Visualizing HIV-1 Assembly at the T-Cell Plasma Membrane Using Single-Molecule Localization Microscopy.

The 20-year revolution in optical fluorescence microscopy, supported by the optimization of both spatial resolution and timely acquisition, allows the visualization of nanoscaled objects in cell biology. Currently, the use of a recent generation of super-resolution fluorescence microscope coupled with improved fluorescent probes gives the possibility to study the replicative cycle of viruses in living cells, at the single-virus particle or protein level. Here, we highlight the protocol for visualizing HIV-1 Gag assembly at the host T-cell plasma membrane using super-resolution light microscopy. Total internal reflection fluorescence microscopy (TIRF-M) coupled with single-molecule localization microscopy (SMLM) enables the detection and characterization of the assembly of viral proteins at the plasma membrane of infected host cells at the single protein level. Here, we describe the TIRF equipment, the T-cell culture for HIV-1, the sample preparation for single-molecule localization microscopies such as PALM and STORM, acquisition protocols, and Gag assembling cluster analysis.

HIV-1 Gag MA domain binds to cardiolipin in a binding mode distinct from virus assemble mediator PI(4,5)P2.

The human immunodeficiency virus type 1 (HIV-1) Gag protein is responsible for facilitating HIV-1 virion assembly and budding. Our study demonstrates that cardiolipin (CL), a component found in the inner mitochondrial membrane, exhibits the highest binding affinity to the N-terminal MA domain of the HIV-1 Gag protein within the lipid group of host cells. To assess this binding interaction, we synthesized short acyl chain derivatives of CL and employed surface plasmon resonance (SPR) analysis to determine the dissociation constants (Kd) for CL and the MA domain. Simultaneously, we examined the Kd of D-myo-phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2 ) derivatives, known to play a crucial role in virion formation. Among all the derivatives, Tetra-C7 -CL exhibited the lowest Kd value (Kd = 30.8 ± 6.9 µM) for MA binding on the CL analog-immobilized sensorchip, indicating a higher affinity. Similarly, the Kd value of Di-C7 -PIP2 (Kd = 36.6 ± 4.7 µM) was the lowest on the PI(4,5)P2 analog-immobilized sensorchip. Thus, Tetra-C7 -CL binds to the MA domain using a distinct binding mode while displaying a comparable binding affinity to Di-C7 -PIP2. This discovery holds significant implications for comprehending the virological importance of CL-MA domain binding, such as its subcellular distribution, including mitochondrial translocation, and involvement in viral particle formation in concert with PI(4,5)P2 . Furthermore, this study has the potential to contribute to the development of drugs in the future.

Structure of the HIV immature lattice allows for essential lattice remodeling within budded virions.

For HIV virions to become infectious, the immature lattice of Gag polyproteins attached to the virion membrane must be cleaved. Cleavage cannot initiate without the protease formed by the homo-dimerization of domains linked to Gag. However, only 5% of the Gag polyproteins, termed Gag-Pol, carry this protease domain, and they are embedded within the structured lattice. The mechanism of Gag-Pol dimerization is unknown. Here, we use spatial stochastic computer simulations of the immature Gag lattice as derived from experimental structures, showing that dynamics of the lattice on the membrane is unavoidable due to the missing 1/3 of the spherical protein coat. These dynamics allow for Gag-Pol molecules carrying the protease domains to detach and reattach at new places within the lattice. Surprisingly, dimerization timescales of minutes or less are achievable for realistic binding energies and rates despite retaining most of the large-scale lattice structure. We derive a formula allowing extrapolation of timescales as a function of interaction free energy and binding rate, thus predicting how additional stabilization of the lattice would impact dimerization times. We further show that during assembly, dimerization of Gag-Pol is highly likely and therefore must be actively suppressed to prevent early activation. By direct comparison to recent biochemical measurements within budded virions, we find that only moderately stable hexamer contacts (-12kBT<∆G<-8kBT) retain both the dynamics and lattice structures that are consistent with experiment. These dynamics are likely essential for proper maturation, and our models quantify and predict lattice dynamics and protease dimerization timescales that define a key step in understanding formation of infectious viruses.

The unique activity of saponin: Induction of cytotoxicity in HTLV-1 infected cells.

Infection with the retrovirus human T-cell leukemia virus type 1 (HTLV-1) sometimes causes diseases that are difficult to cure. To find anti-HTLV-1 natural compounds, we opted to screen using the HTLV-1-infected T-cell line, MT-2. Based on our results, an extract of the pulp/seeds of Akebia quinata Decaisne fruit killed MT-2 cells but did not affect the Jurkat cell line that was not infected with virus. To determine the active ingredients, seven saponins with one-six sugar moieties were isolated from A. quinata seeds, and their activities against the two cell lines were examined. Both cell lines were killed in a similar manner by Akebia saponins A and B. Further, Akebia saponins D, E, PK and G did not exhibit cytotoxicity. Akebia saponin C had a similar activity to the extract found in the screening. This compound was found to enhance Gag aggregation, induce the abnormal cleavage of Gag, suppress virion release, and preferentially kill HTLV-1 infected cells; however, their relationship remains elusive. Our findings may lead to the development of new therapies for infectious diseases based on the removal of whole-virus-infected cells.

The Rous sarcoma virus Gag Polyprotein Forms Biomolecular Condensates Driven by Intrinsically-disordered Regions.

Biomolecular condensates (BMCs) play important roles incellular structures includingtranscription factories, splicing speckles, and nucleoli. BMCs bring together proteins and other macromolecules, selectively concentrating them so that specific reactions can occur without interference from the surrounding environment. BMCs are often made up of proteins that contain intrinsically disordered regions (IDRs), form phase-separated spherical puncta, form liquid-like droplets that undergo fusion and fission, contain molecules that are mobile, and are disrupted with phase-dissolving drugs such as 1,6-hexanediol. In addition to cellular proteins, many viruses, including influenza A, SARS-CoV-2, and human immunodeficiency virus type 1 (HIV-1) encode proteins that undergo phase separation and rely on BMC formation for replication. In prior studies of the retrovirus Rous sarcoma virus (RSV), we observed that the Gag protein forms discrete spherical puncta in the nucleus, cytoplasm, and at the plasma membrane that co-localize with viral RNA and host factors, raising the possibility that RSV Gag forms BMCs that participate in the intracellular phase of the virion assembly pathway. In our current studies, we found that Gag contains IDRs in the N-terminal (MAp2p10) and C-terminal (NC) regions of the protein and fulfills many criteria of BMCs. Although the role of BMC formation in RSV assembly requires further study, our results suggest the biophysical properties of condensates are required for the formation of Gag complexes in the nucleus and the cohesion of these complexes as they traffic through the nuclear pore, into the cytoplasm, and to the plasma membrane, where the final assembly and release of virus particles occurs.

Molecular physiology of Arc/Arg3.1: The oligomeric state hypothesis of synaptic plasticity.

The immediate early gene, Arc, is a pivotal regulator of synaptic plasticity, memory, and cognitive flexibility. But what is Arc protein? How does it work? Inside the neuron, Arc is a protein interaction hub and dynamic regulator of intra-cellular signaling in synaptic plasticity. In remarkable contrast, Arc can also self-assemble into retrovirus-like capsids that are released in extracellular vesicles and capable of intercellular transfer of RNA. Elucidation of the molecular basis of Arc hub and capsid functions, and the relationship between them, is vital for progress. Here, we discuss recent findings on Arc structure-function and regulation of oligomerization that are giving insight into the molecular physiology of Arc. The unique features of mammalian Arc are emphasized, while drawing comparisons with Drosophila Arc and retroviral Gag. The Arc N-terminal domain, found only in mammals, is proposed to play a key role in regulating Arc hub signaling, oligomerization, and formation of capsids. Bringing together several lines of evidence, we hypothesize that Arc function in synaptic plasticity-long-term potentiation (LTP) and long-term depression (LTD)-are dictated by different oligomeric forms of Arc. Specifically, monomer/dimer function in LTP, tetramer function in basic LTD, and 32-unit oligomer function in enhanced LTD. The role of mammalian Arc capsids is unclear but likely depends on the cross-section of captured neuronal activity-induced RNAs. As the functional states of Arc are revealed, it may be possible to selectively manipulate specific forms of Arc-dependent plasticity and intercellular communication involved in brain function and dysfunction.

Viral and Host Factors Regulating HIV-1 Envelope Protein Trafficking and Particle Incorporation.

The HIV-1 envelope glycoprotein (Env) is an essential structural component of the virus, serving as the receptor-binding protein and principal neutralizing determinant. Env trimers are incorporated into developing particles at the plasma membrane of infected cells. Incorporation of HIV-1 Env into particles in T cells and macrophages is regulated by the long Env cytoplasmic tail (CT) and the matrix region of Gag. The CT incorporates motifs that interact with cellular factors involved in endosomal trafficking. Env follows an unusual pathway to arrive at the site of particle assembly, first traversing the secretory pathway to the plasma membrane (PM), then undergoing endocytosis, followed by directed sorting to the site of particle assembly on the PM. Many aspects of Env trafficking remain to be defined, including the sequential events that occur following endocytosis, leading to productive recycling and particle incorporation. This review focuses on the host factors and pathways involved in Env trafficking, and discusses leading models of Env incorporation into particles.

Advances in HIV-1 Assembly.

The assembly of HIV-1 particles is a concerted and dynamic process that takes place on the plasma membrane of infected cells. An abundance of recent discoveries has advanced our understanding of the complex sequence of events leading to HIV-1 particle assembly, budding, and release. Structural studies have illuminated key features of assembly and maturation, including the dramatic structural transition that occurs between the immature Gag lattice and the formation of the mature viral capsid core. The critical role of inositol hexakisphosphate (IP6) in the assembly of both the immature and mature Gag lattice has been elucidated. The structural basis for selective packaging of genomic RNA into virions has been revealed. This review will provide an overview of the HIV-1 assembly process, with a focus on recent advances in the field, and will point out areas where questions remain that can benefit from future investigation.

Thermodynamic Affinity and Nature of Forces Defining Glycosaminoglycan-Protein Systems Using Fluorescence Spectroscopy.

Among the biophysical techniques used to study glycosaminoglycan (GAG)-protein interactions, fluorescence spectroscopy is a quantitative tool that has been extensively used to provide structural and dynamical information. Its advantages include high sensitivity, relative ease of applicability, and wide range of available fluorescence labels and probes. A large majority of protein-GAG systems have been studied using either intrinsic (e.g., Trp) or extrinsic (e.g., a noncovalent fluorophore) probes. It forms the basis for measurement of dissociation constant and stoichiometry of GAG-protein complexes. We describe step-by-step procedures to measure the affinity of GAG-protein complexes, parse the ionic and non-ionic components of the free energy of binding, and identify the site of GAG binding through competitive binding experiments.

Advances in Studying Glycosaminoglycan-Protein Interactions Using Capillary Electrophoresis.

Methods for studying interactions between glycosaminoglycans (GAGs) and proteins have assumed considerable significance as their biological importance increases. Capillary electrophoresis (CE) is a powerful method to study these interactions due to its speed, high efficiency, and low sample/reagent consumption. In addition, CE works effectively under a wide range of physiologically relevant conditions. This chapter presents the state of the art on CE methods for studying GAG-protein interactions including affinity capillary electrophoresis (ACE), capillary zone electrophoresis (CZE), frontal analysis (FA)/frontal analysis continuous capillary electrophoresis (FACCE), and capillary electrokinetic chromatography (CEC) with detailed experimental protocols for ACE and CZE methods.

Rab3a, a small GTP-binding protein, is required for the stabilization of the murine leukaemia virus Gag protein.

We recently identified a CD63-interacting protein to understand the role of CD63 in virion production of the human immunodeficiency virus type 1, and we have found that Rab3a forms a complex with CD63. In this study, we analysed the effect of Rab3a on virion production of the murine leukaemia virus (MLV), which is another member of the retrovirus family. We found that Rab3a silencing induced lysosomal degradation of the MLV Gag protein, and recovery of the Rab3a expression restored the level of the Gag protein through a complex formation of MLV Gag and Rab3a, indicating that Rab3a is required for MLV Gag protein expression. In contrast, CD63 silencing decreased the infectivity of released virions but had no effect on virion production, indicating that CD63 facilitates the infectivity of released MLV particles. Although Rab3a induced CD63 degradation in uninfected cells, the complex of MLV Gag and Rab3a suppressed the Rab3a-mediated CD63 degradation in MLV-infected cells. Finally, we found that the MLV Gag protein interacts with Rab3a to stabilize its own protein and CD63 that facilitates the infectivity of released MLV particles. Considering the involvement of Rab3a in lysosome trafficking to the plasma membrane, it may also induce cell surface transport of the MLV Gag protein.

A Background Assessable and Correctable Bimolecular Fluorescence Complementation System for Nanoscopic Single-Molecule Imaging of Intracellular Protein-Protein Interactions.

Bimolecular Fluorescence Complementation (BiFC) is a versatile approach for intracellular analysis of protein-protein interactions (PPIs), but the tendency of the split fluorescent protein (FP) fragments to self-assemble when brought into close proximity of each other by random collision can lead to generation of false-positive signals that hamper high-definition imaging of PPIs occurring on the nanoscopic level. While it is thought that expressing the fusion proteins at a low level can remove false positives without impacting specific signals, there has been no effective strategy to test this possibility. Here, we present a system capable of assessing and removing BiFC false positives, termed Background Assessable and Correctable-BiFC (BAC-BiFC), in which one of the split FP fragments is fused with an optically distinct FP that serves as a reference marker, and the single-cell fluorescence ratio of the BiFC signal to the reference signal is used to gauge an optimal transfection condition. We showed that when BAC-BiFC is designed to image PPIs regulating Human Immunodeficiency Virus type 1 (HIV-1) assembly, the fluorescence ratio could decrease with decreasing probe quantity, and ratios approaching the limit of detection could allow physiologically relevant characterization of the assembly process on the nanoscale by single-molecule localization microscopy (SMLM). With much improved clarity, previously undescribed features of HIV-1 assembly were revealed.

Insights into structure, affinity, specificity, and function of GAG-protein interactions through the chemoenzymatic preparation of defined sulfated oligohyaluronans.

High amounts of glycosaminoglycans (GAG) such as hyaluronan (HA) occur in connective tissues. There is nowadays increasing evidence that a "sulfation code" exists which mediates numerous GAG functions. High molecular weight and inhomogeneity of GAG, however, aggravated detailed studies. Thus, synthetic oligosaccharides were urgently required. We will review here chemoenzymatic and analytic strategies to provide defined sulfated and anomerically modified GAG oligosaccharides of the HA type. Representative studies of protein/GAG interactions by (bio)chemical and biophysical methods are reported yielding novel insights into GAG-protein binding. Finally, the biological conclusions and in vivo applications of defined sulfated GAG oligosaccharides will be discussed.

Phosphorylation-dependent control of Activity-regulated cytoskeleton-associated protein (Arc) protein by TNIK.

Activity-regulated cytoskeleton-associated protein (Arc) is an immediate early gene product that support neuroplastic changes important for cognitive function and memory formation. As a protein with homology to the retroviral Gag protein, a particular characteristic of Arc is its capacity to self-assemble into virus-like capsids that can package mRNAs and transfer those transcripts to other cells. Although a lot has been uncovered about the contributions of Arc to neuron biology and behavior, very little is known about how different functions of Arc are coordinately regulated both temporally and spatially in neurons. The answer to this question we hypothesized must involve the occurrence of different protein post-translational modifications acting to confer specificity. In this study, we used mass spectrometry and sequence prediction strategies to map novel Arc phosphorylation sites. Our approach led us to recognize serine 67 (S67) and threonine 278 (T278) as residues that can be modified by TNIK, which is a kinase abundantly expressed in neurons that shares many functional overlaps with Arc and has, along with its interacting proteins such as the NMDA receptor, and been implicated as a risk factor for psychiatric disorders. Furthermore, characterization of each residue using site-directed mutagenesis to create S67 and T278 mutant variants revealed that TNIK action at those amino acids can strongly influence Arc's subcellular distribution and self-assembly as capsids. Together, our findings reveal an unsuspected connection between Arc and TNIK. Better understanding of the interplay between these two proteins in neuronal cells could lead to new insights about apparition and progression of psychiatric disorders. Cover Image for this issue: https://doi.org/10.1111/jnc.15077.

Production of Vesicular Stomatitis Virus Glycoprotein-Pseudotyped Lentiviral Vector Is Enhanced by Ezrin Silencing.

Human immunodeficiency virus type 1 (HIV-1)-based viral vector is widely used as a biomaterial to transfer a gene of interest into target cells in many biological study fields including gene therapy. Vesicular stomatitis virus glycoprotein (VSV-G)-containing HIV-1 vector much more efficiently transduces various mammalian cells than other viral envelope proteins-containing vectors. Understanding the mechanism would contribute to development of a novel method of efficient HIV-1 vector production. HIV-1 vector is generally constructed by transient transfection of human 293T or African green monkey COS7 cells. It was found in this study that HIV-1 Gag protein is constitutively digested in lysosomes of African green monkey cells. Surprisingly, VSV-G elevated HIV-1 Gag protein levels, suggesting that VSV-G protects Gag protein from the lysosomal degradation. Unphosphorylated ezrin, but not phosphorylated ezrin, was detected in COS7 cells, and ezrin silencing elevated Gag protein levels in the presence of VSV-G. Expression of unphosphorylated ezrin reduced Gag protein amounts. These results indicate that unphosphorylated ezrin proteins inhibit the VSV-G-mediated stabilization of HIV-1 Gag protein. Trafficking of HIV-1 Gag-associated intracellular vesicles may be controlled by ezrin. Finally, this study found that ezrin silencing yields higher amount of VSV-G-pseudotyped HIV-1 vector.

Structural and biophysical characterizations of HIV-1 matrix trimer binding to lipid nanodiscs shed light on virus assembly.

During the late phase of the HIV-1 replication cycle, the viral Gag polyproteins are targeted to the plasma membrane for assembly. The Gag-membrane interaction is mediated by binding of Gag's N-terminal myristoylated matrix (MA) domain to phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). The viral envelope (Env) glycoprotein is then recruited to the assembly sites and incorporated into budding particles. Evidence suggests that Env incorporation is mediated by interactions between Gag's MA domain and the cytoplasmic tail of the gp41 subunit of Env (gp41CT). MA trimerization appears to be an obligatory step for this interaction. Insufficient production of a recombinant MA trimer and unavailability of a biologically relevant membrane system have been barriers to detailed structural and biophysical characterization of the putative MA-gp41CT-membrane interactions. Here, we engineered a stable recombinant HIV-1 MA trimer construct by fusing a foldon domain (FD) of phage T4 fibritin to the MA C terminus. Results from NMR experiments confirmed that the FD attachment does not adversely alter the MA structure. Employing hydrogen-deuterium exchange MS, we identified an MA-MA interface in the MA trimer that is implicated in Gag assembly and Env incorporation. Utilizing lipid nanodiscs as a membrane mimetic, we show that the MA trimer binds to membranes 30-fold tighter than does the MA monomer and that incorporation of PI(4,5)P2 and phosphatidylserine enhances the binding of MA to nanodiscs. These findings advance our understanding of a fundamental mechanism in HIV-1 assembly and provide a template for investigating the interaction of MA with gp41CT.

Purification and Functional Characterization of a Biologically Active Full-Length Feline Immunodeficiency Virus (FIV) Pr50Gag.

The feline immunodeficiency virus (FIV) full-length Pr50Gag precursor is a key player in the assembly of new viral particles. It is also a critical component of the efficient selection and packaging of two copies of genomic RNA (gRNA) into the newly formed virus particles from a wide pool of cellular and spliced viral RNA. To understand the molecular mechanisms involved during FIV gRNA packaging, we expressed the His6-tagged and untagged recombinant FIV Pr50Gag protein both in eukaryotic and prokaryotic cells. The recombinant Pr50Gag-His6-tag fusion protein was purified from soluble fractions of prokaryotic cultures using immobilized metal affinity chromatography (IMAC). This purified protein was able to assemble in vitro into virus-like particles (VLPs), indicating that it preserved its ability to oligomerize/multimerize. Furthermore, VLPs formed in eukaryotic cells by the FIV full-length Pr50Gag both in the presence and absence of His6-tag could package FIV sub-genomic RNA to similar levels, suggesting that the biological activity of the recombinant full-length Pr50Gag fusion protein was retained in the presence of His6-tag at the carboxy terminus. Successful expression and purification of a biologically active, recombinant full-length Pr50Gag-His6-tag fusion protein will allow study of the intricate RNA-protein interactions involved during FIV gRNA encapsidation.

Insulin-induced gene 1 (INSIG1) inhibits HIV-1 production by degrading Gag via activity of the ubiquitin ligase TRC8.

Insulin-induced gene 1 (INSIG1) regulates sterol synthesis by mediating the activation of sterol regulatory element-binding protein (SREBP) and the degradation of the HMG-CoA reductase (HMGCR). INSIG1 is up-regulated during HIV-1 infection, but its role in HIV-1 infection is unknown. In this report, using pseudovirus production, protein overexpression, and gene knockouts, we found that INSIG1 inhibits HIV-1 production by accelerating the degradation of the HIV-1 Gag protein. Unlike the degradation of HMGCR via the E3 ubiquitin ligase autocrine motility factor receptor (AMFR), a process that depends on the proteasome, INSIG1 coordinated with another ligase, translocation in renal carcinoma chromosome 8 (TRC8), and promoted Gag degradation through the lysosome pathway. We conclude that INSIG1 functions as a sentinel responsive to HIV-1 production and inhibits HIV-1 replication by degrading Gag, a process occurring at intracellular membrane sites such as the endoplasmic reticulum and endosomes where both INSIG1 and Gag may be located.

The matrix domain of the Gag protein from avian sarcoma virus contains a PI(4,5)P2-binding site that targets Gag to the cell periphery.

The Gag protein of avian sarcoma virus (ASV) lacks an N-myristoyl (myr) group, but contains structural domains similar to those of HIV-1 Gag. Similarly to HIV-1, ASV Gag accumulates on the plasma membrane (PM) before egress; however, it is unclear whether the phospholipid PI(4,5)P2 binds directly to the matrix (MA) domain of ASV Gag, as is the case for HIV-1 Gag. Moreover, the role of PI(4,5)P2 in ASV Gag localization and budding has been controversial. Here, we report that substitution of residues that define the PI(4,5)P2-binding site in the ASV MA domain (reported in an accompanying paper) interfere with Gag localization to the cell periphery and inhibit the production of virus-like particles (VLPs). We show that co-expression of Sprouty2 (Spry2) or the pleckstrin homology domain of phospholipase Cδ (PH-PLC), two proteins that bind PI(4,5)P2, affects ASV Gag trafficking to the PM and budding. Replacement of the N-terminal 32 residues of HIV-1 MA, which encode its N-terminal myr signal and its PI(4,5)P2-binding site, with the structurally equivalent N-terminal 24 residues of ASV MA created a chimera that localized at the PM and produced VLPs. In contrast, the homologous PI(4,5)P2-binding signal in ASV MA could target HIV-1 Gag to the PM when substituted, but did not support budding. Collectively, these findings reveal a basic patch in both ASV and HIV-1 Gag capable of mediating PM binding and budding for ASV but not for HIV-1 Gag. We conclude that PI(4,5)P2 is a strong determinant of ASV Gag targeting to the PM and budding.

Structural basis for targeting avian sarcoma virus Gag polyprotein to the plasma membrane for virus assembly.

For most retroviruses, including HIV-1, binding of the Gag polyprotein to the plasma membrane (PM) is mediated by interactions between Gag's N-terminal myristoylated matrix (MA) domain and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) in the PM. The Gag protein of avian sarcoma virus (ASV) lacks the N-myristoylation signal but contains structural domains having functions similar to those of HIV-1 Gag. The molecular mechanism by which ASV Gag binds to the PM is incompletely understood. Here, we employed NMR techniques to elucidate the molecular determinants of the membrane-binding domain of ASV MA (MA87) to lipids and liposomes. We report that MA87 binds to the polar head of phosphoinositides such as PI(4,5)P2 We found that MA87 binding to inositol phosphates (IPs) is significantly enhanced by increasing the number of phosphate groups, indicating that the MA87-IP binding is governed by charge-charge interactions. Using a sensitive NMR-based liposome-binding assay, we show that binding of MA87 to liposomes is enhanced by incorporation of PI(4,5)P2 and phosphatidylserine. We also show that membrane binding is mediated by a basic surface formed by Lys-6, Lys-13, Lys-23, and Lys-24. Substitution of these residues to glutamate abolished binding of MA87 to both IPs and liposomes. In an accompanying paper, we further report that mutation of these lysine residues diminishes Gag assembly on the PM and inhibits ASV particle release. These findings provide a molecular basis for ASV Gag binding to the inner leaflet of the PM and advance our understanding of the basic mechanisms of retroviral assembly.