Computational Insights into the Conformational Dynamics of HIV-1 Vpr in a Lipid Bilayer for Ion Channel Modeling.

HIV-1 Vpr is a multifunctional accessory protein consisting of 96 amino acids that play a critical role in viral pathogenesis. Among its diverse range of activities, Vpr can create a cation-selective ion channel within the plasma membrane. However, the oligomeric state of this channel has not yet been elucidated. In this study, we investigated the conformational dynamics of Vpr helices to model the ion channel topology. First, we employed a series of multiscale simulations to investigate the specific structure of monomeric Vpr in a membrane model. During the lipid bilayer self-assembly coarse grain simulation, the C-terminal helix (residues 56-77) effectively formed the transmembrane region, while the N-terminal helix exhibited an amphipathic nature by associating horizontally with a single leaflet. All-atom molecular dynamics (MD) simulations of full-length Vpr inside a phospholipid bilayer show that the C-terminal helix remains very stable inside the bilayer core in a vertical orientation. Subsequently, using the predicted C-terminal helix orientation and conformation, various oligomeric states (ranging from tetramer to heptamer) possibly forming the Vpr ion channel were built and further evaluated. Among these models, the pentameric form exhibited consistent stability in MD simulations and displayed a compatible conformation for a water-assisted ion transport mechanism. This study provides structural insights into the ion channel activity of the Vpr protein and the foundation for developing therapeutics against HIV-1 Vpr-related conditions.

High-throughput virtual search of small molecules for controlling the mechanical stability of human CD4.

Protein mechanical stability determines the function of a myriad of proteins, especially proteins from the extracellular matrix. Failure to maintain protein mechanical stability may result in diseases and disorders such as cancer, cardiomyopathies, or muscular dystrophy. Thus, developing mutation-free approaches to enhance and control the mechanical stability of proteins using pharmacology-based methods may have important implications in drug development and discovery. Here, we present the first approach that employs computational high-throughput virtual screening and molecular docking to search for small molecules in chemical libraries that function as mechano-regulators of the stability of human cluster of differentiation 4, receptor of HIV-1. Using single-molecule force spectroscopy, we prove that these small molecules can increase the mechanical stability of CD4D1D2 domains over 4-fold in addition to modifying the mechanical unfolding pathways. Our experiments demonstrate that chemical libraries are a source of mechanoactive molecules and that drug discovery approaches provide the foundation of a new type of molecular function, that is, mechano-regulation, paving the way toward mechanopharmacology.

A two-step mechanism for the binding of the HIV-1 MPER epitope by the 10E8 antibody onto biosensor-supported lipid bilayers.

HIV-1 antibodies targeting the carboxy-terminal area of the membrane-proximal external region (ctMPER) are close to exerting viral pan-neutralization. Here, we reconstituted the ctMPER epitope as the N-terminal extremity of the Env glycoprotein transmembrane domain helix and immobilized it onto biosensor-supported lipid bilayers. We assessed the binding mechanism of anti-MPER antibody 10E8 through Surface Plasmon Resonance, and found, through equilibrium and kinetic binding analyses as a function of bilayer thickness, peptide length, and paratope mutations, that 10E8 engages first with the epitope peptide (encounter), limited by ctMPER helix accessibility at the membrane surface, and then inserts into the lipid bilayer assisted by favorable Fab-membrane interactions (docking). This mechanistic information may help in devising new strategies to develop more efficient MPER-targeting vaccines.

Hexagonal Lattices of HIV Capsid Proteins Explored by Simulations Based on a Thermodynamically Consistent Model.

HIV capsid proteins (CAs) may self-assemble into a variety of shapes under in vivo and in vitro conditions. Here, we employed simulations based on a residue-level coarse-grained (CG) model with full conformational flexibility to investigate hexagonal lattices, which are the underlying structural pattern for CA aggregations. Facilitated by enhanced sampling simulations to rigorously calculate CA dimerization and polymerization affinities, we calibrated our model to reproduce the experimentally measured affinities. Using the calibrated model, we performed unbiased simulations on several large systems consisting of 1512 CA subunits, allowing reversible binding and unbinding of the CAs in a thermodynamically consistent manner. In one simulation, a preassembled hexagonal CA sheet developed spontaneous curvatures reminiscent of those observed in experiments, and the edges of the sheet exhibited local curvatures larger than those of the interior. In other simulations starting with randomly distributed CAs at different concentrations, existing CA assemblies grew by binding free capsomeres to the edges and by merging with other assemblies. At high CA concentrations, rapid establishment of predominant aggregates was followed by much slower adjustments toward more regular hexagonal lattices, with increasing numbers of intact CA hexamers and pentamers being formed. Our approach of adapting a general CG model to specific systems by using experimental binding data represents a practical and effective strategy for simulating and elucidating intricate protein aggregations.

HIV-1 capsids enter the FG phase of nuclear pores like a transport receptor.

HIV-1 infection requires nuclear entry of the viral genome. Previous evidence suggests that this entry proceeds through nuclear pore complexes (NPCs), with the 120 × 60 nm capsid squeezing through an approximately 60-nm-wide central channel1 and crossing the permeability barrier of the NPC. This barrier can be described as an FG phase2 that is assembled from cohesively interacting phenylalanine-glycine (FG) repeats3 and is selectively permeable to cargo captured by nuclear transport receptors (NTRs). Here we show that HIV-1 capsid assemblies can target NPCs efficiently in an NTR-independent manner and bind directly to several types of FG repeats, including barrier-forming cohesive repeats. Like NTRs, the capsid readily partitions into an in vitro assembled cohesive FG phase that can serve as an NPC mimic and excludes much smaller inert probes such as mCherry. Indeed, entry of the capsid protein into such an FG phase is greatly enhanced by capsid assembly, which also allows the encapsulated clients to enter. Thus, our data indicate that the HIV-1 capsid behaves like an NTR, with its interior serving as a cargo container. Because capsid-coating with trans-acting NTRs would increase the diameter by 10 nm or more, we suggest that such a 'self-translocating' capsid undermines the size restrictions imposed by the NPC scaffold, thereby bypassing an otherwise effective barrier to viral infection.

Molecular Determinants of PQBP1 Binding to the HIV-1 Capsid Lattice.

Human immunodeficiency virus type 1 (HIV-1) stimulates innate immune responses upon infection, including cyclic GMP-AMP synthase (cGAS) signaling that results in type I interferon production. HIV-1-induced activation of cGAS requires the host cell factor polyglutamine binding protein 1 (PQBP1), an intrinsically disordered protein that bridges capsid recognition and cGAS recruitment. However, the molecular details of PQBP1 interactions with the HIV-1 capsid and their functional implications remain poorly understood. Here, we show that PQBP1 binds to HIV-1 capsids through charge complementing contacts between acidic residues in the N-terminal region of PQBP1 and an arginine ring in the central channel of the HIV-1 CA hexamer that makes up the viral capsid. These studies reveal the molecular details of PQBP1's primary interaction with the HIV-1 capsid and suggest that additional elements are likely to contribute to stable capsid binding.

HIV-1 Env trimers asymmetrically engage CD4 receptors in membranes.

Human immunodeficiency virus 1 (HIV-1) infection is initiated by binding of the viral envelope glycoprotein (Env) to the cell-surface receptor CD41-4. Although high-resolution structures of Env in a complex with the soluble domains of CD4 have been determined, the binding process is less understood in native membranes5-13. Here we used cryo-electron tomography to monitor Env-CD4 interactions at the membrane-membrane interfaces formed between HIV-1 and CD4-presenting virus-like particles. Env-CD4 complexes organized into clusters and rings, bringing the opposing membranes closer together. Env-CD4 clustering was dependent on capsid maturation. Subtomogram averaging and classification revealed that Env bound to one, two and finally three CD4 molecules, after which Env adopted an open state. Our data indicate that asymmetric HIV-1 Env trimers bound to one and two CD4 molecules are detectable intermediates during virus binding to host cell membranes, which probably has consequences for antibody-mediated immune responses and vaccine immunogen design.

Intermediate conformations of CD4-bound HIV-1 Env heterotrimers.

HIV-1 envelope (Env) exhibits distinct conformational changes in response to host receptor (CD4) engagement. Env, a trimer of gp120 and gp41 heterodimers, has been structurally characterized in a closed, prefusion conformation with closely associated gp120s and coreceptor binding sites on gp120 V3 hidden by V1V2 loops1-4 and in fully saturated CD4-bound open Env conformations with changes including outwardly rotated gp120s and displaced V1V2 loops3-9. To investigate changes resulting from substoichiometric CD4 binding, we solved single-particle cryo-electron microscopy (cryo-EM) structures of soluble, native-like heterotrimeric Envs bound to one or two CD4 molecules. Most of the Env trimers bound to one CD4 adopted the closed, prefusion Env state, with a minority exhibiting a heterogeneous partially open Env conformation. When bound to two CD4s, the CD4-bound gp120s exhibited an open Env conformation including a four-stranded gp120 bridging sheet and displaced gp120 V1V2 loops that expose the coreceptor sites on V3. The third gp120 adopted an intermediate, occluded-open state10 that showed gp120 outward rotation but maintained the prefusion three-stranded gp120 bridging sheet with only partial V1V2 displacement and V3 exposure. We conclude that most of the engagements with one CD4 molecule were insufficient to stimulate CD4-induced conformational changes, whereas binding two CD4 molecules led to Env opening in CD4-bound protomers only. The substoichiometric CD4-bound soluble Env heterotrimer structures resembled counterparts derived from a cryo-electron tomography study of complexes between virion-bound Envs and membrane-anchored CD4 (ref. 11), validating their physiological relevance. Together, these results illuminate intermediate conformations of HIV-1 Env and illustrate its structural plasticity.

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

IMPORTANCE: CD4-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.

Broadly neutralizing antibody epitopes on HIV-1 particles are exposed after virus interaction with host cells.

The envelope (Env) glycoproteins on HIV-1 virions are the sole target of broadly neutralizing antibodies (bNAbs) and the focus of vaccines. However, many cross-reactive conserved epitopes are often occluded on virus particles, contributing to the evasion of humoral immunity. This study aimed to identify the Env epitopes that are exposed/occluded on HIV-1 particles and to investigate the mechanisms contributing to their masking. Using a flow cytometry-based assay, three HIV-1 isolates, and a panel of antibodies, we show that only select epitopes, including V2i, the gp120-g41 interface, and gp41-MPER, are accessible on HIV-1 particles, while V3, V2q, and select CD4bs epitopes are masked. These epitopes become accessible after allosteric conformational changes are induced by the pre-binding of select Abs, prompting us to test if similar conformational changes are required for these Abs to exhibit their neutralization capability. We tested HIV-1 neutralization where the virus-mAb mix was pre-incubated/not pre-incubated for 1 hour prior to adding the target cells. Similar levels of neutralization were observed under both assay conditions, suggesting that the interaction between virus and target cells sensitizes the virions for neutralization via bNAbs. We further show that lectin-glycan interactions can also expose these epitopes. However, this effect is dependent on the lectin specificity. Given that, bNAbs are ideal for providing sterilizing immunity and are the goal of current HIV-1 vaccine efforts, these data offer insight on how HIV-1 may occlude these vulnerable epitopes from the host immune response. In addition, the findings can guide the formulation of effective antibody combinations for therapeutic use. IMPORTANCE The human immunodeficiency virus (HIV-1) envelope (Env) glycoprotein mediates viral entry and is the sole target of neutralizing antibodies. Our data suggest that antibody epitopes including V2q (e.g., PG9, PGT145), CD4bs (e.g., VRC01, 3BNC117), and V3 (2219, 2557) are masked on HIV-1 particles. The PG9 and 2219 epitopes became accessible for binding after conformational unmasking was induced by the pre-binding of select mAbs. Attempts to understand the masking mechanism led to the revelation that interaction between virus and host cells is needed to sensitize the virions for neutralization by broadly neutralizing antibodies (bNAbs). These data provide insight on how bNAbs may gain access to these occluded epitopes to exert their neutralization effects and block HIV-1 infection. These findings have important implications for the way we evaluate the neutralizing efficacy of antibodies and can potentially guide vaccine design.

Asymmetric conformations of cleaved HIV-1 envelope glycoprotein trimers in styrene-maleic acid lipid nanoparticles.

During virus entry, the pretriggered human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) trimer initially transits into a default intermediate state (DIS) that remains structurally uncharacterized. Here, we present cryo-EM structures at near-atomic resolution of two cleaved full-length HIV-1 Env trimers purified from cell membranes in styrene-maleic acid lipid nanoparticles without antibodies or receptors. The cleaved Env trimers exhibited tighter subunit packing than uncleaved trimers. Cleaved and uncleaved Env trimers assumed remarkably consistent yet distinct asymmetric conformations, with one smaller and two larger opening angles. Breaking conformational symmetry is allosterically coupled with dynamic helical transformations of the gp41 N-terminal heptad repeat (HR1N) regions in two protomers and with trimer tilting in the membrane. The broken symmetry of the DIS potentially assists Env binding to two CD4 receptors-while resisting antibody binding-and promotes extension of the gp41 HR1 helical coiled-coil, which relocates the fusion peptide closer to the target cell membrane.

HIV-1 Group M Capsid Amino Acid Variability: Implications for Sequence Quality Control of Genotypic Resistance Testing.

BACKGROUND: With the approval of the HIV-1 capsid inhibitor, lenacapavir, capsid sequencing will be required for managing lenacapavir-experienced individuals with detectable viremia. Successful sequence interpretation will require examining new capsid sequences in the context of previously published sequence data. METHODS: We analyzed published HIV-1 group M capsid sequences from 21,012 capsid-inhibitor naïve individuals to characterize amino acid variability at each position and influence of subtype and cytotoxic T lymphocyte (CTL) selection pressure. We determined the distributions of usual mutations, defined as amino acid differences from the group M consensus, with a prevalence ≥ 0.1%. Co-evolving mutations were identified using a phylogenetically-informed Bayesian graphical model method. RESULTS: 162 (70.1%) positions had no usual mutations (45.9%) or only conservative usual mutations with a positive BLOSUM62 score (24.2%). Variability correlated independently with subtype-specific amino acid occurrence (Spearman rho = 0.83; p < 1 × 10-9) and the number of times positions were reported to contain an HLA-associated polymorphism, an indicator of CTL pressure (rho = 0.43; p = 0.0002). CONCLUSIONS: Knowing the distribution of usual capsid mutations is essential for sequence quality control. Comparing capsid sequences from lenacapavir-treated and lenacapavir-naïve individuals will enable the identification of additional mutations potentially associated with lenacapavir therapy.

Novel role of HIV-1 Nef in regulating the ubiquitination of cellular proteins.

Our recent data established that HIV-1 Nef is pivotal in determining the fate of cellular proteins by modulating ubiquitination. However, it is unknown which proteins are ubiquitinated in the presence of Nef, a question critical for understanding the proliferation/restriction strategies of HIV-1 in infected cells. To identify cellular proteins ubiquitinated by Nef, we conducted a proteomic analysis of cellular proteins in the presence and absence of Nef. Proteomic analysis in HEK293T cells indicated that 93 proteins were upregulated and 232 were downregulated in their ubiquitination status by Nef. Computational analysis classified these proteins based on molecular function, biological process, subcellular localization, and biological pathway. Of those proteins, we found a majority of molecular functions to be involved in binding and catalytic activity. With respect to biological processes, a significant portion of the proteins identified were related to cellular and metabolic processes. Subcellular localization analysis showed the bulk of proteins to be localized to the cytosol and cytosolic compartments, which is consistent with the known function and location of Nef during HIV-1 infection. As for biological pathways, the wide range of affected proteins was denoted by the multiple modes to fulfill function, as distinguished from a strictly singular means, which was not detected. Among these ubiquitinated proteins, six were found to directly interact with Nef, wherein two were upregulated and four downregulated. We also identified 14 proteins involved in protein stability through directly participating in the Ubiquitin Proteasome System (UPS)-mediated proteasomal degradation pathway. Of those proteins, we found six upregulated and eight downregulated. Taken together, these analyses indicate that HIV-1 Nef is integral to regulating the stability of various cellular proteins via modulating ubiquitination. The molecular mechanisms directing Nef-triggered regulation of cellular protein ubiquitination are currently under investigation.

A molecular switch modulates assembly and host factor binding of the HIV-1 capsid.

The HIV-1 capsid is a fullerene cone made of quasi-equivalent hexamers and pentamers of the viral CA protein. Typically, quasi-equivalent assembly of viral capsid subunits is controlled by a molecular switch. Here, we identify a Thr-Val-Gly-Gly motif that modulates CA hexamer/pentamer switching by folding into a 310 helix in the pentamer and random coil in the hexamer. Manipulating the coil/helix configuration of the motif allowed us to control pentamer and hexamer formation in a predictable manner, thus proving its function as a molecular switch. Importantly, the switch also remodels the common binding site for host factors that are critical for viral replication and the new ultra-potent HIV-1 inhibitor lenacapavir. This study reveals that a critical assembly element also modulates the post-assembly and viral replication functions of the HIV-1 capsid and provides new insights on capsid function and inhibition.

Neutron Reflectometry and Molecular Simulations Demonstrate HIV-1 Nef Homodimer Formation on Model Lipid Bilayers.

The HIV-1 Nef protein plays a critical role in viral infectivity, high-titer replication in vivo, and immune escape of HIV-infected cells. Nef lacks intrinsic biochemical activity, functioning instead through interactions with diverse host cell signaling proteins and intracellular trafficking pathways. Previous studies have established an essential role for Nef homodimer formation at the plasma membrane for most if not all its functions. Here we combined neutron reflectometry of full-length myristoylated Nef bound to model lipid bilayers with molecular simulations based on previous X-ray crystal structures of Nef homodimers. This integrated approach provides direct evidence that Nef associates with the membrane as a homodimer with its structured core region displaced from the membrane for partner protein engagement. Parallel studies of a dimerization-defective mutant, Nef-L112D, demonstrate that the helical dimerization interface present in previous crystal structures stabilizes the membrane-bound dimer. X-ray crystallography of the Nef-L112D mutant in complex with the SH3 domain of the Nef-associated host cell kinase Hck revealed a monomeric 1:1 complex instead of the 2:2 dimer complex formed with wild-type Nef. Importantly, the crystal structure of the Nef-L112D core and SH3 interface are virtually identical to the wild-type complex, indicating that this mutation does not affect the overall Nef fold. These findings support the intrinsic capacity of Nef to homodimerize at lipid bilayers using structural features present in X-ray crystal structures of dimeric complexes.

Insights into the oligomeric structure of the HIV-1 Vpu protein.

The HIV-1-encoded protein Vpu forms an oligomeric ion channel/pore in membranes and interacts with host proteins to support the virus lifecycle. However, Vpu molecular mechanisms are currently not well understood. Here, we report on the Vpu oligomeric organization under membrane and aqueous conditions and provide insights into how the Vpu environment affects the oligomer formation. For these studies, we designed a maltose-binding protein (MBP)-Vpu chimera protein and produced it in E. coli in soluble form. We analyzed this protein using analytical size-exclusion chromatography (SEC), negative staining electron microscopy (nsEM), and electron paramagnetic resonance (EPR) spectroscopy. Surprisingly, we found that MBP-Vpu formed stable oligomers in solution, seemingly driven by Vpu transmembrane domain self-association. A coarse modeling of nsEM data as well as SEC and EPR data suggests that these oligomers most likely are pentamers, similar to what was reported regarding membrane-bound Vpu. We also noticed reduced MBP-Vpu oligomer stability upon reconstitution of the protein in ß-DDM detergent and mixtures of lyso-PC/PG or DHPC/DHPG. In these cases, we observed greater oligomer heterogeneity, with MBP-Vpu oligomeric order generally lower than in solution; however, larger oligomers were also present. Notably, we found that in lyso-PC/PG, above a certain protein concentration, MBP-Vpu assembles into extended structures, which had not been reported for Vpu. Therefore, we captured various Vpu oligomeric forms, which can shed light on Vpu quaternary organization. Our findings could be useful in understanding Vpu organization and function in cellular membranes and could provide information regarding the biophysical properties of single-pass transmembrane proteins.

Peptide Triazole Inhibitors of HIV-1: Hijackers of Env Metastability.

With 1.5 million new infections and 690,000 AIDS-related deaths globally each year, HIV- 1 remains a pathogen of significant public health concern. Although a wide array of effective antiretroviral drugs have been discovered, these largely target intracellular stages of the viral infectious cycle, and inhibitors that act at or before the point of viral entry still require further advancement. A unique class of HIV-1 entry inhibitors, called peptide triazoles (PTs), has been developed, which irreversibly inactivates Env trimers by exploiting the protein structure's innate metastable nature. PTs, and a related group of inhibitors called peptide triazole thiols (PTTs), are peptide compounds that dually engage the CD4 receptor and coreceptor binding sites of Env's gp120 subunit. This triggers dramatic conformational rearrangements of Env, including the shedding of gp120 (PTs and PTTs) and lytic transformation of the gp41 subunit to a post-fusion-like arrangement (PTTs). Due to the nature of their dual receptor site engagement, PT/PTT-induced conformational changes may elucidate mechanisms behind the native fusion program of Env trimers following receptor and coreceptor engagement, including the role of thiols in fusion. In addition to inactivating Env, PTT-induced structural transformation enhances the exposure of important and conserved neutralizable regions of gp41, such as the membrane proximal external region (MPER). PTT-transformed Env could present an intriguing potential vaccine immunogen prototype. In this review, we discuss the origins of the PT class of peptide inhibitors, our current understanding of PT/PTT-induced structural perturbations and viral inhibition, and prospects for using these antagonists for investigating Env structural mechanisms and for vaccine development.

Molecular recognition of a membrane-anchored HIV-1 pan-neutralizing epitope.

Antibodies against the carboxy-terminal section of the membrane-proximal external region (C-MPER) of the HIV-1 envelope glycoprotein (Env) are considered as nearly pan-neutralizing. Development of vaccines capable of producing analogous broadly neutralizing antibodies requires deep understanding of the mechanism that underlies C-MPER recognition in membranes. Here, we use the archetypic 10E8 antibody and a variety of biophysical techniques including single-molecule approaches to study the molecular recognition of C-MPER in membrane mimetics. In contrast to the assumption that an interfacial MPER helix embodies the entire C-MPER epitope recognized by 10E8, our data indicate that transmembrane domain (TMD) residues contribute to binding affinity and specificity. Moreover, anchoring to membrane the helical C-MPER epitope through the TMD augments antibody binding affinity and relieves the effects exerted by the interfacial MPER helix on the mechanical stability of the lipid bilayer. These observations support that addition of TMD residues may result in more efficient and stable anti-MPER vaccines.

Genome sequence analysis suggests coevolution of the DIS, SD, and Psi hairpins in HIV-1 genomes.

HIV-1 RNA dimerization is a critical step in viral life cycle. It is a prerequisite for genome packaging and plays an important role in reverse transcription and recombination. Dimerization is promoted by the DIS (dimerization initiation site) hairpin located in the 5' leader of HIV-1 genome. Despite the high genetic diversity in HIV-1 group M, only five apical loops (AAGCGCGCA, AAGUGCGCA, AAGUGCACA, AGGUGCACA and AGUGCAC) are commonly found in DIS hairpins. We refer to the parent DISes with these apical loops as DISLai, DISTrans, DISF, DISMal, and DISC, respectively. Based on identity or similarity of DIS hairpins to parent DISes, we distributed HIV-1 M genomes into five dimerization groups. Comparison of the primary and secondary structures of DIS, SD and Psi hairpins in about 3000 HIV-1 M genomes showed that the mutation frequencies at particular nucleotide positions of these hairpins differ among the dimerization groups, and DISF may be an origin of other parent DISes. We found that DIS, SD and Psi hairpins have hundreds of variants, only some of them occurring rather frequently. The lower part of DIS hairpin with G x AGG internal loop is highly conserved in both HIV-1 and SIV genomes. We supposed that the G-quadruplex, located 56 nts downstream of the Gag start codon, may participate in switching of HIV-1 leader RNA from BMH (branched multiple hairpins) to LDI (long distance interaction) conformation.

Structural and Mechanistic Evidence for Calcium Interacting Sites in the HIV Transmembrane Protein gp41 Involved in Membrane Fusion.

The HIV envelope protein gp160 comprises two subunits, gp120 and gp41, responsible for receptor binding and membrane fusion during viral entry, respectively. In the course of the membrane fusion process, gp41 undergoes a conformational change, leading to the formation of a six-helix bundle (SHB), which ultimately drives membrane fusion. The gp41 C-terminal and N-terminal heptad repeats (CHR and NHR) interact with one another to form the SHB, and this step can be targeted by peptide inhibitors, which are used in the clinic to mitigate HIV infection. Here, we discover the calcium interaction motifs (CIMs) in the gp41 CHR and NHR regions via NMR spectroscopy. We find that the assembly of the CHR-NHR SHB is facilitated in Ca2+-containing media and impaired in CIM mutants. Of note, the clinically approved, gp41-derived fusion inhibitor T20, which does not contain the CIM motif, exhibits reduced inhibitory efficiency when challenged with calcium. This finding could have important implications for the development of better fusion inhibitors for HIV.