Biology subjects

Kappes, J. C.

Publications and source records attributed to Kappes, J. C..

13 recordsLinked to original sources

bioRxiv2026

Performance of Rilpivirine-Based Hydrophobic Tags and PROTACs Directed Against HIV-1 Reverse Transcriptase

We aimed to repurpose the non-nucleoside reverse transcriptase (RT) inhibitor (NNRTI) rilpivirine (RPV) as a targeted protein degrader (TPD) of HIV-1 RT. Structure-guided modeling identified a TPD bifunctional linker exit trajectory from RPV that threads the NNRTI entrance channel. Ten RPV analogs (RPV') with varied warhead-linker attachments retained RT inhibition and antiviral activity, guiding selection of an amide-linked connector for degrader construction. We synthesized Proteolysis Targeting Chimeras (PROTACs) designed to recruit CUL4CRBN and CUL2VHL, alongside adamantyl acetic acid-based Hydrophobic Tags (HyTs). TPDs were evaluated for RT inhibition, virus inhibition, biophysical target engagement, and proteasome-dependent degradation. Some PROTACs showed minimal antiviral activity or limited solubility for our assays, whereas the HyT with a tetraethylene glycol (PEG4) linker (PEG4-Ad) exhibited low nanomolar potency to suppress HIV-1 replication and acceptable solubility. PEG4-Ad reduced RT levels in a proteasome-dependent manner without cytotoxicity but did not exhibit superior potency against RPV resistance mutations. MD simulations suggested that PEG4 linkers maximize episodic exposure of the hydrophobic tag, consistent with PEG4-Ad efficacy. These data highlight the promise - and constraints - of antiviral degraders, indicating that linker-controlled hydrophobic tag exposure and subcellular target accessibility may be critical design parameters for prospective therapeutics.

microbiology↗

bioRxiv2025

Nanobody-Driven Stabilization Synergistically Rescues F508del-CFTR and Reveals an Alternative Active State of the Channel

Defects in protein trafficking underlie many genetic diseases, including cystic fibrosis (CF), where the predominant F508del mutation destabilizes the cystic fibrosis transmembrane conductance regulator (CFTR) channel, leading to its degradation. To provide a protein-specific chaperone, we used lipid nanoparticles to deliver mRNA encoding T2a, a nanobody that thermally stabilizes CFTR via high-affinity binding to nucleotide-binding domain 1 (NBD1). When combined with clinically-approved correctors, T2a considerably improved F508del-CFTR maturation, plasma membrane expression, and channel activity. Single-channel recordings revealed that nanobody binding sustained channel activity by promoting both full open and sub-conductance gating states and protecting F508del-CFTR against thermal deactivation. Because T2a binding to NBD1 prevents the ATP-dependent NBD1-NBD2 association that drives canonical channel opening, the observation of channel activity in the presence of the nanobody suggested adoption of a novel open-channel CFTR structure. This inference was confirmed by cryo-EM analyses of CFTR in the presence of T2a, which revealed a novel open-channel conformation in which NBD1 binds to an alternative site on CFTR incompatible with NBD dimerization. Our findings establish a new paradigm to correct protein trafficking by stabilizing misfolded domains with targeted nanobodies and demonstrate a broadly applicable framework to treat CF and related protein misfolding diseases.

biochemistry↗

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↗

bioRxiv2023

Folding correctors can restore CFTR posttranslational folding landscape by allosteric domain-domain coupling

AbstractThe folding/misfolding and pharmacological rescue of multidomain ATP-binding cassette (ABC) C-subfamily transporters, essential for organismal health, remain incompletely understood. The ABCC transporters core consists of two nucleotide binding domains (NBD1,2) and transmembrane domains (TMD1,2). Using molecular dynamic simulations, biochemical and hydrogen deuterium exchange approaches, we show that the mutational uncoupling or stabilization of NBD1-TMD1/2 interfaces can compromise or facilitate the CFTR(ABCC7)-, MRP1(ABCC1)-, and ABCC6-transporters posttranslational coupled domain-folding in the endoplasmic reticulum. Allosteric or orthosteric binding of VX-809 and/or VX-445 folding correctors to TMD1/2 can rescue kinetically trapped CFTR post-translational folding intermediates of cystic fibrosis (CF) mutants of NBD1 or TMD1 by global rewiring inter-domain allosteric-networks. We propose that dynamic allosteric domain-domain communications not only regulate ABCC-transporters function but are indispensable to tune the folding landscape of their post-translational intermediates. These allosteric networks can be compromised by CF-mutations, and reinstated by correctors, offering a framework for mechanistic understanding of ABCC-transporters (mis)folding. One-Sentence SummaryAllosteric interdomain communication and its modulation are critical determinants of ABCC-transporters post-translational conformational biogenesis, misfolding, and pharmacological rescue.

biophysics↗

bioRxiv2023

Conformations of the Human Immunodeficiency Virus (HIV-1) Envelope Glycoproteins in Detergents and Styrene-Maleic Acid Lipid Particles (SMALPs)

The mature human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) trimer, which consists of non-covalently associated gp120 exterior and gp41 transmembrane subunits, mediates virus entry into cells. The pretriggered (State-1) Env conformation is the major target for broadly neutralizing antibodies (bNAbs), whereas receptor-induced downstream Env conformations elicit immunodominant, poorly neutralizing antibody (pNAb) responses. To examine the contribution of membrane anchorage to the maintenance of the metastable pretriggered Env conformation, we compared wild-type and State-1-stabilized Envs solubilized in detergents or in styrene-maleic acid (SMA) copolymers. SMA directly incorporates membrane lipids and resident membrane proteins into lipid nanodiscs (SMALPs). The integrity of the Env trimer in SMALPs was maintained at both 4{degrees}C and room temperature. By contrast, Envs solubilized in Cymal-5, a non-ionic detergent, were unstable at room temperature, although their stability was improved at 4{degrees}C and after incubation with the entry inhibitor BMS-806. Envs solubilized in ionic detergents were relatively unstable at either temperature. Comparison of Envs solubilized in Cymal-5 and SMA at 4{degrees}C revealed subtle differences in bNAb binding to the gp41 membrane-proximal external region (MPER), consistent with these distinct modes of Env solubilization. Otherwise, the antigenicity of the Cymal-5- and SMA- solubilized Envs was remarkably similar, both in the absence and presence of BMS-806. However, both solubilized Envs were recognized differently from the mature membrane Env by specific bNAbs and pNAbs. Thus, detergent-based and detergent-free solubilization at 4{degrees}C alters the pretriggered membrane Env conformation in consistent ways, indicating that loss of Env association with the membrane results in default state(s). IMPORTANCEThe human immunodeficiency virus (HIV-1) envelope glycoproteins (Envs) in the viral membrane mediate virus entry into the host cell and are targeted by neutralizing antibodies elicited by natural infection or vaccines. Detailed studies of membrane proteins rely on purification procedures that allow the proteins to maintain their natural conformation. In this study, we show that a styrene-maleic acid (SMA) copolymer can extract HIV-1 Env from a membrane without the use of detergents. The Env in SMA is more stable at room temperature than Env in detergents. The purified Env in SMA maintains many but not all of the characteristics expected of the natural membrane Env. Our results underscore the importance of the membrane environment to the native conformation of HIV-1 Env. Purification methods that bypass the need for detergents could be useful tools for future studies of HIV-1 Env structure and its interaction with receptors and antibodies.

microbiology↗

bioRxiv2022

Mechanism of dual pharmacological correction and potentiation of human CFTR

Cystic fibrosis (CF) is caused by mutations in a chloride channel called the human Cystic Fibrosis Transmembrane Conductance Regulator (hCFTR). We used cryo-EM global conformational ensemble reconstruction to characterize the mechanism by which the breakthrough drug VX445 (Elexacaftor) simultaneously corrects both protein-folding and channel-gating defects caused by CF mutations. VX445 drives hCFTR molecules harboring the gating-defective G551D mutation towards the open-channel conformation by binding to a site in the first transmembrane domain. This binding interaction reverses the usual pathway of allosteric structural communication by which ATP binding activates channel conductance, which is blocked by the G551D mutation. Our ensemble reconstructions include a 3.4 [A] non-native structure demonstrating that detachment of the first nucleotide-binding domain of hCFTR is directly coupled to local unfolding of the VX445 binding site. Reversal of this unfolding transition likely contributes to its corrector activity by cooperatively stabilizing NBD1 and the transmembrane domains of hCFTR during biogenesis. SummaryCryo-EM global conformational ensemble reconstruction has been used to characterize the mechanism-of-action of a breakthrough pharmaceutical that corrects fatal protein-folding and channel-gating defects in the human cystic fibrosis transmembrane conductance regulator (CFTR).

biophysics↗

bioRxiv2022

Small CD4 mimetics sensitize HIV-1-infected macrophages to antibody-dependent cellular cytotoxicity

HIV-1 Envelope (Env) conformation determines the susceptibility of infected CD4+ T cells to Antibody Dependent Cellular Cytotoxicity (ADCC). Upon interaction with CD4, Env adopts more "open" conformations, exposing ADCC epitopes. HIV-1 limits Env-CD4 interaction and protects infected cells against ADCC by downregulating CD4 via Nef, Vpu and Env. Limited data exists however of the role of these proteins in downmodulating CD4 on infected macrophages and how this impacts Env conformation. While Nef, Vpu and Env are all required to efficiently downregulate CD4 on infected CD4+T cells, we show here that any one of these proteins is sufficient to downmodulate most CD4 from the surface of infected macrophages. Consistent with this finding, Nef and Vpu have a lesser impact on Env conformation and ADCC sensitivity in infected macrophages compared to CD4+T cells. However, treatment of infected macrophages with small CD4-mimetics expose vulnerable CD4-induced Env epitopes and sensitize them to ADCC.

microbiology↗

bioRxiv2021

Detection of the HIV-1 accessory proteins Nef and Vpu by flow cytometry represents a new tool to study their functional interplay within a single infected CD4+ T cell

The HIV-1 Nef and Vpu accessory proteins are known to protect infected cells from antibody-dependent cellular cytotoxicity (ADCC) responses by limiting exposure of CD4-induced (CD4i) envelope (Env) epitopes at the cell surface. Although both proteins target the host receptor CD4 for degradation, the extent of their functional redundancy is unknown. Here, we developed an intracellular staining technique that permits the intracellular detection of both Nef and Vpu in primary CD4+ T cells by flow cytometry. Using this method, we show that the combined expression of Nef and Vpu predicts the susceptibility of HIV-1-infected primary CD4+ T cells to ADCC by HIV+ plasma. We also show that Vpu cannot compensate for the absence of Nef, thus providing an explanation for why some infectious molecular clones that carry a LucR reporter gene upstream of Nef render infected cells more susceptible to ADCC responses. Our method thus represents a new tool to dissect the biological activity of Nef and Vpu in the context of other host and viral proteins within single infected CD4+ T cells. IMPORTANCEHIV-1 Nef and Vpu exert several biological functions that are important for viral immune evasion, release and replication. Here, we developed a new method allowing simultaneous detection of these accessory proteins in their native form together with some of their cellular substrates. This allowed us to show that Vpu cannot compensate the lack of a functional Nef, which has implication for studies that use Nef-defective viruses to study ADCC responses.

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↗

bioRxiv2021

Asymmetric structures and conformational plasticity of the uncleaved full-length human immunodeficiency virus (HIV-1) envelope glycoprotein trimer

The functional human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) trimer [(gp120/gp41)3] is produced by cleavage of a conformationally flexible gp160 precursor. Gp160 cleavage or the binding of BMS-806, an entry inhibitor, stabilizes the pre-triggered, "closed" (State-1) conformation recognized by rarely elicited broadly neutralizing antibodies. Poorly neutralizing antibodies (pNAbs) elicited at high titers during natural infection recognize more "open" Env conformations (States 2 and 3) induced by binding the receptor, CD4. We found that BMS-806 treatment and crosslinking decreased the exposure of pNAb epitopes on cell-surface gp160; however, after detergent solubilization, crosslinked and BMS-806-treated gp160 sampled non-State-1 conformations that could be recognized by pNAbs. Cryo-electron microscopy of the purified BMS-806-bound gp160 revealed two hitherto unknown asymmetric trimer conformations, providing insights into the allosteric coupling between trimer opening and structural variation in the gp41 HR1N region. The individual protomer structures in the asymmetric gp160 trimers resemble those of other genetically modified or antibody-bound cleaved HIV-1 Env trimers, which have been suggested to assume State-2-like conformations. Asymmetry of the uncleaved Env potentially exposes surfaces of the trimer to pNAbs. To evaluate the effect of stabilizing a State-1-like conformation of the membrane Env precursor, we treated cells expressing wild-type HIV-1 Env with BMS-806. BMS-806 treatment decreased both gp160 cleavage and the addition of complex glycans, implying that gp160 conformational flexibility contributes to the efficiency of these processes. Selective pressure to maintain flexibility in the precursor of functional Env allows the uncleaved Env to sample asymmetric conformations that potentially skew host antibody responses toward pNAbs. IMPORTANCEThe envelope glycoprotein (Env) trimers on the surface of human immunodeficiency virus (HIV-1) mediate the entry of the virus into host cells and serve as targets for neutralizing antibodies. The functional Env trimer is produced by cleavage of the gp160 precursor in the infected cell. We found that the HIV-1 Env precursor is highly plastic, allowing it to assume different asymmetric shapes. This conformational plasticity is potentially important for Env cleavage and proper modification by sugars. Having a flexible, asymmetric Env precursor that can misdirect host antibody responses without compromising virus infectivity would be an advantage to a persistent virus like HIV-1.

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↗

bioRxiv2018

Structural insights into the conformational plasticity of the full-length trimeric HIV-1 envelope glycoprotein precursor

The human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) trimer mediates viral entry into cells and is the major target for the host antibody response. In infected cells, the mature Env [(gp120/gp41)3] is produced by cleavage of a trimeric gp160 precursor. Proteolytic cleavage decreases Env conformational flexibility, allowing the mature Env to resist antibody binding to conserved elements. The conformational plasticity of the Env precursor skews the humoral immune response towards the elicitation of ineffectual antibodies, contributing to HIV-1 persistence in the infected host. The structural basis for the plasticity of the Env precursor remains elusive. Here we use cryo-electron microscopy to visualize two coexisting conformational states of the full-length Env precursor at nominal resolutions of 5.5 and 8.0 [A]. The State-P2 conformation features a three-helix bundle of the gp41 heptad repeat region in the core, but has disordered membrane-interactive regions. State-P1 trimers lack the three-helix bundle and instead retain ordered transmembrane and membrane-proximal external regions embracing a central cavity. Our structural data shed light on the unusual plasticity of the Env precursor and provide new clues to Env immunogen discovery.

microbiology↗