Biology subjects

Chiu, T.-J.

Publications and source records attributed to Chiu, T.-J..

8 recordsLinked to original sources

bioRxiv2026

Nef and Vpu protect infected cells from ADCP mediated by plasma from people with HIV-1

The HIV-1 envelope glycoprotein (Env) represents the only viral antigen at the surface of infected cells, making it an ideal target for antibody-based therapies. Most antibodies elicited in people with HIV (PWH) do not recognize Env in its native "closed" conformation but readily bind to Env when it samples the CD4-bound "open" conformation. Downregulation of CD4 at the surface of infected cells by the viral accessory proteins Nef and Vpu prevents the premature opening of Env and has been shown to protect infected cells from antibody-dependent cellular cytotoxicity (ADCC) mediated by PWH plasma. Here, we report that deletion of Nef and Vpu from primary infectious molecular clones renders infected cells vulnerable to antibody-dependent cellular phagocytosis (ADCP) mediated by PWH plasma. This is in part linked to the premature engagement of Env with CD4. In agreement with an "open" Env being vulnerable to ADCP, small CD4 mimetic compounds (CD4mc) sensitize in vitro-infected cells and ex vivo-expanded CD4 T cells to ADCP mediated by autologous monocytes in presence of PWH plasma. This effect was further improved by increasing cell surface Env through IFN-induced BST-2 upregulation. IMPORTANCEDeveloping new therapies to eliminate HIV-1-infected cells is essential to decrease the size of the HIV-1 reservoir. Fc-effector functions such as antibody dependent cellular cytotoxicity (ADCC) have shown potential in eliminating in vitro-infected cells and ex vivo-expanded infected cells from people with HIV, decreasing the size of the reservoir and delaying viral rebound in humanized mice. Here, we report that antibody dependent cellular phagocytosis (ADCP) can also be harnessed to eliminate HIV-1-infected cells. We show that infected cells harboring "open" Env conformations are susceptible to ADCP-mediated killing in the presence of plasma from people with HIV. A better understanding of the contribution of different Fc-effector functions in the elimination of infected cells could help guide the development of new therapeutic approaches toward an HIV-1 cure.

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↗

bioRxiv2024

The asymmetric opening of HIV-1 Env by a potent CD4 mimetic enables anti-coreceptor binding site antibodies to mediate ADCC

HIV-1 envelope glycoproteins (Env) from primary HIV-1 isolates typically adopt a pretriggered "closed" conformation that resists to CD4-induced (CD4i) non-neutralizing antibodies (nnAbs) mediating antibody-dependent cellular cytotoxicity (ADCC). CD4-mimetic compounds (CD4mcs) "open-up" Env allowing binding of CD4i nnAbs, thereby sensitizing HIV-1-infected cells to ADCC. Two families of CD4i nnAbs, the anti-cluster A and anti-coreceptor binding site (CoRBS) Abs, are required to mediate ADCC in combination with the indane CD4mc BNM-III-170. Recently, new indoline CD4mcs with improved potency and breadth have been described. Here, we show that the lead indoline CD4mc, CJF-III-288, sensitizes HIV-1-infected cells to ADCC mediated by anti-CoRBS Abs alone, contributing to improved ADCC activity. Structural and conformational analyses reveal that CJF-III-288, in combination with anti-CoRBS Abs, potently stabilizes an asymmetric "open" State-3 Env conformation, This Env conformation orients the anti-CoRBS Ab to improve ADCC activity and therapeutic potential.

microbiology↗

bioRxiv2024

The combination of three CD4-induced antibodies targeting highly conserved Env regions with a small CD4-mimetic achieves potent ADCC activity

The majority of naturally-elicited antibodies against the HIV-1 envelope glycoproteins (Env) are non-neutralizing (nnAbs), because they are unable to recognize the Env timer in its native "closed" conformation. Nevertheless, it has been shown that nnAbs have the potential to eliminate HIV-1-infected cells by Antibody-Dependent Cellular Cytotoxicity (ADCC) provided that Env is present on the cell surface in its "open" conformation. This is because most nnAbs recognize epitopes that become accessible only after Env interaction with CD4 and the exposure of epitopes that are normally occluded in the closed trimer. HIV-1 limits this vulnerability by downregulating CD4 from the surface of infected cells, thus preventing a premature encounter of Env with CD4. Small CD4-mimetics (CD4mc) sensitize HIV-1-infected cells to ADCC by opening the Env glycoprotein and exposing CD4-induced (CD4i) epitopes. There are two families of CD4i nnAbs, termed anti-cluster A and anti-CoRBS Abs, which are known to mediate ADCC in the presence of CD4mc. Here, we performed Fab competition experiments and found that anti-gp41 cluster I antibodies comprise a major fraction of the plasma ADCC activity in people living with HIV (PLWH). Moreover, addition of gp41 cluster I antibodies to cluster A and CoRBS antibodies greatly enhanced ADCC mediated cell killing in the presence of a potent indoline CD4mc, CJF-III-288. This cocktail outperformed broadly-neutralizing antibodies and even showed activity against HIV-1 infected monocyte-derived macrophages. Thus, combining CD4i antibodies with different specificities achieves maximal ADCC activity, which may be of utility in HIV cure strategies. IMPORTANCEThe elimination of HIV-1-infected cells remains an important medical goal. While current antiretroviral therapy decreases viral loads below detection levels, it does not eliminate latently infected cells which form the viral reservoir. Here, we developed a cocktail of non-neutralizing antibodies targeting highly conserved Env regions and combined it with a potent indoline CD4mc. This combination exhibited very potent ADCC activity against HIV-1-infected primary CD4+ T cells as well as monocyte-derived macrophages, suggesting its potential utility in decreasing the size of the viral reservoir.

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 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

Global Increases in Human Immunodeficiency Virus Neutralization Sensitivity Due to Alterations in the Membrane-Proximal External Region of the Envelope Glycoprotein Can be Minimized by Distant State 1-Stabilizing Changes

Binding to the receptor, CD4, drives the pretriggered, "closed" (State-1) conformation of the human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) trimer ((gp120/gp41)3) into more "open" conformations. HIV-1 Env on the viral membrane is maintained in a State-1 conformation that resists binding and neutralization by commonly elicited antibodies. Premature triggering of Env before the virus engages a target cell typically leads to increased susceptibility to spontaneous inactivation or ligand-induced neutralization. Here, we show that single amino acid substitutions in the gp41 membrane-proximal external region (MPER) of a primary HIV-1 strain result in viral phenotypes indicative of premature triggering of Env to downstream conformations. Specifically, the MPER changes reduced viral infectivity and globally increased virus sensitivity to poorly neutralizing antibodies, soluble CD4, a CD4-mimetic compound, and exposure to cold. By contrast, the MPER mutants exhibited decreased sensitivity to the State-1 preferring inhibitor, BMS-806, and to the PGT151 broadly neutralizing antibody. Depletion of cholesterol from virus particles did not produce the same State 1-destabilizing phenotypes as MPER alterations. Notably, State 1-stabilizing changes in Env distant from the MPER could minimize the phenotypic effects of MPER alteration, but did not affect virus sensitivity to cholesterol depletion. Thus, membrane-proximal gp41 elements contribute to the maintenance of the pretriggered Env conformation. The conformationally disruptive effects of MPER changes can be minimized by distant State 1-stabilizing Env modifications, a strategy that may be useful in preserving the native pretriggered state of Env. IMPORTANCEThe pretriggered shape of the human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) is a major target for antibodies that can neutralize many strains of the virus. An effective HIV-1 vaccine may need to raise these types of antibodies, but this goal has proven difficult. One reason is that the pretriggered shape of Env is unstable and dependent on interactions near the viral membrane. Here we show that the membrane-proximal external region (MPER) of Env plays an important role in maintaining Env in a pretriggered shape. Alterations in the MPER resulted in global changes in Env conformation that disrupted its pretriggered shape. We also found that these disruptive effects of MPER changes can be minimized by distant Env modifications that stabilize the pretriggered shape. These modifications may be useful for preserving the native shape of Env for structural and vaccine studies.

microbiology↗