HIV-1 Envelope Glycoproteins Proteolytic Cleavage Protects Infected Cells from ADCC Mediated by Plasma from Infected Individuals

The HIV-1 envelope glycoprotein (Env) is synthesized in the endoplasmic reticulum as a trimeric gp160 precursor, which requires proteolytic cleavage by a cellular furin protease to mediate virus-cell fusion. Env is conformationally flexible but controls its transition from the unbound “closed” conformation (State 1) to downstream CD4-bound conformations (States 2/3), which are required for fusion. In particular, HIV-1 has evolved several mechanisms that reduce the premature “opening” of Env which exposes highly conserved epitopes recognized by non-neutralizing antibodies (nnAbs) capable of mediating antibody-dependent cellular cytotoxicity (ADCC). Env cleavage decreases its conformational transitions favoring the adoption of the “closed” conformation. Here we altered the gp160 furin cleavage site to impair Env cleavage and to examine its impact on ADCC responses mediated by plasma from HIV-1-infected individuals. We found that infected primary CD4+ T cells expressing uncleaved, but not wildtype, Env are efficiently recognized by nnAbs and become highly susceptible to ADCC responses mediated by plasma from HIV-1-infected individuals. Thus, HIV-1 limits the exposure of uncleaved Env at the surface of HIV-1-infected cells at least in part to escape ADCC responses.

HIV-1 envelope glycoproteins proteolytic cleavage protects infected cells from ADCC mediated by plasma from infected individuals

The HIV-1 envelope glycoprotein (Env) is synthesized in the endoplasmic reticulum as a trimeric gp160 precursor, which requires proteolytic cleavage by a cellular furin protease to mediate virus-cell fusion. Env is conformationally flexible, but controls its transition from the unbound closed conformation (State 1) to downstream CD4-bound conformations (States 2/3), which are required for fusion. In particular, HIV-1 has evolved several mechanisms that reduce the premature opening of Env which exposes highly conserved epitopes recognized by non-neutralizing antibodies (nnAbs) capable of mediating antibody-dependent cellular cytotoxicity (ADCC). Env cleavage decreases its conformational transitions favoring the adoption of the closed conformation. Here we altered the gp160 furin cleavage site to impair Env cleavage and to examine its impact on ADCC responses mediated by plasma from HIV-1-infected individuals. We found that infected primary CD4+ T cells expressing uncleaved, but not wildtype, Env are efficiently recognized by nnAbs and become highly susceptible to ADCC responses mediated by plasma from HIV-1-infected individuals. Thus, HIV-1 limits the exposure of uncleaved Env at the surface of HIV-1-infected cells at least in part to escape ADCC responses.

Stabilizing the HIV-1 envelope glycoprotein State 2A conformation

The HIV-1 envelope glycoprotein (Env) trimer [(gp120/gp41)3] is a metastable complex expressed at the surface of viral particles and infected cells that samples different conformations. Before engaging CD4, Env adopts an antibody-resistant “closed” conformation (State 1). CD4 binding triggers an intermediate conformation (State 2) and then a more “open” conformation (State 3) that can be recognized by non-neutralizing antibodies (nnAbs) such as those that recognize the coreceptor binding site (CoRBS). Binding of antibodies to the CoRBS permits another family of nnAbs, the anti-cluster A family of Abs which target the gp120 inner domain, to bind and stabilize an asymmetric conformation (State 2A). Cells expressing Env in this conformation are susceptible to antibody-dependent cellular cytotoxicity (ADCC). This conformation can be stabilized by small-molecule CD4 mimetics (CD4mc) or soluble CD4 (sCD4) in combination with anti-CoRBS Ab and anti-cluster A antibodies. The precise stoichiometry of each component that permits this sequential opening of Env remains unknown. Here, we used a cell-based ELISA (CBE) assay to evaluate each component individually. In this assay we used a “trimer mixing” approach by combining wild-type (wt) subunits with subunits impaired for CD4 or CoRBS Ab binding. This enabled us to show that State 2A requires all three gp120 subunits to be bound by sCD4/CD4mc and anti-CoRBS Abs. Two of these subunits can then bind anti-cluster A Abs. Altogether, our data suggests how this antibody vulnerable Env conformation is stabilized. Importance Stabilization of HIV-1 Env State 2A has been shown to sensitize infected cells to ADCC. State 2A can be stabilized by a “cocktail” composed of CD4mc, anti-CoRBS and anti-cluster A Abs. We present evidence that optimal State 2A stabilization requires all three gp120 subunits to be bound by both CD4mc and anti-CoRBS Abs. Our study provides valuable information on how to stabilize this ADCC-vulnerable conformation. Strategies aimed at stabilizing State 2A might have therapeutic utility.

Shedding-Resistant HIV-1 Envelope Glycoproteins Adopt Downstream Conformations That Remain Responsive to Conformation-Preferring Ligands

ABSTRACT The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) trimer of gp120-gp41 heterodimers mediates virus entry into CD4-positive (CD4+) cells. Single-molecule fluorescence resonance energy transfer (smFRET) has revealed that native Env on the surface of viruses predominantly exists in a pretriggered conformation (state 1) that is preferentially recognized by many broadly neutralizing antibodies (bNAbs). Env is activated by binding receptor CD4, which drives transitions through a default intermediate conformation (state 2) into the three-CD4-bound open conformation (state 3). The application of smFRET to assess the conformational state of existing Env constructs and ligand complexes recently revealed that all current high-resolution structures correspond to downstream states 2 and 3. The structure of state 1, therefore, remains unknown. We sought to identify conditions whereby HIV-1 Env could be stabilized in the pretriggered state 1 for possible structural characterization. Shedding of gp120, known to severely complicate structural studies, can be prevented by using the uncleaved gp160JR-FL precursor with alterations in the protease cleavage site (R508S/R511S) or by introducing a disulfide bridge between gp120 and gp41 designated “SOS” (A501C/T605C). smFRET demonstrated that both shedding-preventing modifications shifted the conformational landscape of Env downstream toward states 2 and 3. However, both membrane-bound Env proteins on the surface of intact viruses remained conformationally dynamic, responsive to state-stabilizing ligands, and able to be stabilized in state 1 by specific ligands such as the Bristol-Myers Squibb (BMS) entry inhibitors. The here-described identification of state 1-stabilizing conditions may enable structural characterization of the state 1 conformation of HIV-1 Env. IMPORTANCE The HIV-1 envelope glycoprotein (Env) opens in response to receptor CD4 binding from a pretriggered (state 1) conformation through a necessary intermediate to the three-CD4-bound conformation. The application of smFRET to test the conformational state of existing Env constructs and ligand complexes used for high-resolution structures recently revealed that they correspond to the downstream conformations. The structure of the pretriggered Env conformation, preferentially recognized by broadly neutralizing antibodies, remains unknown. Here, we identify experimental conditions that stabilize membrane-bound and shedding-resistant virus Env trimers in state 1, potentially facilitating structural characterization of this unknown conformational state.

Exposing HIV-1 Env: Implications for therapeutic strategies

The human immunodeficiency virus (HIV-1) envelope glycoprotein trimer (Env) is exposed on the surfaces of both virions and infected cells. Thus, Env is the principal target for neutralizing antibodies and antibodies able to mediate antibodydependent cellular cytotoxicity (ADCC). The HIV-1 Env is a flexible molecule known to exist in at least three different conformational states: states 1, 2 and 3. Before interacting with the primary receptor, CD4, Env preferentially adopts a compact, “closed” conformation (state 1) that is largely antibody-resistant. The CD4 binding “opens” Env increasing the vulnerability of infected cells to ADCC mediated by non-neutralizing antibodies, as these easily-elicited antibodies preferentially recognize epitopes exposed in the open conformational states (states 2/3). These antibodies include the anti-coreceptor binding site and the anti-cluster A families of antibodies that, in combination with small CD4-mimetic compounds, stabilize a new asymmetric Env conformation (state 2A) that is vulnerable to ADCC. Approaches aimed at stabilizing this “open” conformation represent new interventional approaches to fight HIV-1 infection.

Minute-scale persistence of a GPCR conformation state triggered by non-cognate G protein interactions primes signaling

Despite the crowded nature of the cellular milieu, ligand–GPCR–G protein interactions are traditionally viewed as spatially and temporally isolated events. In contrast, recent reports suggest the spatial and temporal coupling of receptor–effector interactions, with the potential to diversify downstream responses. In this study, we combine protein engineering of GPCR–G protein interactions with affinity sequestration and photo-manipulation of the crucial Gα C terminus, to demonstrate the temporal coupling of cognate and non-cognate G protein interactions through priming of the GPCR conformation. We find that interactions of the Gαs and Gαq C termini with the β2-adrenergic receptor (β2-AR), targeted at the G-protein-binding site, enhance Gs activation and cyclic AMP levels. β2-AR–Gα C termini interactions alter receptor conformation, which persists for ~90 s following Gα C terminus dissociation. Non-cognate G-protein expression levels impact cognate signaling in cells. Our study demonstrates temporal allostery in GPCRs, with implications for the modulation of downstream responses through the canonical G-protein-binding interface.

PTP-3(LAR PTPR) promotes intramolecular folding of SYD-2(liprin-α) to inactivate UNC-104(KIF1A) in neurons

This study aims to demonstrate how PTP-3 regulates SYD-2 to control UNC-104-mediated axonal transport. UNC-104 is the C. elegans homolog of kinesin-3 KIF-1A known for its fast shuttling of STVs (synaptic vesicle protein transport vesicles) in axons. SYD-2 is the homolog of liprin-α in C. elegans known to directly regulate UNC-104 as well as being a substrate of LAR PTPR (leukocyte common antigen-related (LAR) protein tyrosine phosphatase (PTP) transmembrane receptor) with PTP-3 as the closest homolog in C. elegans. CoIP assays revealed increased interaction between UNC-104 and SYD-2 in lysates from ptp-3 knockout worms. Intramolecular FRET analysis revealed that SYD-2 predominantly exists in an open conformation state in ptp-3 mutants. These assays also revealed that non-phosphorylatable SYD-2 (Y741F) exists predominately in folded conformations while phosphomimicking SYD-2 (Y741E) exists predominantly in open conformations. In ptp-3 mutants, SNB-1 cargo accumulates in soma while at the same time UNC-104 motors increasingly cluster along initial segments of axons. Interestingly, the unc-104 gene is downregulated in ptp-3 mutants that might explain the vesicle retention phenotype. More strikingly, the few visibly moving motors and STVs were overly active in neurons of these mutants. We propose a model in which the lack of PTP-3 promotes increased open conformations of SYD-2 that in turn facilitates UNC-104/SYD-2 interactions boosting motor and STVs moving speeds.

Crystal structures of the closed form ofMycobacterium tuberculosisdihydrofolate reductase in complex with dihydrofolate and antifolates

Tuberculosis is a disease caused byMycobacterium tuberculosisand is the leading cause of death from a single infectious pathogen, with a high prevalence in developing countries in Africa and Asia. There still is a need for the development or repurposing of novel therapies to combat this disease owing to the long-term nature of current therapies and because of the number of reported resistant strains. Here, structures of dihydrofolate reductase fromM. tuberculosis(MtDHFR), which is a key target of the folate pathway, are reported in complex with four antifolates, pyrimethamine, cycloguanil, diaverdine and pemetrexed, and its substrate dihydrofolate in order to understand their binding modes. The structures of all of these complexes were obtained in the closed-conformation state of the enzyme and a fine structural analysis indicated motion in key regions of the substrate-binding site and different binding modes of the ligands. In addition, the affinities, throughKdmeasurement, of diaverdine and methotrexate have been determined; MtDHFR has a lower affinity (highestKd) for diaverdine than pyrimethamine and trimethoprim, and a very high affinity for methotrexate, as expected. The structural comparisons and analysis described in this work provide new information about the plasticity of MtDHFR and the binding effects of different antifolates.