Explore the vast range of titles available.

CRISPR-associated protein 9 (Cas9)-mediated genome editing provides a promising cure for HIV-1/AIDS; however, gene delivery efficiency in vivo remains an obstacle to overcome. Here, we demonstrate the feasibility and efficiency of excising the HIV-1 provirus in three different animal models using an all-in-one adeno-associated virus (AAV) vector to deliver multiplex single-guide RNAs (sgRNAs) plus Staphylococcus aureus Cas9 (saCas9). The quadruplex sgRNAs/saCas9 vector outperformed the duplex vector in excising the integrated HIV-1 genome in cultured neural stem/progenitor cells from HIV-1 Tg26 transgenic mice. Intravenously injected quadruplex sgRNAs/saCas9 AAV-DJ/8 excised HIV-1 proviral DNA and significantly reduced viral RNA expression in several organs/tissues of Tg26 mice. In EcoHIV acutely infected mice, intravenously injected quadruplex sgRNAs/saCas9 AAV-DJ/8 reduced systemic EcoHIV infection, as determined by live bioluminescence imaging. Additionally, this quadruplex vector induced efficient proviral excision, as determined by PCR genotyping in the liver, lungs, brain, and spleen. Finally, in humanized bone marrow/liver/thymus (BLT) mice with chronic HIV-1 infection, successful proviral excision was detected by PCR genotyping in the spleen, lungs, heart, colon, and brain after a single intravenous injection of quadruplex sgRNAs/saCas9 AAV-DJ/8. In conclusion, in vivo excision of HIV-1 proviral DNA by sgRNAs/saCas9 in solid tissues/organs can be achieved via AAV delivery, a significant step toward human clinical trials. [Display omitted] Yin et al. use multiplex CRISPR/Cas9 genome editing technology to excise the HIV-1 provirus in a precise manner in three different HIV-1 animal models via in vivo AAV gene delivery. The feasibility of HIV excision in infected cells in vivo paves the way toward human clinical trials to cure HIV-1 infection.

A short, 14-amino-acid segment called SP1, located in the Gag structural protein 1 , has a critical role during the formation of the HIV-1 virus particle. During virus assembly, the SP1 peptide and seven preceding residues fold into a six-helix bundle, which holds together the Gag hexamer and facilitates the formation of a curved immature hexagonal lattice underneath the viral membrane 2 , 3 . Upon completion of assembly and budding, proteolytic cleavage of Gag leads to virus maturation, in which the immature lattice is broken down; the liberated CA domain of Gag then re-assembles into the mature conical capsid that encloses the viral genome and associated enzymes. Folding and proteolysis of the six-helix bundle are crucial rate-limiting steps of both Gag assembly and disassembly, and the six-helix bundle is an established target of HIV-1 inhibitors 4 , 5 . Here, using a combination of structural and functional analyses, we show that inositol hexakisphosphate (InsP6, also known as IP 6 ) facilitates the formation of the six-helix bundle and assembly of the immature HIV-1 Gag lattice. IP 6 makes ionic contacts with two rings of lysine residues at the centre of the Gag hexamer. Proteolytic cleavage then unmasks an alternative binding site, where IP 6 interaction promotes the assembly of the mature capsid lattice. These studies identify IP 6 as a naturally occurring small molecule that promotes both assembly and maturation of HIV-1. Inositol hexakisphosphate, which is found in all mammalian cells, binds to two separate sites to promote the assembly and maturation of HIV-1 virus particles.

The HIV-1 envelope glycoprotein (Env) is sparsely incorporated onto assembling virus particles on the host cell plasma membrane in order for the virus to balance infectivity and evade the immune response. Env becomes trapped in a nascent particle on encounter with the polymeric viral protein Gag, which forms a dense protein lattice on the inner leaflet of the plasma membrane. While Env incorporation efficiency is readily measured biochemically from released particles, very little is known about the spatiotemporal dynamics of Env trapping events. Herein, we demonstrate, via high-resolution single-molecule tracking, that retention of Env trimers within single virus assembly sites requires the Env cytoplasmic tail (CT) and the L12 residue in the matrix (MA) domain of Gag but does not require curvature of the viral lattice. We further demonstrate that Env trimers are confined to subviral regions of a budding Gag lattice, supporting a model where direct interactions and/or steric corralling between the Env-CT and a lattice of MA trimers promote Env trapping and infectious HIV-1 assembly.

Retrovirus assembly is driven by the multidomain structural protein Gag. Interactions between the capsid domains (CA) of Gag result in Gag multimerization, leading to an immature virus particle that is formed by a protein lattice based on dimeric, trimeric, and hexameric protein contacts. Among retroviruses the inter- and intra-hexamer contacts differ, especially in the N-terminal sub-domain of CA (CANTD). For HIV-1 the cellular molecule inositol hexakisphosphate (IP6) interacts with and stabilizes the immature hexamer, and is required for production of infectious virus particles. We have used in vitro assembly, cryo-electron tomography and subtomogram averaging, atomistic molecular dynamics simulations and mutational analyses to study the HIV-related lentivirus equine infectious anemia virus (EIAV). In particular, we sought to understand the structural conservation of the immature lentivirus lattice and the role of IP6 in EIAV assembly. Similar to HIV-1, IP6 strongly promoted in vitro assembly of EIAV Gag proteins into virus-like particles (VLPs), which took three morphologically highly distinct forms: narrow tubes, wide tubes, and spheres. Structural characterization of these VLPs to sub-4Å resolution unexpectedly showed that all three morphologies are based on an immature lattice with preserved key structural components, highlighting the structural versatility of CA to form immature assemblies. A direct comparison between EIAV and HIV revealed that both lentiviruses maintain similar immature interfaces, which are established by both conserved and non-conserved residues. In both EIAV and HIV-1, IP6 regulates immature assembly via conserved lysine residues within the CACTD and SP. Lastly, we demonstrate that IP6 stimulates in vitro assembly of immature particles of several other retroviruses in the lentivirus genus, suggesting a conserved role for IP6 in lentiviral assembly.

HIV-1 protease (PR) cleavage of the Gag polyprotein triggers the assembly of mature, infectious particles. Final cleavage of Gag occurs at the junction helix between the capsid protein CA and the SP1 spacer peptide. Here we used MicroED to delineate the binding interactions of the maturation inhibitor bevirimat (BVM) using very thin frozen-hydrated, 3D microcrystals of a CTD-SP1 Gag construct with and without bound BVM. The 2.9-Å MicroED structure revealed that a single BVM molecule stabilizes the six-helix bundle via both electrostatic interactions with the dimethylsuccinyl moiety and hydrophobic interactions with the pentacyclic triterpenoid ring. These results provide insight into the mechanism of action of BVM and related maturation inhibitors that will inform further drug discovery efforts. This study also demonstrates the capabilities of MicroED for structure-based drug design.

Understanding the molecular mechanisms involved in the assembly of viruses is essential for discerning how viruses transmit from cell to cell and host to host. Although molecular aspects of assembly have been studied for many viruses, we still have little information about these events in real time. Enveloped viruses such as HIV that assemble at, and bud from, the plasma membrane have been studied in some detail using live cell fluorescence imaging techniques; however, these approaches provide little information about the real-time morphological changes that take place as viral components come together to form individual virus particles. Here we used correlative scanning ion conductance microscopy and fluorescence confocal microscopy to measure the topological changes, together with the recruitment of fluorescently labeled viral proteins such as Gag and Vpr, during the assembly and release of individual HIV virus-like particles (VLPs) from the top, nonadherent surfaces of living cells. We show that 1) labeling of viral proteins with green fluorescent protein affects particle formation, 2) the kinetics of particle assembly on different plasma membrane domains can vary, possibly as a consequence of differences in membrane biophysical properties, and 3) VLPs budding from the top, unimpeded surface of cells can reach full size in 20 s and disappear from the budding site in 0.5 to 3 min from the moment curvature is initially detected, significantly faster than has been previously reported.

A reservoir of HIV-infected cells that persists despite suppressive antiretroviral therapy (ART) is the source of viral rebound upon ART cessation and the major barrier to a cure. Understanding reservoir seeding dynamics will help identify the best timing for HIV cure strategies. Here we characterize reservoir seeding using longitudinal samples from before and after ART initiation in individuals who sequentially became infected with genetically distinct HIV variants (superinfected). We previously identified cases of superinfection in a cohort of Kenyan women, and the dates of both initial infection and superinfection were determined. Six women, superinfected 0.2-5.2 years after initial infection, were subsequently treated with ART 5.4-18.0 years after initial infection. We performed next-generation sequencing of HIV gag and env RNA from plasma collected during acute infection as well as every ~2 years thereafter until ART initiation, and of HIV DNA from PBMCs collected 0.9-4.8 years after viral suppression on ART. We assessed phylogenetic relationships between HIV DNA reservoir sequences and longitudinal plasma RNA sequences prior to ART, to determine proportions of initial and superinfecting variants in the reservoir. The proportions of initial and superinfection lineage variants present in the HIV DNA reservoir were most similar to the proportions present in HIV RNA immediately prior to ART initiation. Phylogenetic analysis confirmed that the majority of HIV DNA reservoir sequences had the smallest pairwise distance to RNA sequences from timepoints closest to ART initiation. Our data suggest that while reservoir cells are created throughout pre-ART infection, the majority of HIV-infected cells that persist during ART entered the reservoir near the time of ART initiation. We estimate the half-life of pre-ART DNA reservoir sequences to be ~25 months, which is shorter than estimated reservoir decay rates during suppressive ART, implying continual decay and reseeding of the reservoir up to the point of ART initiation.

Human immunodeficiency virus type 1 (HIV-1) assembles as immature particles, which require the proteolytic cleavage of structural polyprotein Gag and the clustering of envelope glycoprotein Env for infectivity. The details of mechanisms underlying Env clustering remain unknown. Here, we determine molecular dynamics of Env on the surface of individual HIV-1 particles using scanning fluorescence correlation spectroscopy on a super-resolution STED microscope. We find that Env undergoes a maturation-induced increase in mobility, highlighting diffusion as one cause for Env clustering. This mobility increase is dependent on Gag-interacting Env tail but not on changes in viral envelope lipid order. Diffusion of Env and other envelope incorporated proteins in mature HIV-1 is two orders of magnitude slower than in the plasma membrane, indicating that HIV-1 envelope is intrinsically a low mobility environment, mainly due to its general high lipid order. Our results provide insights into dynamic properties of proteins on the surface of individual virus particles. To become infectious, HIV-1 particles undergo a maturation process involving the clustering of envelope glycoprotein Env. Here, Chojnacki et al. employ super-resolution STED-FCS microscopy to study dynamics of Env molecules on HIV-1 particles and show that Env undergoes a maturation-induced increase in mobility.

Inositol hexakisphosphate (IP6) promotes HIV-1 assembly by stabilizing the immature Gag lattice and becomes enriched within virions, where it is required for mature capsid assembly. Previously, we identified Gag mutants that package little IP6 yet assemble particles, though they are non-infectious due to defective capsid formation. Here, we report a compensatory mutation, G225R, in the C-terminus of capsid protein (CA) that restores capsid assembly and infectivity in these IP6-deficient mutants. G225R also enhances in vitro assembly of CA into capsid-like particles at far lower IP6 concentrations than required for wild-type CA. CryoEM structures of G225R CA hexamers and lattices at 2.7 Å resolution reveal that the otherwise disordered C-terminus becomes structured, stabilizing hexamer-hexamer interfaces. Molecular dynamics simulations support this mechanism. These findings uncover how HIV-1 can adapt to IP6 deficiency and highlight a previously unrecognized structural role of the CA C-terminus, while offering tools for capsid-related studies. IP6 is a critical host cofactor for HIV-1 assembly and infectivity. In this study, the authors uncover the structural basis by which HIV-1 adapts to a deficiency in IP6 packaging through a G225R mutation at the C-terminus of the capsid protein.

Virus-like particles (VLPs) are non-infective vaccine candidates that have gained interest given their natural ability to elicit strong immune responses. Particularly, HIV-1 Gag-based VLPs are one of the most described platforms for vaccine development, provided their ability for successful pseudotyping either by genetic engineering or click chemistry. When Gag polyprotein is recombinantly expressed, VLPs are naturally assembled in the vicinity of the cell membrane and then secreted by cell budding, taking part of the host cell membrane. Their properties are dependent upon the cell line and manufacturing method. Although great advancements toward the implementation of analytical methods have been made, VLP quality attributes are quite unclear whenever production is enhanced by metabolic engineering or process intensification strategies. This work offers a comparative study of VLP quality attributes upon transient gene expression (TGE) in HEK293 cell cultures operated in batch and perfusion mode. Moreover, the impact of specific productivity is also studied by ataxia telangiectasia mutated (ATM) gene silencing, which has been reported to enhance fourfold VLP production. A linear negative correlation was found between the ratio of Gag monomers/VLP and specific productivity. 3100 ± 100 monomers/VLP were obtained for the standard batch production, dropping to 1900 ± 100 and 800 ± 60 for the perfusion and batch ATM-knockdown conditions, respectively. Furthermore, functionalization rates were measured in terms of Cy5 per total particles (TP). Both perfusion-derived nanoparticles achieved functionalization rates of 2800 Cy5/TP. On the contrary, those nanoparticles produced in batch yielded functionalization rates below 1000 Cy5/TP. Moreover, a complete lipidome analysis revealed a relative decrease in the quantity of lipid/particle for all studied conditions in comparison to the standard batch production. Finally, all VLP samples were characterized to assess the impact of the differential physicochemical properties upon purification and stability rates. Key points • VLP quality inversely correlates with Gag-specific productivity and operation mode. • Functionalization and lipid content drop with metabolic burden or ATM silencing. • Perfusion enables high VLP recovery and lyophilization with preserved morphology.