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.

Strategies for eliciting multiple lineages of broadly neutralizing antibodies to HIV by vaccination.

A prophylactic vaccine would be a powerful tool in the fight against HIV. Passive immunization of animals with broadly neutralizing antibodies (bnAbs) affords protection against viral challenge, and recent data from the Antibody Mediated Prevention clinical trials support the concept of bnAbs providing protection against HIV in humans, albeit only at broad and potent neutralizing antibody titers. Moreover, it is now clear that a successful vaccine will also need to induce bnAbs against multiple neutralizing epitopes on the HIV envelope (Env) glycoprotein. Here, we review recent clinical trials evaluating bnAb-based vaccines, and discuss key issues in the development of an HIV vaccine capable of targeting multiple Env neutralizing epitopes.

Photoswitches for controllable RNA binding: a future approach in the RNA-targeting therapy.

RNA is an emerging drug target that opens new perspectives in the treatment of viral and bacterial infections, cancer and a range of so far incurable genetic diseases. Among the various strategies towards the design and development of selective and efficient ligands for targeting and detection of therapeutically relevant RNA, photoswitchable RNA binders represent a very promising approach due to the possibility to control the ligand-RNA and protein-RNA interactions by light with high spatiotemporal resolution. However, the field of photoswitchable RNA binders still remains underexplored due to challenging design of lead structures that should combine high RNA binding selectivity with efficient photochemical performance. The aim of this highlight article is to describe the development of photoswitchable noncovalent RNA binders and to outline the current situation and perspectives of this emerging interdisciplinary field.

Impact of the Endosomal Escape Activity of Cell-Penetrating Peptides on the Endocytic Pathway.

Cell-penetrating peptides (CPPs) are routinely used for the delivery of macromolecules into live human cells. To enter the cytosolic space of cells, CPPs typically permeabilize the membrane of endosomes. In turn, several approaches have been developed to increase the endosomal membrane permeation activity of CPPs so as to improve delivery efficiencies. The endocytic pathway is, however, important in maintaining cellular homeostasis, and understanding how endosomal permeation impacts cells is now critical to define the general utility of CPPs. Herein, we investigate how CPP-based delivery protocols affect the endocytic network. We detect that, in some cases, cell penetration induces the activation of Chmp1b, Galectin-3, and TFEB, which are components of endosomal repair, organelle clearance, and biogenesis pathways, respectively. We also detect that cellular delivery modulates endocytosis and endocytic proteolysis. Remarkably, a multimeric analogue of the prototypical CPP TAT permeabilizes endosomes efficiently without inducing membrane damage responses. These results challenge the notion that reagents that make endosomes leaky are generally toxic. Instead, our data indicates that it is possible to enter cells with minimal deleterious effects.

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.

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.

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.

Structural basis for strand-transfer inhibitor binding to HIV intasomes.

The HIV intasome is a large nucleoprotein assembly that mediates the integration of a DNA copy of the viral genome into host chromatin. Intasomes are targeted by the latest generation of antiretroviral drugs, integrase strand-transfer inhibitors (INSTIs). Challenges associated with lentiviral intasome biochemistry have hindered high-resolution structural studies of how INSTIs bind to their native drug target. Here, we present high-resolution cryo-electron microscopy structures of HIV intasomes bound to the latest generation of INSTIs. These structures highlight how small changes in the integrase active site can have notable implications for drug binding and design and provide mechanistic insights into why a leading INSTI retains efficacy against a broad spectrum of drug-resistant variants. The data have implications for expanding effective treatments available for HIV-infected individuals.

HIV biosensors for early diagnosis of infection: The intertwine of nanotechnology with sensing strategies.

Human immunodeficiency virus (HIV) is a lentivirus that leads to acquired immunodeficiency syndrome (AIDS). With increasing awareness of AIDS emerging as a global public health threat, different HIV testing kits have been developed to detect antibodies (Ab) directed toward different parts of HIV. A great limitation of these tests is that they can not detect HIV antibodies during early virus infection. Therefore, to overcome this challenge, a wide range of biosensors have been developed for early diagnosis of HIV infection. A significant amount of these studies have been focused on the application of nanomaterials for improving the sensitivity and accuracy of the sensing methods. Following an introduction into this field, a first section of this review covers the synthesis and applicability of such nanomaterials as metal nanoparticles (NPs), quantum dots (QDs), carbon-based nanomaterials and metal nanoclusters (NCs). A second larger section covers the latest developments concerning nanomaterial-based biosensors for HIV diagnosis, with paying a special attention to the determination of CD4+ cells as a hall mark of HIV infection, HIV gene, HIV p24 core protein, HIV p17 peptide, HIV-1 virus-like particles (VLPs) and HIV related enzymes, particularly those that are passed on from the virus to the CD4+ T lymphocytes and are necessary for viral reproduction within the host cell. These studies are described in detail along with their diverse principles/mechanisms (e.g. electrochemistry, fluorescence, electromagnetic-piezoelectric, surface plasmon resonance (SPR), surface enhanced Raman spectroscopy (SERS) and colorimetry). Despite the significant progress in HIV biosensing in the last years, there is a great need for the development of point-of-care (POC) technologies which are affordable, robust, easy to use, portable, and possessing sufficient quantitative accuracy to enable clinical decision making. In the final section, the focus is on the portable sensing devices as a new standard of POC and personalized diagnostics.

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.

Development of a Cyclic Peptide Inhibitor of the p6/UEV Protein-Protein Interaction.

The budding of HIV from infected cells is driven by the protein-protein interaction between the p6 domain of the HIV Gag protein and the UEV domain of the human TSG101 protein. We report the development of a cyclic peptide inhibitor of the p6/UEV interaction, from a non cell-permeable parent that was identified in a SICLOPPS screen. Amino acids critical for the activity of the parent cyclic peptide were uncovered using alanine-scanning, and a series of non-natural analogues synthesized and assessed. The most potent molecule disrupts the p6/UEV interaction with an IC50 of 6.17 ± 0.24 µM by binding to UEV with a Kd of 11.9 ± 2.8 µM. This compound is cell permeable and active in a cellular virus-like particle budding assay with an IC50 of ∼2 µM. This work further demonstrates the relative simplicity with which the potency and activity of cyclic peptides identified from SICLOPPS libraries can be optimized.

Smartphone assisted immunodetection of HIV p24 antigen using reusable, centrifugal microchannel array chip.

An integrated immunodetection platform employing a simple, reusable, centrifugal microchannel array chip and a smartphone as detection unit was developed. The applicability of the platform to the detection of HIV p24 antigen was demonstrated. The microchip was made of polycarbonate and contained 4 × 8 zigzag microchannels. After the monoclonal antibody of HIV p24 was adsorbed onto the channel surfaces, HIV p24 was introduced into the microchannel to react with the antibody. A biotin linked polyclonal antibody was then brought in to react with HIV p24, and SP80 (containing streptavidin and horseradish peroxidase) was introduced to react with the biotin. Finally, a solution containing 3,3',5,5'-tetramethylbenzidine and other reagents was passed through the above channels, horseradish peroxidase catalyzed the oxidation of tetramethylbenzidine (to 3,3',5,5'- tetramethylbenzidine diamine) forming a dark color. The color intensity, indicating HIV p24 antigen quantity, was then photographed via a smartphone, and the color of each microchannel was processed via a computer to determine the HIV p24 antigen concentration. Under the optimized conditions, limits of detection (LODs) of 0.17 ng/ml and 0.11 ng/ml were obtained for p24 antigen in a buffer solution and human serum, respectively. Channel washing/rinsing was implemented via a centrifugal force. An economic portable centrifugal device that could accommodate up to 4 microchips was assembled, and multi-step solution loading and rinsing involved in this sandwich immunoassay were performed conveniently. The microchip could be reused after a simple regeneration process. The low-cost polycarbonate microchip and centrifugal device together with the simple but efficient operation make the method a promising tool for HIV screening in resource limited areas.

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.

Automatically Fixing Errors in Glycoprotein Structures with Rosetta.

Recent advances in single-particle cryo-electron microscopy (cryoEM) have resulted in determination of an increasing number of protein structures with resolved glycans. However, existing protocols for the refinement of glycoproteins at low resolution have failed to keep up with these advances. As a result, numerous deposited structures contain glycan stereochemical errors. Here, we describe a Rosetta-based approach for both cryoEM and X-ray crystallography refinement of glycoproteins that is capable of correcting conformational and configurational errors in carbohydrates. Building upon a previous Rosetta framework, we introduced additional features and score terms enabling automatic detection, setup, and refinement of glycan-containing structures. We benchmarked this approach using 12 crystal structures and showed that glycan geometries can be automatically improved while maintaining good fit to the crystallographic data. Finally, we used this method to refine carbohydrates of the human coronavirus NL63 spike glycoprotein and of an HIV envelope glycoprotein, demonstrating its usefulness for cryoEM refinement.

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.

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.

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.

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.