The HIV capsid mimics karyopherin engagement of FG-nucleoporins.

HIV can infect non-dividing cells because the viral capsid can overcome the selective barrier of the nuclear pore complex and deliver the genome directly into the nucleus1,2. Remarkably, the intact HIV capsid is more than 1,000 times larger than the size limit prescribed by the diffusion barrier of the nuclear pore3. This barrier in the central channel of the nuclear pore is composed of intrinsically disordered nucleoporin domains enriched in phenylalanine-glycine (FG) dipeptides. Through multivalent FG interactions, cellular karyopherins and their bound cargoes solubilize in this phase to drive nucleocytoplasmic transport4. By performing an in vitro dissection of the nuclear pore complex, we show that a pocket on the surface of the HIV capsid similarly interacts with FG motifs from multiple nucleoporins and that this interaction licences capsids to penetrate FG-nucleoporin condensates. This karyopherin mimicry model addresses a key conceptual challenge for the role of the HIV capsid in nuclear entry and offers an explanation as to how an exogenous entity much larger than any known cellular cargo may be able to non-destructively breach the nuclear envelope.

Temporal control by cofactors prevents kinetic trapping in retroviral Gag lattice assembly.

For retroviruses like HIV to proliferate, they must form virions shaped by the self-assembly of Gag polyproteins into a rigid lattice. This immature Gag lattice has been structurally characterized and reconstituted in vitro, revealing the sensitivity of lattice assembly to multiple cofactors. Due to this sensitivity, the energetic criterion for forming stable lattices is unknown, as are their corresponding rates. Here, we use a reaction-diffusion model designed from the cryo-ET structure of the immature Gag lattice to map a phase diagram of assembly outcomes controlled by experimentally constrained rates and free energies, over experimentally relevant timescales. We find that productive assembly of complete lattices in bulk solution is extraordinarily difficult due to the large size of this ∼3700 monomer complex. Multiple Gag lattices nucleate before growth can complete, resulting in loss of free monomers and frequent kinetic trapping. We therefore derive a time-dependent protocol to titrate or "activate" the Gag monomers slowly within the solution volume, mimicking the biological roles of cofactors. This general strategy works remarkably well, yielding productive growth of self-assembled lattices for multiple interaction strengths and binding rates. By comparing to the in vitro assembly kinetics, we can estimate bounds on rates of Gag binding to Gag and the cellular cofactor IP6. Our results show that Gag binding to IP6 can provide the additional time delay necessary to support smooth growth of the immature lattice with relatively fast assembly kinetics, mostly avoiding kinetic traps. Our work provides a foundation for predicting and disrupting formation of the immature Gag lattice via targeting specific protein-protein binding interactions.

Broadly Neutralizing Antibodies to Highly Antigenically Variable Viruses as Templates for Vaccine Design.

Development of vaccines to highly variable viruses such as Human Immunodeficiency Virus and influenza A viruses faces multiple challenges. In this article, these challenges are described and reverse vaccinology approaches to generate universal vaccines against both pathogens are laid out and compared.

Increasing the Assembly Efficacy of Peptidic ß-Sheets for a Highly-Sensitive HIV Detection.

Our recent publication illustrates the critical role of phenylalanine-mediated aromatic-aromatic interactions in determining the assembly of peptidic ß-sheets. However, the effect of phenylalanine number on regulating the assembly efficacy of peptidic ß-sheets remains poorly understood. We herein evaluate the assembly efficacy of ß-sheets of a series of oligopeptides which contain 0, 1, 2, or 3 phenylalanine in their molecular backbones. In our assembly system, two phenylalanine (2F) is the minimum number for driving the assembly of ß-sheets of oligopeptides. Oligopeptides with three phenylalanine (3F) show significantly increased assembly efficacy of ß-sheets compared to that with 2F. These results suggest a positive correlation between the phenylalanine number and assembly efficacy of ß-sheets. By improving the assembly efficacy of ß-sheets, we further develop a highly sensitive HIV analytical system in which the specific binding of ß-sheets with Congo Red induces enhanced fluorescence. For HIV p24 detection, the 3F-based analytical system (0.61 pg/mL) shows a significantly lower limit of detection (LOD) than the 2F-based analytical system (2.44 pg/mL), both of which are more sensitive than commercial ELISA (5 pg/mL) used in the clinic. This work not only illustrates the effect of phenylalanine number on regulating the assembly efficacy of ß-sheets but also provides a guideline for the construction of a highly sensitive analytical system of disease diagnosis.

Simple, General Criterion for Onset of Disclination Disorder on Curved Surfaces.

Determining the positions of lattice defects on bounded elastic surfaces with Gaussian curvature is a nontrivial task of mechanical energy optimization. We introduce a simple way to predict the onset of disclination disorder from the shape of the surface. The criterion fixes the value of a weighted integral Gaussian curvature to a universal constant and proves accurate across a great variety of shapes. It provides improved understanding of the limitations to crystalline order in many natural and engineering contexts, such as the assembly of viral capsids.

Structural basis of lentiviral subversion of a cellular protein degradation pathway.

Lentiviruses contain accessory genes that have evolved to counteract the effects of host cellular defence proteins that inhibit productive infection. One such restriction factor, SAMHD1, inhibits human immunodeficiency virus (HIV)-1 infection of myeloid-lineage cells as well as resting CD4(+) T cells by reducing the cellular deoxynucleoside 5'-triphosphate (dNTP) concentration to a level at which the viral reverse transcriptase cannot function. In other lentiviruses, including HIV-2 and related simian immunodeficiency viruses (SIVs), SAMHD1 restriction is overcome by the action of viral accessory protein x (Vpx) or the related viral protein r (Vpr) that target and recruit SAMHD1 for proteasomal degradation. The molecular mechanism by which these viral proteins are able to usurp the host cell's ubiquitination machinery to destroy the cell's protection against these viruses has not been defined. Here we present the crystal structure of a ternary complex of Vpx with the human E3 ligase substrate adaptor DCAF1 and the carboxy-terminal region of human SAMHD1. Vpx is made up of a three-helical bundle stabilized by a zinc finger motif, and wraps tightly around the disc-shaped DCAF1 molecule to present a new molecular surface. This adapted surface is then able to recruit SAMHD1 via its C terminus, making it a competent substrate for the E3 ligase to mark for proteasomal degradation. The structure reported here provides a molecular description of how a lentiviral accessory protein is able to subvert the cell's normal protein degradation pathway to inactivate the cellular viral defence system.

Enhanced neonatal Fc receptor function improves protection against primate SHIV infection.

To protect against human immunodeficiency virus (HIV-1) infection, broadly neutralizing antibodies (bnAbs) must be active at the portals of viral entry in the gastrointestinal or cervicovaginal tracts. The localization and persistence of antibodies at these sites is influenced by the neonatal Fc receptor (FcRn), whose role in protecting against infection in vivo has not been defined. Here, we show that a bnAb with enhanced FcRn binding has increased gut mucosal tissue localization, which improves protection against lentiviral infection in non-human primates. A bnAb directed to the CD4-binding site of the HIV-1 envelope (Env) protein (denoted VRC01) was modified by site-directed mutagenesis to increase its binding affinity for FcRn. This enhanced FcRn-binding mutant bnAb, denoted VRC01-LS, displayed increased transcytosis across human FcRn-expressing cellular monolayers in vitro while retaining FcγRIIIa binding and function, including antibody-dependent cell-mediated cytotoxicity (ADCC) activity, at levels similar to VRC01 (the wild type). VRC01-LS had a threefold longer serum half-life than VRC01 in non-human primates and persisted in the rectal mucosa even when it was no longer detectable in the serum. Notably, VRC01-LS mediated protection superior to that afforded by VRC01 against intrarectal infection with simian-human immunodeficiency virus (SHIV). These findings suggest that modification of FcRn binding provides a mechanism not only to increase serum half-life but also to enhance mucosal localization that confers immune protection. Mutations that enhance FcRn function could therefore increase the potency and durability of passive immunization strategies to prevent HIV-1 infection.

Trade-offs between enzyme fitness and solubility illuminated by deep mutational scanning.

Proteins are marginally stable, and an understanding of the sequence determinants for improved protein solubility is highly desired. For enzymes, it is well known that many mutations that increase protein solubility decrease catalytic activity. These competing effects frustrate efforts to design and engineer stable, active enzymes without laborious high-throughput activity screens. To address the trade-off between enzyme solubility and activity, we performed deep mutational scanning using two different screens/selections that purport to gauge protein solubility for two full-length enzymes. We assayed a TEM-1 beta-lactamase variant and levoglucosan kinase (LGK) using yeast surface display (YSD) screening and a twin-arginine translocation pathway selection. We then compared these scans with published experimental fitness landscapes. Results from the YSD screen could explain 37% of the variance in the fitness landscapes for one enzyme. Five percent to 10% of all single missense mutations improve solubility, matching theoretical predictions of global protein stability. For a given solubility-enhancing mutation, the probability that it would retain wild-type fitness was correlated with evolutionary conservation and distance to active site, and anticorrelated with contact number. Hybrid classification models were developed that could predict solubility-enhancing mutations that maintain wild-type fitness with an accuracy of 90%. The downside of using such classification models is the removal of rare mutations that improve both fitness and solubility. To reveal the biophysical basis of enhanced protein solubility and function, we determined the crystallographic structure of one such LGK mutant. Beyond fundamental insights into trade-offs between stability and activity, these results have potential biotechnological applications.

Targeting RNA with Small Molecules To Capture Opportunities at the Intersection of Chemistry, Biology, and Medicine.

The biology of healthy and disease-affected cells is often mediated by RNA structures, desirable targets for small molecule chemical probes and lead medicines. Although structured regions are found throughout the transcriptome, some even with demonstrated functionality, human RNAs are considered recalcitrant to small molecule targeting. However, targeting structured regions with small molecules provides an important alternative to oligonucleotides that target sequence. In this Perspective, we describe challenges and progress in developing small molecules interacting with RNA (SMIRNAs) to capture their significant opportunities at the intersection of chemistry, biology, and medicine. Key to establishing a new paradigm in chemical biology and medicine is the development of methods to obtain, preferably by design, bioactive compounds that modulate RNA targets and companion methods that validate their direct effects in cells and pre-clinical models. While difficult, demonstration of direct target engagement in the complex cellular milieu, along with methods to establish modes of action, is required to push this field forward. We also describe frameworks for accelerated advancements in this burgeoning area, their implications, key new technologies for development of SMIRNAs, and milestones that have led to broader acceptance of RNA as a small molecule druggable target.

Exploiting glycan topography for computational design of Env glycoprotein antigenicity.

Mounting evidence suggests that glycans, rather than merely serving as a "shield", contribute critically to antigenicity of the HIV envelope (Env) glycoprotein, representing critical antigenic determinants for many broadly neutralizing antibodies (bNAbs). While many studies have focused on defining the role of individual glycans or groups of proximal glycans in bNAb binding, little is known about the effects of changes in the overall glycan landscape in modulating antibody access and Env antigenicity. Here we developed a systems glycobiology approach to reverse engineer the complexity of HIV glycan heterogeneity to guide antigenicity-based de novo glycoprotein design. bNAb binding was assessed against a panel of 94 recombinant gp120 monomers exhibiting defined glycan site occupancies. Using a Bayesian machine learning algorithm, bNAb-specific glycan footprints were identified and used to design antigens that selectively alter bNAb antigenicity as a proof-of concept. Our approach provides a new design strategy to predictively modulate antigenicity via the alteration of glycan topography, thereby focusing the humoral immune response on sites of viral vulnerability for HIV.

A rapid label- and enzyme-free G-quadruplex-based fluorescence strategy for highly-sensitive detection of HIV DNA.

Because rapid, convenient, and selective methods for HIV detection are urgently needed, herein, a simple label-free and enzyme-free strategy is constructed for sensitive fluorescence detection of HIV DNA using the fluorescent intercalating dye thioflavin T (THT) as the detection signal source. This strategy utilizes a hairpin DNA sequence (H1) and two assistant strands. H1 is wisely designed with a G-quadruplex sequence in the stem. Target DNA, when present in solution, will hybridize with H1 to form H1/target duplexes and release the G-quadruplexes. Additionally, the assistant probes hybridize with the unfolded H1 to form a stable DNA double strand, resulting in the displacement of the target to participate in another similar reaction cycle. Consequently, many G-quadruplex structures are generated, leading to a significantly amplified fluorescence signal of THT. The linear range is from 0.1 nM to 50.0 nM with a limit of detection of 13 pM. Results can be achieved within 40 min, because the cyclic amplification involves only one DNA hairpin and two auxiliary chains. Furthermore, this platform exhibited good selectivity with one base mismatch or other DNA sequences. This strategy could be used as a simple, sensitive, and selective tool to detect other DNA biomarkers.

High-Precision Single-Molecule Characterization of the Folding of an HIV RNA Hairpin by Atomic Force Microscopy.

The folding of RNA into a wide range of structures is essential for its diverse biological functions from enzymatic catalysis to ligand binding and gene regulation. The unfolding and refolding of individual RNA molecules can be probed by single-molecule force spectroscopy (SMFS), enabling detailed characterization of the conformational dynamics of the molecule as well as the free-energy landscape underlying folding. Historically, high-precision SMFS studies of RNA have been limited to custom-built optical traps. Although commercial atomic force microscopes (AFMs) are widely deployed and offer significant advantages in ease-of-use over custom-built optical traps, traditional AFM-based SMFS lacks the sensitivity and stability to characterize individual RNA molecules precisely. Here, we developed a high-precision SMFS assay to study RNA folding using a commercial AFM and applied it to characterize a small RNA hairpin from HIV that plays a key role in stimulating programmed ribosomal frameshifting. We achieved rapid data acquisition in a dynamic assay, unfolding and then refolding the same individual hairpin more than 1,100 times in 15 min. In comparison to measurements using optical traps, our AFM-based assay featured a stiffer force probe and a less compliant construct, providing a complementary measurement regime that dramatically accelerated equilibrium folding dynamics. Not only did kinetic analysis of equilibrium trajectories of the HIV RNA hairpin yield the traditional parameters used to characterize folding by SMFS (zero-force rate constants and distances to the transition state), but we also reconstructed the full 1D projection of the folding free-energy landscape comparable to state-of-the-art studies using dual-beam optical traps, a first for this RNA hairpin and AFM studies of nucleic acids in general. Looking forward, we anticipate that the ease-of-use of our high-precision assay implemented on a commercial AFM will accelerate studying folding of diverse nucleic acid structures.

Oligomeric viral proteins: small in size, large in presence.

Viruses are obligate parasites that rely heavily on host cellular processes for replication. The small number of proteins typically encoded by a virus is faced with selection pressures that lead to the evolution of distinctive structural properties, allowing each protein to maintain its function under constraints such as small genome size, high mutation rate, and rapidly changing fitness conditions. One common strategy for this evolution is to utilize small building blocks to generate protein oligomers that assemble in multiple ways, thereby diversifying protein function and regulation. In this review, we discuss specific cases that illustrate how oligomerization is used to generate a single defined functional state, to modulate activity via different oligomeric states, or to generate multiple functional forms via different oligomeric states.

Ultrasensitive Detection of HIV DNA with Polymerase Chain Reaction-Dynamic Light Scattering.

Early diagnosis of HIV biomarkers or genes is the key to reducing acquired immunodeficiency syndrome (AIDS) mortality. In our work, we developed a novel polymerase chain reaction-dynamic light scattering (PCR-DLS) assay for one-step sensitive detection of HIV DNA based on the average-diameter change of gold nanoparticles (AuNPs). This is the first PCR assay that makes use of the DLS technique as a signal read-out, with the particle size measured by DLS increasing with the concentration of target DNA. With the help of the AuNP probes, this PCR-DLS assay can effectively improve the specificity of PCR reactions, which can greatly increase the detection sensitivity, with a detection limit of 1.8 aM (S/N = 3). In addition, the proposed strategy was successfully used to analyze target DNA in human serum samples, indicating that the PCR-DLS assay has a promising potential application for rapid and early clinical diagnosis of HIV infection.

Gold-Catalyzed C-S Aryl-Group Transfer in Zinc Finger Proteins.

Reaction of the Au-C N chelate [Au(bnpy)Cl2 ] with the full-length zinc finger (ZnF; ZnCys3 His) of HIV nucleocapsid protein NCp7 results in C-S aryl transfer from the AuIII organometallic species to a cysteine of the ZnF. The reaction is general and occurs even for finger 3 of the transcription factor Sp1, containing a ZnCys2 His2 coordination sphere. This reaction is the first demonstration of group transfer from a coordination compound to biologically important zinc fingers, and is especially noteworthy for the ZnCys2 His2 transcription factors. The work expands the corpus of organometallic species which can efficiently modify biomolecules through C-atom transfer. The electronic features of the gold compound leading to this unexpected reaction were explored by X-ray absorption spectroscopy.

Rationally Designed Peptidyl Virus-Like Particles Enable Targeted Delivery of Genetic Cargo.

We report a strategy to construct peptidyl virus-like particles (pVLPs) by mimicking the human immunodeficiency virus and simian virus 40. We designed two viral peptides with cell/nucleus-targeting capabilities that can co-assemble in their active conformations into well-defined nanoparticles. The self-assembled nanoparticles can encapsulate the DNA of clustered regularly interspaced short palindromic repeat associated proteins 9 (CRISPR/Cas9) to form biodegradable pVLPs with excellent cell-targeting ability and biocompatibility. The pVLPs can penetrate the cellular membrane and deliver genetic cargos into the nucleus through the viral entry route. The results provide a promising pathway for engineering artificial viruses with desired functions.

Deep sequencing of HIV: clinical and research applications.

Human immunodeficiency virus (HIV) exhibits remarkable diversity in its genomic makeup and exists in any given individual as a complex distribution of closely related but nonidentical genomes called a viral quasispecies, which is subject to genetic variation, competition, and selection. This viral diversity clinically manifests as a selection of mutant variants based on viral fitness in treatment-naive individuals and based on drug-selective pressure in those on antiretroviral therapy (ART). The current standard-of-care ART consists of a combination of antiretroviral agents, which ensures maximal viral suppression while preventing the emergence of drug-resistant HIV variants. Unfortunately, transmission of drug-resistant HIV does occur, affecting 5% to >20% of newly infected individuals. To optimize therapy, clinicians rely on viral genotypic information obtained from conventional population sequencing-based assays, which cannot reliably detect viral variants that constitute <20% of the circulating viral quasispecies. These low-frequency variants can be detected by highly sensitive genotyping methods collectively grouped under the moniker of deep sequencing. Low-frequency variants have been correlated to treatment failures and HIV transmission, and detection of these variants is helping to inform strategies for vaccine development. Here, we discuss the molecular virology of HIV, viral heterogeneity, drug-resistance mutations, and the application of deep sequencing technologies in research and the clinical care of HIV-infected individuals.

The IDP-Specific Force Field ff14IDPSFF Improves the Conformer Sampling of Intrinsically Disordered Proteins.

Intrinsically disordered proteins (IDPs) or intrinsically disordered regions do not have a fixed tertiary structure but play key roles in signal regulation, molecule recognition, and drug targeting. However, it is difficult to study the structure and function of IDPs by traditional experimental methods because of their diverse conformations. Limitations of current generic protein force fields and solvent models were reported in the previous simulations of IDPs. We have also explored overcoming these limitations by developing the ff99IDPs and ff14IDPs force fields to correct the dihedral distribution for eight disorder-promoting residues often observed in IDPs and found encouraging improvements. Here we extend our correction of backbone dihedral terms to all 20 naturally occurring amino acids in the IDP-specific force field ff14IDPSFF to further improve the quality of the modeling of IDPs. Extensive tests of seven IDPs and 14 unstructured short peptides show that the simulated Cα chemical shifts obtained with the ff14IDPSFF force field are in quantitative agreement with those from NMR experiments and are more accurate than those obtained with the base generic force field and also our previous ff14IDPs that only corrects the eight disorder-promoting amino acids. The influence of the solvent model was also investigated and found to be less important. Finally, our explicit-solvent MD simulations further show that ff14IDPSFF can still be used to model structural and dynamical properties of two tested folded proteins, with a slightly better agreement in the loop regions for both structural and dynamical properties. These findings confirm that the newly developed IDP-specific force field ff14IDPSFF can improve the conformer sampling of intrinsically disordered proteins.

Human Immunodeficiency Virus Proteins Mimic Human T Cell Receptors Inducing Cross-Reactive Antibodies.

Human immunodeficiency virus (HIV) hides from the immune system in part by mimicking host antigens, including human leukocyte antigens. It is demonstrated here that HIV also mimics the V-ß-D-J-ß of approximately seventy percent of about 600 randomly selected human T cell receptors (TCR). This degree of mimicry is greater than any other human pathogen, commensal or symbiotic organism studied. These data suggest that HIV may be evolving into a commensal organism just as simian immunodeficiency virus has done in some types of monkeys. The gp120 envelope protein, Nef protein and Pol protein are particularly similar to host TCR, camouflaging HIV from the immune system and creating serious barriers to the development of safe HIV vaccines. One consequence of HIV mimicry of host TCR is that antibodies against HIV proteins have a significant probability of recognizing the corresponding TCR as antigenic targets, explaining the widespread observation of lymphocytotoxic autoantibodies in acquired immunodeficiency syndrome (AIDS). Quantitative enzyme-linked immunoadsorption assays (ELISA) demonstrated that every HIV antibody tested recognized at least one of twelve TCR, and as many as seven, with a binding constant in the 10-8 to 10-9 m range. HIV immunity also affects microbiome tolerance in ways that correlate with susceptibility to specific opportunistic infections.