency virus (HIV-1) virions and virus-like particles

During transport to the surface of infected cells, the human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) trimer is cleaved to produce the mature functional Env trimer ((gp120/gp41)3). Env cleavage stabilizes the pretriggered Env conformation (PTC), the major target for broadly neutralizing antibodies. Although the mature Env is relatively enriched in virions and virus-like particles (VLPs), conformationally flexible uncleaved Envs typically contaminate preparations of these particles. In non-permissive cells, the long [~]149-residue gp41 cytoplasmic tail (CT) is necessary for Env incorporation into virions. In a minority of HIV-1 strains, the gp41 CT is clipped in virions by the viral protease. Here, we compare Envs with CT truncations and CT alterations that increase or decrease protease clipping in permissive cells. Changes in protease clipping affected amphotericin B sensitivity, but did not alter other viral phenotypes. By contrast, a corresponding CT truncation (L748STOP) increased cell-surface and virion Env levels, cell-cell fusion, and virus infectivity and cytotoxicity. Notably, in diverse HIV-1 strains, the ratio of cleaved/uncleaved Envs in preparations of virions and extracellular vesicles was increased by this CT truncation. ESCRT and ALIX-binding region (EABR) vesicles incorporated significantly more uncleaved CT-truncated Env than HIV-1 VLPs. Env CT deletion/truncation did not qualitatively alter the viral neutralization profile; however, increased antibody concentrations were required to neutralize viruses with the higher levels of cleaved Env that resulted from CT truncation. Specific CT truncations provide a means of enriching the PTC and limiting the incorporation of nonfunctional and conformationally heterogeneous uncleaved Envs into preparations of virions and VLPs. IMPORTANCEThe human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) trimer mediates entry of the virus into host cells. The pretriggered conformation (PTC) of Env is the major target for protective broadly neutralizing antibodies, but the PTC is unstable and therefore difficult to study. The cleavage of the flexible Env precursor stabilizes the PTC. Therefore, the presence of uncleaved Env compromises the purity of the PTC in Env preparations. We found that certain truncations of the Env cytoplasmic tail resulted in improved ratios of cleaved:uncleaved Env in preparations of HIV-1 viruses or virus-like particles. In some contexts, cytoplasmic tail truncation increased the level of Env in virus preparations. Although higher concentrations of antibodies were required to neutralize these viruses, Envs with specific truncations of the cytoplasmic tail retained the PTC. Thus, cytoplasmic tail truncation could assist efforts to purify and characterize the Env PTC on the viral membrane.

microbiology↗

bioRxiv2024

Stoichiometry of HIV-1 Envelope Glycoprotein Protomers with Changes That Stabilize or Destabilize the Pretriggered Conformation

During human immunodeficiency virus (HIV-1) entry into host cells, binding to the receptors, CD4 and CCR5/CXCR4, triggers conformational changes in the metastable envelope glycoprotein (Env) trimer ((gp120-gp41)3). CD4 binding induces Env to make transitions from its pretriggered conformation (PTC) to more "open" conformations that are sensitive to inhibition by antibodies, CD4-mimetic compounds (CD4mcs) and exposure to cold. Changes in functional membrane Envs have been identified that either stabilize or destabilize the PTC. Here, we investigate the stoichiometric requirements for the PTC-stabilizing and -destabilizing changes in the Env protomers. To this end, we generated viruses bearing Envs with mixed protomers exhibiting different degrees of PTC stability and determined the sensitivity of the viruses to cold (0{degrees}C) and, in some cases, to a CD4mc. The number of stabilized Env protomers required to achieve stabilization of the PTC was inversely related to the degree of PTC stabilization that resulted from the introduced Env change. For strongly stabilizing Env changes, modification of a single protomer was sufficient to achieve PTC stabilization; given adequate stability, the modified protomer can apparently constrain the conformation of the other two protomers to maintain the PTC. Weakly stabilizing Env changes needed to be present in all three protomers to achieve efficient stabilization of the PTC. In many cases, the PTC was disrupted when destabilizing changes were present in only a single protomer. These complementary results suggest that conformational symmetry among the protomers of the functional Env trimer is conducive to the integrity of the PTC. IMPORTANCEThe human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) trimer consists of three protomers. In response to receptor binding, the flexible Env changes its conformation to mediate virus entry into host cells. The shape-shifting ability of Env also contributes to HIV-1s capacity to evade the host immune system. The pretriggered (State-1) conformation (PTC) of Env is an important target for virus entry inhibitors and host antibodies, but is unstable and therefore incompletely characterized. Changes in Env amino acids that either stabilize or destabilize the PTC have been identified. Here, we define how many Env protomers need to be modified by these changes to achieve stabilization or destabilization of the PTC. These results can guide the placement of changes in the HIV-1 Env protomers to control the movement of the Env trimer from the PTC, allowing better characterization of this elusive conformation and testing of its utility in vaccines.

microbiology↗

bioRxiv2023

V3 tip determinants of susceptibility to inhibition by CD4-mimetic compounds in natural clade A human immunodeficiency virus (HIV-1) envelope glycoproteins

CD4-mimetic compounds (CD4mcs) bind the human immunodeficiency virus (HIV-1) gp120 exterior envelope glycoprotein (Env) and compete for binding to CD4, the host receptor. CD4mcs prematurely trigger conformational changes in Env similar to those induced by CD4, leading to transient activation of infectivity followed by irreversible virus inactivation. Natural HIV-1 variants exhibit a wide range of susceptibilities to CD4mc inhibition, only a small fraction of which can be explained by variation in the gp120 Phe-43 cavity/vestibule where CD4mcs bind. Here, we study Envs from the resistant HIV-1BG505 and the more sensitive HIV-1191955_A4 clade A strains. The major determinant of the relative sensitivity of the HIV-1191955_A4 Env to CD4mcs mapped to a single residue change (F317Y) in the tip of the gp120 V3 variable loop. In the Envs of several HIV-1 strains, replacement of the more prevalent Phe 317 with a tyrosine residue increased virus sensitivity to multiple CD4mcs. Tryptophan substitutions at residues 317 and 316 resulted in increases and decreases, respectively, in sensitivity to CD4mcs. Some of the gp120 V3 changes increased virus sensitivity to inactivation by both CD4mc and cold exposure, phenotypes indicative of increased Env triggerability. Infection of CD4-negative cells expressing the CCR5 coreceptor by these Env variants was triggered more efficiently by CD4mcs. For the panel of studied HIV-1 Envs, resistance to the CD4mcs was associated with decreased ability to support virus entry. These studies illustrate how variation in gp120 outside the CD4mc binding site can influence the sensitivity of natural HIV-1 strains to inhibition by these compounds. IMPORTANCECD4-mimetic compounds (CD4mcs) are small-molecule inhibitors of human immunodeficiency virus (HIV-1) entry into host cells. CD4mcs target a pocket on the viral envelope glycoprotein (Env) spike that is used for binding to the receptor, CD4, and is highly conserved among HIV-1 strains. Nonetheless, naturally occurring HIV-1 strains exhibit a wide range of sensitivities to CD4mcs. Our study identifies changes distant from the binding pocket that can influence the susceptibility of natural HIV-1 strains to the antiviral effects of multiple CD4mcs. We relate the antiviral potency of the CD4mc against this panel of HIV-1 variants to the ability of the CD4mc to activate entry-related changes in Env conformation prematurely. These findings will guide efforts to improve the potency and breadth of CD4mcs against natural HIV-1 variants.

microbiology↗

bioRxiv2023

Indoline CD4-mimetic Compounds Mediate Potent and Broad HIV-1 Inhibition and Sensitization to Antibody-dependent Cellular Cytotoxicity

Binding to the host cell receptors, CD4 and CCR5/CXCR4, triggers large-scale conformational changes in the human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) trimer [(gp120/gp41)3] that promote virus entry into the cell. CD4-mimetic compounds (CD4mcs) comprise small organic molecules that bind in the highly conserved CD4-binding site of gp120 and prematurely induce inactivating Env conformational changes, including shedding of gp120 from the Env trimer. By inducing more "open," antibody-susceptible Env conformations, CD4mcs also sensitize HIV-1 virions to neutralization by antibodies and infected cells to antibody-dependent cellular cytotoxicity (ADCC). Here, we report the design, synthesis and evaluation of novel CD4mcs based on an indoline scaffold. Compared with our current lead indane scaffold CD4mc, BNM-III-170, several indoline CD4mcs exhibit increased potency and breadth against HIV-1 variants from different geographic clades. Viruses that were selected for resistance to the lead indane CD4mc, BNM-III-170, are susceptible to inhibition by the indoline CD4mcs. The indoline CD4mcs also potently sensitize HIV-1-infected cells to ADCC mediated by plasma from HIV-1-infected individuals. Crystal structures indicate that the indoline CD4mcs gain potency compared to the indane CD4mcs through more favorable {pi}-{pi} overlap from the indoline pose and by making favorable contacts with the vestibule of the CD4-binding pocket on gp120. The rational design of indoline CD4mcs thus holds promise for further improvements in antiviral activity, potentially contributing to efforts to treat and prevent HIV-1 infection.

microbiology↗

bioRxiv2022

Characterization of the Human Immunodeficiency Virus (HIV-1) Envelope Glycoprotein Conformational States on Infectious Virus Particles

Human immunodeficiency virus (HIV-1) entry into cells involves triggering of the viral envelope glycoprotein (Env) trimer ((gp120/gp41)3) by the primary receptor, CD4, and coreceptors, CCR5 or CXCR4. The pretriggered (State-1) conformation of the mature (cleaved) Env is targeted by broadly neutralizing antibodies (bNAbs), which are inefficiently elicited compared with poorly neutralizing antibodies (pNAbs). Here we characterize variants of the moderately triggerable HIV-1AD8 Env on virions produced by an infectious molecular proviral clone; such virions contain more cleaved Env than pseudotyped viruses. We identified three types of cleaved wild-type AD8 Env trimers on virions: 1) State-1-like trimers preferentially recognized by bNAbs and exhibiting strong subunit association; 2) trimers recognized by pNAbs directed against the gp120 coreceptor-binding region and exhibiting weak, detergent-sensitive subunit association; and 3) a minor gp41-only population. The first Env population was enriched and the other Env populations reduced by introducing State-1-stabilizing changes in the AD8 Env or by treatment of the virions with crosslinker or the State-1-preferring entry inhibitor, BMS-806. These stabilized AD8 Envs were also more resistant to gp120 shedding induced by a CD4-mimetic compound or by incubation on ice. Conversely, a State-1-destabilized, CD4-independent AD8 Env variant exhibited weaker bNAb recognition and stronger pNAb recognition. Similar relationships between Env triggerability and antigenicity/shedding propensity on virions were observed for other HIV-1 strains. Our results show that State-1 Envs on virions can be significantly enriched by optimizing Env cleavage; stabilizing the pretriggered conformation by Env modification, crosslinking or BMS-806 treatment; strengthening Env subunit interactions; and using CD4-negative producer cells. IMPORTANCEEfforts to develop an effective HIV-1 vaccine have been frustrated by the inability to elicit broad neutralizing antibodies that recognize multiple virus strains. Such antibodies are able to bind a particular shape of the HIV-1 envelope glycoprotein trimer, as it exists on a viral membrane but before engaging receptors on the host cell. Here, we establish simple yet powerful assays to characterize the envelope glycoproteins in a natural context on virus particles. We find that, depending on the HIV-1 strain, some envelope glycoproteins change shape and fall apart, creating decoys that can potentially divert the host immune response. We identify requirements to keep the relevant envelope glycoprotein target for broad neutralizing antibodies intact on virus-like particles. These studies suggest strategies that should facilitate efforts to produce and use virus-like particles as vaccine immunogens.

microbiology↗

bioRxiv2022

Characterization of human immunodeficiency virus (HIV-1) envelope glycoprotein variants selected for resistance to a CD4-mimetic compound

Binding to host cell receptors, CD4 and CCR5/CXCR4, triggers conformational changes in the human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) trimer that promote virus entry. CD4 binding allows the gp120 exterior Env to bind CCR5/CXCR4 and induces a pre-hairpin intermediate conformation in the gp41 transmembrane Env. Small-molecule CD4-mimetic compounds (CD4mcs) bind within the conserved Phe-43 cavity of gp120, near the binding site for CD4. CD4mcs inhibit HIV-1 infection by competing with CD4 and by prematurely activating Env, leading to irreversible inactivation. BNM-III-170 is a CD4mc that inhibits the infection of approximately 70% of HIV-1 strains at micromolar concentrations. We selected and analyzed variants of the primary HIV-1AD8 strain resistant to BNM-III-170. Two changes (S375N and I424T) in gp120 residues that flank the Phe-43 cavity each conferred [~]5-fold resistance to BNM- III-170 with minimal fitness cost. A third change (E64G) in Layer 1 of the gp120 inner domain resulted in [~]100-fold resistance to BNM-III-170, [~]2-3-fold resistance to soluble CD4-Ig, and a moderate decrease in viral fitness. The gp120 changes additively or synergistically contributed to BNM-III-170 resistance. The sensitivity of the Env variants to BNM-III-170 inhibition of virus entry correlated with their sensitivity to BNM-III-170- induced Env activation and shedding of gp120. The S375N and I424T changes, but not the E64G change, conferred resistance to BMS-806, a potent HIV-1 entry inhibitor that blocks Env conformational transitions. These studies identify pathways whereby HIV-1 can develop resistance to CD4mcs and BMS-806 conformational blockers, two classes of entry inhibitors that target the conserved gp120 Phe-43 cavity. IMPORTANCECD4-mimetic compounds (CD4mcs) and BMS-806 are small-molecule inhibitors of human immunodeficiency virus (HIV-1) entry into host cells. Although CD4mcs and BMS-806 inhibit HIV-1 entry by different mechanisms, they both target a pocket on the viral envelope glycoprotein (Env) spike that is used for binding to the receptor, CD4, and is highly conserved among HIV-1 strains. Our study identifies changes near this pocket that can confer various levels of resistance to the antiviral effects of both a CD4mc and BMS-806. We relate the antiviral potency of a CD4mc against this panel of HIV-1 variants to the ability of the CD4mc to activate changes in Env conformation and to induce the shedding of the gp120 exterior Env from the spike. These findings will guide efforts to improve the potency and breadth of small-molecule HIV-1 entry inhibitors.

microbiology↗

bioRxiv2021

Functional properties of the spike glycoprotein of the emerging SARS-CoV-2 variant B.1.1.529

The recently emerged B.1.1.529 (Omicron) SARS-CoV-2 variant has a highly divergent spike (S) glycoprotein. We compared the functional properties of B.1.1.529 S with those of previous globally prevalent SARS-CoV-2 variants, D614G and B.1.617.2. Relative to these variants, B.1.1.529 S exhibits decreased processing, resulting in less efficient syncytium formation and lower S incorporation into virus particles. Nonetheless, B.1.1.529 S supports virus infection equivalently. B.1.1.529 and B.1.617.2 S glycoproteins bind ACE2 with higher affinity than D614G S. The unliganded B.1.1.529 S trimer is less stable at low temperatures than the other SARS-CoV-2 spikes, a property related to spike conformation. Upon ACE2 binding, the B.1.1.529 S trimer sheds S1 at 37 degrees but not at 0 degrees C. B.1.1.529 pseudoviruses are relatively resistant to neutralization by sera from convalescent COVID-19 patients and vaccinees. These properties of the B.1.1.529 spike glycoprotein likely influence the transmission, cytopathic effects and immune evasion of this emerging variant.

microbiology↗

bioRxiv2021

Functional and Highly Crosslinkable HIV-1 Envelope Glycoproteins Enriched in a Pretriggered Conformation

Binding to the receptor, CD4, drives the pretriggered, "closed" (State-1) conformation of the human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) trimer into more "open" conformations (States 2 and 3). Broadly neutralizing antibodies, which are elicited inefficiently, mostly recognize the State-1 Env conformation, whereas the more commonly elicited poorly neutralizing antibodies recognize States 2/3. HIV-1 Env metastability has created challenges for defining the State-1 structure and developing immunogens mimicking this labile conformation. The availability of functional State-1 Envs that can be efficiently crosslinked at lysine and/or acidic amino acid residues might assist these endeavors. To that end, we modified HIV-1AD8 Env, which exhibits an intermediate level of triggerability by CD4. We introduced lysine/acidic residues at positions that exhibit such polymorphisms in natural HIV-1 strains. Env changes that were tolerated with respect to gp120-gp41 processing, subunit association and virus entry were further combined. Two common polymorphisms, Q114E and Q567K, as well as a known variant, A582T, additively rendered pseudoviruses resistant to cold, soluble CD4 and a CD4-mimetic compound, phenotypes indicative of stabilization of the pretriggered State-1 Env conformation. Combining these changes resulted in two lysine-rich HIV-1AD8 Env variants (E.2 and AE.2) with neutralization- and cold-resistant phenotypes comparable to those of natural, less triggerable Tier 2/3 HIV-1 isolates. Compared with these and the parental Envs, the E.2 and AE.2 Envs were cleaved more efficiently and exhibited stronger gp120-trimer association in detergent lysates. These highly crosslinkable Envs enriched in a pretriggered conformation should assist characterization of the structure and immunogenicity of this labile state. IMPORTANCEThe development of an efficient vaccine is critical for combating HIV-1 infection worldwide. However, the instability of the pretriggered shape (State 1) of the viral envelope glycoprotein (Env) makes it difficult to raise neutralizing antibodies against HIV-1. Here, by introducing multiple changes in Env, we derived two HIV-1 Env variants that are enriched in State 1 and can be efficiently crosslinked to maintain this shape. These Env complexes are more stable in detergent, assisting their purification. Thus, our study provides a path to a better characterization of the native pretriggered Env, which should assist vaccine development.

molecular biology↗

bioRxiv2021

Analysis of glycosylation and disulfide bonding of wild-type SARS-CoV-2 spike glycoprotein

The SARS-CoV-2 coronavirus, the etiologic agent of COVID-19, uses its spike (S) glycoprotein anchored in the viral membrane to enter host cells. The S glycoprotein is the major target for neutralizing antibodies elicited by natural infection and by vaccines. Approximately 35% of the SARS-CoV-2 S glycoprotein consists of carbohydrate, which can influence virus infectivity and susceptibility to antibody inhibition. We found that virus-like particles produced by coexpression of SARS-CoV-2 S, M, E and N proteins contained spike glycoproteins that were extensively modified by complex carbohydrates. We used a fucose-selective lectin to enrich the Golgi-resident fraction of a wild-type SARS-CoV-2 S glycoprotein trimer, and determined its glycosylation and disulfide bond profile. Compared with soluble or solubilized S glycoproteins modified to prevent proteolytic cleavage and to retain a prefusion conformation, more of the wild-type S glycoprotein N-linked glycans are processed to complex forms. Even Asn 234, a significant percentage of which is decorated by high-mannose glycans on soluble and virion S trimers, is predominantly modified in the Golgi by processed glycans. Three incompletely occupied sites of O-linked glycosylation were detected. Viruses pseudotyped with natural variants of the serine/threonine residues implicated in O-linked glycosylation were generally infectious and exhibited sensitivity to neutralization by soluble ACE2 and convalescent antisera comparable to that of the wild-type virus. Unlike other natural cysteine variants, a Cys15Phe (C15F) mutant retained partial, but unstable, infectivity. These findings enhance our understanding of the Golgi processing of the native SARS-CoV-2 S glycoprotein carbohydrates and could assist the design of interventions.

molecular biology↗

bioRxiv2020

Spike glycoprotein and host cell determinants of SARS-CoV-2 entry and cytopathic effects

SARS-CoV-2, a betacoronavirus, is the cause of the COVID-19 pandemic. The SARS-CoV-2 spike (S) glycoprotein trimer mediates virus entry into host cells and cytopathic effects. We studied the contribution of several S glycoprotein features to these functions, focusing on those that differ among related coronaviruses. Acquisition of the furin cleavage site by the SARS-CoV-2 S glycoprotein decreased virus stability and infectivity, but greatly enhanced the ability to form lethal syncytia. Notably, the D614G change found in globally predominant SARS-CoV-2 strains restored infectivity, modestly enhanced responsiveness to the ACE2 receptor and susceptibility to neutralizing sera, and tightened association of the S1 subunit with the trimer. Apparently, two unique features of the SARS-CoV-2 S glycoprotein, the furin cleavage site and D614G, have evolved to balance virus infectivity, stability, cytopathicity and antibody vulnerability. Although the endodomain (cytoplasmic tail) of the S2 subunit was not absolutely required for virus entry or syncytium formation, alteration of palmitoylated cysteine residues in the cytoplasmic tail decreased the efficiency of these processes. As proteolytic cleavage contributes to the activation of the SARS-CoV-2 S glycoprotein, we evaluated the ability of protease inhibitors to suppress S glycoprotein function. Matrix metalloprotease inhibitors suppressed S-mediated cell-cell fusion, but not virus entry. Synergy between inhibitors of matrix metalloproteases and TMPRSS2 suggests that both proteases can activate the S glycoprotein during the process of syncytium formation. These results provide insights into SARS-CoV-2 S glycoprotein-host cell interactions that likely contribute to the transmission and pathogenicity of this pandemic agent. IMPORTANCEThe development of an effective and durable SARS-CoV-2 vaccine is essential for combating the growing COVID-19 pandemic. The SARS-CoV-2 spike (S) glycoprotein is the main target of neutralizing antibodies elicited during virus infection or following vaccination. Knowledge of the spike glycoprotein evolution, function and interactions with host factors will help researchers to develop effective vaccine immunogens and treatments. Here we identify key features of the spike glycoprotein, including the furin cleavage site and the D614G natural mutation, that modulate viral cytopathic effects, infectivity and sensitivity to inhibition. We also identify two inhibitors of host metalloproteases that block S-mediated cell-cell fusion, which contributes to the destruction of the virus-infected cell.

microbiology↗