The SARS-CoV-2 nucleocapsid (N) protein is essential for viral RNA packaging, replication, and immune modulation. Despite its central role, the mechanistic contributions of its individual domains, the N-terminal domain (NTD), C-terminal domain (CTD), and the intrinsically flexible linker (LINK), remain poorly defined, largely due to the protein's structural complexity. In this study, we developed a panel of twelve alpaca-derived nanobodies (VHHs) targeting the NTD, CTD, and LINK regions of N. Using ELISA and biolayer interferometry, we characterized their binding affinities, and we mapped their epitopes via hydrogen-deuterium exchange-mass spectrometry (HDX-MS) and structural modeling. When expressed intracellularly, these VHHs inhibited SARS-CoV-2 infection. In vitro, they disrupted phase separation of the N protein, a critical step in viral replication. Strikingly, VHHs targeting each domain independently blocked both phase condensation and viral replication, underscoring the functional importance of all three regions. These findings establish domain-specific VHHs as versatile tools for dissecting N biology, with promising therapeutic potential.

Lateral flow assays (LFAs) are among the most successful technologies for point-of-care and at-home testing, but further advances are needed to reduce costs and accelerate development. Alpaca-derived nanobodies (Nbs), single-domain antibody fragments, are promising immunoassay reagents across diverse applications. Their small size and ease of recombinant production make them particularly well suited for diagnostics. Here, we present a paper-based LFA targeting the SARS-CoV-2 nucleocapsid (N) protein that exclusively uses Nbs for direct antigen detection. We also demonstrate in-house synthesis of Nb-coated gold nanoparticles, enabling instrument-free visual readout and detection of N protein down to 40 ng/mL. This design avoids components that require mammalian cell culture and can be produced entirely from in-house reagents, simplifying manufacturing and lowering component costs. Because the assay is read visually without an external reader, it is well suited for deployment in resource-limited settings. Together, these results highlight the speed and practicality of developing Nb-based LFAs and suggest a broadly applicable strategy for detecting other clinically important disease biomarkers.

Based on observations that HIV-1 envelope (Env) proteins on the surfaces of cells have the capacity to fuse with neighboring cells or enveloped viruses that express CD4 receptors and CXCR4 co-receptors, we tested factors that affect the capacities of lentiviral vectors pseudotyped with CD4 and CXCR4 variants to infect Env-expressing cells. The process, which we refer to as fusion in reverse, involves the binding and activation of cellular Env proteins to fuse membranes with lentiviruses carrying CD4 and CXCR4 proteins. We have found that infection via fusion in reverse depends on cell surface Env levels, is inhibitable by an HIV-1-specific fusion inhibitor, and preferentially requires lentiviral pseudotyping with a glycosylphosphatidylinositol (GPI)-anchored CD4 variant and a cytoplasmic tail-truncated CXCR4 protein. We have demonstrated that latently HIV-1-infected cells can be specifically infected using this mechanism, and that activation of latently infected cells increases infection efficiency. The fusion in reverse approach allowed us to characterize how alteration of CD4 plus CXCR4 lipid membranes affected Env protein activities. In particular, we found that perturbation of membrane cholesterol levels did not affect Env activity. In contrast, viruses assembled in cells deficient for long-chain sphingolipids showed increased infectivities, while viruses that incorporated a lipid scramblase were non-infectious. Our results yield new insights into factors that influence envelope protein functions.

Mycobacterium tuberculosis (Mtb) is known to survive within macrophages by compromising the integrity of the phagosomal compartment in which it resides. This activity primarily relies on the ESX-1 secretion system, predominantly involving the protein duo ESAT-6 and CFP-10. CFP-10 likely acts as a chaperone, while ESAT-6 likely disrupts phagosomal membrane stability via a largely unknown mechanism. we employ a series of biochemical analyses, protein modeling techniques, and a novel ESAT-6-specific nanobody to gain insight into the ESAT-6’s mode of action. First, we measure the binding kinetics of the tight 1:1 complex formed by ESAT-6 and CFP-10 at neutral pH. Subsequently, we demonstrate a rapid self-association of ESAT-6 into large complexes under acidic conditions, leading to the identification of a stable tetrameric ESAT-6 species. Using molecular dynamics simulations, we pinpoint the most probable interaction interface. Furthermore, we show that cytoplasmic expression of an anti-ESAT-6 nanobody blocks Mtb replication, thereby underlining the pivotal role of ESAT-6 in intracellular survival. Together, these data suggest that ESAT-6 acts by a pH dependent mechanism to establish two-way communication between the cytoplasm and the Mtb-containing phagosome.

While investigating methods to target gene delivery vectors to specific cell types, we examined the potential of using a nanobody against the SARS-CoV-2 Spike protein receptor-binding domain to direct lentivirus infection of Spike-expressing cells. Using four different approaches, we found that lentiviruses with surface-exposed nanobody domains selectively infect Spike-expressing cells. Targeting is dependent on the fusion function of the Spike protein, and conforms to a model in which nanobody binding to the Spike protein triggers the Spike fusion machinery. The nanobody-Spike interaction also is capable of directing cell-cell fusion and the selective infection of nanobody-expressing cells by Spike-pseudotyped lentivirus vectors. Significantly, cells infected with SARS-CoV-2 are efficiently and selectively infected by lentivirus vectors pseudotyped with a chimeric nanobody protein. Our results suggest that cells infected by any virus that forms syncytia may be targeted for gene delivery by using an appropriate nanobody or virus receptor mimic. Vectors modified in this fashion may prove useful in the delivery of immunomodulators to infected foci to mitigate the effects of viral infections.IMPORTANCEWe have discovered that lentiviruses decorated on their surfaces with a nanobody against the SARS-CoV-2 Spike protein selectively infect Spike-expressing cells. Infection is dependent on the specificity of the nanobody and the fusion function of the Spike protein and conforms to a reverse fusion model, in which nanobody binding to Spike triggers the Spike fusion machinery. The nanobody-Spike interaction also can drive cell-cell fusion and infection of nanobody-expressing cells with viruses carrying the Spike protein. Importantly, cells infected with SARS-CoV-2 are selectively infected with nanobody-decorated lentiviruses. These results suggest that cells infected by any virus that expresses an active receptor-binding fusion protein may be targeted by vectors for delivery of cargoes to mitigate infections.

How HIV-1 Env trimers assemble into virus particles remains incompletely understood. In restrictive cells, viral incorporation of Env is dependent on the Env CT and on the MA protein, which assembles lattices composed of hexamers of trimers in immature and mature viruses. Recent evidence indicates that CT assembles trimeric baseplate structures that require membrane-proximal residues to interface with trimeric transmembrane domains and C-terminal helices in the CT. We found that mutations of these membrane-proximal residues impaired replication in restrictive cells. This defect was countered by a MA mutation that does not localize to any obvious interprotein regions but was only inefficiently suppressed by a MA mutation that stabilizes MA trimers and has been shown to suppress other CT-dependent Env defects. Our results suggest that efficient suppression of baseplate mutations involves stabilization of MA inter-trimer contacts and/or direct MA-CT associations. These observations shed new light on how Env assembles into virions.

Trimers of the HIV-1 envelope (Env) protein perform receptor binding and virus-cell fusion functions during the virus life cycle. The cytoplasmic tail (CT) of Env forms an unusual baseplate structure, and is palmitoylated, rich in arginines, carries trafficking motifs, binds cholesterol, and interacts with host proteins. To dissect CT activities, we examined a panel of Env variants, including CT truncations, mutations, and an extension. We found that whereas all variants could replicate in permissive cells, viruses with CT truncations or baseplate mutations were defective in restrictive cells. We also identified a determinant in HIV-1 amphotericin sensitivity, and characterized variants that escape amphotericin inhibition via viral protease-mediated CT cleavage. Results additionally showed that full-length, his tagged Env can oligomerize and be co-assembled with CT truncations that delete portions of the baseplate, host protein binding sites, and trafficking signals. Our observations illuminate novel aspects of HIV-1 CT structure, interactions, and functions.

The spike glycoprotein of SARS-CoV-2 engages with human ACE 2 to facilitate infection. Here, we describe an alpaca-derived heavy chain antibody fragment (VHH), saRBD-1, that disrupts this interaction by competitively binding to the spike protein receptor-binding domain. We further generated an engineered bivalent nanobody construct engineered by a flexible linker and a dimeric Fc conjugated nanobody construct. Both multivalent nanobodies blocked infection at picomolar concentrations and demonstrated no loss of potency against emerging variants of concern including Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Epsilon (B.1.427/429), and Delta (B.1.617.2). saRBD-1 tolerates elevated temperature, freeze-drying, and nebulization, making it an excellent candidate for further development into a therapeutic approach for COVID-19.

The capsid (CA) domain of the HIV-1 precursor Gag (PrGag) protein plays multiple roles in HIV-1 replication, and is central to the assembly of immature virions, and mature virus cores. CA proteins themselves are composed of N-terminal domains (NTDs) and C-terminal domains (CTDs). We have investigated the interactions of CA with anti-CA nanobodies, which derive from the antigen recognition regions of camelid heavy chain-only antibodies. The one CA NTD-specific and two CTD-specific nanobodies we analyzed proved sensitive and specific HIV-1 CA detection reagents in immunoassays. When co-expressed with HIV-1 Gag proteins in cells, the NTD-specific nanobody was efficiently assembled into virions and did not perturb virus assembly. In contrast, the two CTD-specific nanobodies reduced PrGag processing, virus release and HIV-1 infectivity. Our results demonstrate the feasibility of Gag-targeted nanobody inhibition of HIV-1.

The lipid composition of HIV-1 virions is enriched in sphingomyelin (SM), but the roles that SM or other sphingolipids (SLs) might play in the HIV-1 replication pathway have not been elucidated. In human cells, SL levels are regulated by ceramide synthase (CerS) enzymes that produce ceramides, which can be converted to SMs, hexosylceramides, and other SLs. In many cell types, CerS2, which catalyzes the synthesis of very long chain ceramides, is the major CerS. We have examined how CerS2 deficiency affects the assembly and infectivity of HIV-1. As expected, we observed that very long chain ceramide, hexosylceramide, and SM were reduced in CerS2 knockout cells. CerS2 deficiency did not affect HIV-1 assembly or the incorporation of the HIV-1 envelope (Env) protein into virus particles, but it reduced the infectivites of viruses produced in the CerS2-deficient cells. The reduced viral infection levels were dependent on HIV-1 Env, since HIV-1 particles that were pseudotyped with the vesicular stomatitis virus glycoprotein did not exhibit reductions in infectivity. Moreover, cell-cell fusion assays demonstrated that the functional defect of HIV-1 Env in CerS2-deficient cells was independent of other viral proteins. Overall, our results indicate that the altered lipid composition of CerS2-deficient cells specifically inhibit the HIV-1 Env receptor binding and/or fusion processes.

Zika virus (ZIKV), an arbovirus of global concern, remodels intracellular membranes to form replication sites. How ZIKV dysregulates lipid networks to allow this, and consequences for disease, is poorly understood. Here, we perform comprehensive lipidomics to create a lipid network map during ZIKV infection. We find that ZIKV significantly alters host lipid composition, with the most striking changes seen within subclasses of sphingolipids. Ectopic expression of ZIKV NS4B protein results in similar changes, demonstrating a role for NS4B in modulating sphingolipid pathways. Disruption of sphingolipid biosynthesis in various cell types, including human neural progenitor cells, blocks ZIKV infection. Additionally, the sphingolipid ceramide redistributes to ZIKV replication sites, and increasing ceramide levels by multiple pathways sensitizes cells to ZIKV infection. Thus, we identify a sphingolipid metabolic network with a critical role in ZIKV replication and show that ceramide flux is a key mediator of ZIKV infection.

The matrix (MA) domains of HIV-1 precursor Gag (PrGag) proteins direct PrGag proteins to plasma membrane (PM) assembly sites where envelope (Env) protein trimers are incorporated into virus particles. MA targeting to PM sites is facilitated by its binding to phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2], and MA binding to cellular RNAs appears to serve a chaperone function that prevents MA from associating with intracellular membranes prior to arrival at the PI(4,5)P2-rich PM. Investigations have shown genetic evidence of an interaction between MA and the cytoplasmic tails (CTs) of Env trimers that contributes to Env incorporation into virions, but demonstrations of direct MA-CT interactions have proven more difficult. In direct binding assays, we show here that MA binds to Env CTs. Using MA mutants, matrix-capsid (MACA) proteins, and MA proteins incubated in the presence of inositol polyphosphate, we show a correlation between MA trimerization and CT binding. RNA ligands with high affinities for MA reduced MA-CT binding levels, suggesting that MA-RNA binding interferes with trimerization and/or directly or indirectly blocks MA-CT binding. Rough-mapping studies indicate that C-terminal CT helices are involved in MA binding and are in agreement with cell culture studies with replication-competent viruses. Our results support a model in which full-length HIV-1 Env trimers are captured in assembling PrGag lattices by virtue of their binding to MA trimers. IMPORTANCE The mechanism by which HIV-1 envelope (Env) protein trimers assemble into virus particles is poorly understood but involves an interaction between Env cytoplasmic tails (CTs) and the matrix (MA) domain of the structural precursor Gag (PrGag) proteins. We show here that direct binding of MA to Env CTs correlates with MA trimerization, suggesting models where MA lattices regulate CT interactions and/or MA-CT trimer-trimer associations increase the avidity of MA-CT binding. We also show that MA binding to RNA ligands impairs MA-CT binding, potentially by interfering with MA trimerization and/or directly or allosterically blocking MA-CT binding sites. Rough mapping implicated CT C-terminal helices in MA binding, in agreement with cell culture studies on MA-CT interactions. Our results indicate that targeting HIV-1 MA-CT interactions may be a promising avenue for antiviral therapy.

The matrix (MA) domain of HIV-1 Gag plays key roles in virus assembly by targeting the Gag precursor to the plasma membrane and directing the incorporation of the viral envelope (Env) glycoprotein into virions. The latter function appears to be in part dependent on trimerization of the MA domain of Gag during assembly, as disruption of the MA trimer interface impairs Env incorporation. Conversely, many MA mutations that impair Env incorporation can be rescued by compensatory mutations in the trimer interface. In this study, we sought to investigate further the biological significance of MA trimerization by isolating and characterizing compensatory mutations that rescue MA trimer interface mutants with severely impaired Env incorporation. By serially propagating MA trimerization-defective mutants in T cell lines, we identified a number of changes in MA, both within and distant from the trimer interface. The compensatory mutations located within or near the trimer interface restored Env incorporation and particle infectivity and permitted replication in culture. The structure of the MA lattice was interrogated by measuring the cleavage of the murine leukemia virus (MLV) transmembrane Env protein by the viral protease in MLV Env-pseudotyped HIV-1 particles bearing the MA mutations and by performing crystallographic studies of in vitro-assembled MA lattices. These results demonstrate that rescue is associated with structural alterations in MA organization and rescue of MA domain trimer formation. Our data highlight the significance of the trimer interface of the MA domain of Gag as a critical site of protein-protein interaction during HIV-1 assembly and establish the functional importance of trimeric MA for Env incorporation.IMPORTANCE The immature Gag lattice is a critical structural feature of assembling HIV-1 particles, which is primarily important for virion formation and release. While Gag forms a hexameric lattice, driven primarily by the capsid domain, the MA domain additionally trimerizes where three Gag hexamers meet. MA mutants that are defective for trimerization are deficient for Env incorporation and replication, suggesting a requirement for trimerization of the MA domain of Gag in Env incorporation. This study used a gain-of-function, forced viral evolution approach to rescue HIV-1 mutants that are defective for MA trimerization. Compensatory mutations that rescue virus replication do so by restoring Env incorporation and MA trimer formation. This study supports the importance of MA domain trimerization in HIV-1 replication and the potential of the trimer interface as a therapeutic target.

Mutations of the Ras proteins HRAS, KRAS4A, KRAS4B, and NRAS are associated with a high percentage of all human cancers. The proteins are composed of highly homologous N-terminal catalytic or globular domains, plus C-terminal hypervariable regions (HVRs). Post-translational modifications of all RAS HVRs helps target RAS proteins to cellular membrane locations where they perform their signaling functions. For the predominant KRAS4 isoform, KRAS4B, post-translational farnesylation and carboxymethylation, along with a patch of HVR basic residues help foster membrane binding. Recent investigations implicate membrane-bound RAS dimers, oligomers, and nanoclusters as landing pads for effector proteins that relay RAS signals. The details of these RAS signaling platforms have not been elucidated completely, in part due to the difficulties in preparing modified proteins. We have employed properly farnesylated and carboxymethylated KRAS4B in lipid monolayer incubations to examine how the proteins assemble on membranes. Our results reveal novel insights into to how KRAS4B may organize on membranes.

The flavivirus family includes Zika virus (ZIKV), Dengue virus, West Nile virus, Japanese Encephalitis and Yellow Fever virus, each of which causes a major global health threat. During their long‐lasting co‐existence with their host, these viruses have developed sophisticated strategies to co‐opt thecellular machinery including, host lipid metabolism, to effectively establish infection. They enter the host cells through receptor‐mediated endocytosis. In endosomes, the acidic environment induces fusion between the virus and the host membrane resulting in viral genome release. The viral RNA is translated into a polyprotein, which is post‐translationally cleaved by cellular and viral proteases into three structural proteins including capsid, membrane, and envelope, and seven nonstructural proteins. During viral infection cell membranes are co‐opted for virtually every step of the viral life cycle: entry, replication, assembly and release. The role of host factors, especially that of lipids, in pathogenesis of flaviviruses is largely unknown, and there exists a gap in understanding the basic biology of these viruses. This is especially true for ZIKV, which until recently was one of the most understudied flaviviruses. Because lipids are an important interface in host‐virus interactions, studying these biomolecules may provide strategies to combat diseases caused by these pathogens. Zika virus (ZIKV) is a positive‐sense RNA virus that remodels host membrane to establish sites of replication and assembly. To determine how ZIKV modulates the host lipid repertoire in an unbiased manner, we performed comprehensive lipid profiling of human cells infected with ZIKV. We found that ZIKV replication triggers significant changes in the cellular sphingolipidomes especially that of ceramide and sphingomyelin highly enriched during infection. Disruption of sphingolipid biosynthesis with CRISPR/Cas9 gene‐editing or small molecule inhibitors effectively blocks ZIKV replication. Both sphingomyelin and ceramide redistribute to ZIKV replication sites, and the sphingomyelinase‐catalyzed degradation of sphingomyelin to ceramide is the critical pathway to establish infection effectively. Inhibition of sphingomyelinase attenuated ZIKV replication, while the accumulation of intracellular ceramide through knockout of sphingomyelin synthesis enhanced ZIKV infection. Thus, we identify a sphingolipid salvage pathway with a critical role in ZIKV replication and show that ceramide is a key mediator of ZIKV pathogenesis.Support or Funding InformationThis work was supported by the Collins Medical Trust and NIH grant R21AI133631. W.B.M. and J.B.W. were supported by the Medical Research Foundation of Oregon, the Oregon Clinical and Translational Research Institute, and the Sunlin and Priscilla Chou Foundation. D.K. was supported by the Summer Equity Internship Program of the OHSU Center for Diversity and Inclusion. E.B. was supported by NIH grants R01GM060170 and R01GM101983. E.A.D. and A.R.N. were supported by NIH grants R01DK105961, U19AI106754, and U54GM069338.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Wild type (WT) HIV-1 envelope (Env) protein cytoplasmic tails (CTs) appear to be composed of membrane-proximal, N-terminal unstructured regions, and three C-terminal amphipathic helices. Previous studies have shown that WT and CT-deleted (ΔCT) Env proteins are incorporated into virus particles via different mechanisms. WT Env proteins traffic to cell plasma membranes (PMs), are rapidly internalized, recycle to PMs, and are incorporated into virions in permissive and restrictive cells in a Gag matrix (MA) protein-dependent fashion. In contrast, previously described ΔCT proteins do not appear to be internalized after their arrival to PMs, and do not require MA, but are only incorporated into virions in permissive cell lines. We have analyzed a new set of HIV-1 CT variants with respect to their replication in permissive and restrictive cells. Our results provide novel details as to how CT elements regulate HIV-1 Env protein function.

The matrix (MA) domain of the HIV-1 precursor Gag protein (PrGag) has been shown interact with the HIV-1 envelope (Env) protein, and to direct PrGag proteins to plasma membrane (PM) assembly sites by virtue of its affinity to phosphatidylinositol-4,5-bisphosphate (PI[4,5]P2). Unexpectedly, HIV-1 viruses with large MA deletions (ΔMA) have been shown to be conditionally infectious as long as they are matched with Env truncation mutant proteins or alternative viral glycoproteins. To characterize the interactions of wild type (WT) and ΔMA Gag proteins with PI(4,5)P2 and other acidic phospholipids, we have employed a set of lipid biosensors as probes. Our investigations showed marked differences in WT and ΔMA Gag colocalization with biosensors, effects on biosensor release, and association of biosensors with virus-like particles. These results demonstrate an alternative approach to the analysis of viral protein-lipid associations, and provide new data as to the lipid compositions of HIV-1 assembly sites.

BACKGROUND:Mutations of the human K-Ras 4B (K-Ras) G protein are associated with a significant proportion of all human cancers. Despite this fact, a comprehensive analysis of K-Ras interactions is lacking. Our investigations focus on characterization of the K-Ras interaction network.MATERIALS AND METHODS:We employed a biotin ligase-tagging approach, in which tagged K-Ras proteins biotinylate neighbor proteins in a proximity-dependent fashion, and proteins are identified via mass spectrometry (MS) sequencing.RESULTS:In transfected cells, a total of 748 biotinylated proteins were identified from cells expressing biotin ligase-tagged K-Ras variants. Significant differences were observed between membrane-associated variants and a farnesylation-defective mutant. In pancreatic cancer cells, 56 K-Ras interaction partners were identified. Most of these were cytoskeletal or plasma membrane proteins, and many have been identified previously as potential cancer biomarkers.CONCLUSION:Biotin ligase tagging offers a rapid and convenient approach to the characterization of K-Ras interaction networks.

We have analyzed a set of quinolinequinones with respect to their reactivities, cytotoxicities, and anti-HIV-1 properties. Most of the quinolinequinones were reactive with glutathione, and several acted as sulfhydryl crosslinking agents. Quinolinequinones inhibited binding of the HIV-1 matrix protein to RNA to varying degrees, and several quinolinequinones showed the capacity to crosslink HIV-1 matrix proteins in vitro, and HIV-1 structural proteins in virus particles. Cytotoxicity assays yielded quinolinequinone CC50 values in the low micromolar range, reducing the potential therapeutic value of these compounds. However, one compound, 6,7-dichloro-5,8-quinolinequinone potently inactivated HIV-1, suggesting that quinolinequinones may prove useful in the preparation of inactivated virus vaccines or for other virucidal purposes.

ABSTRACT The HIV-1 matrix (MA) protein is the amino-terminal domain of the HIV-1 precursor Gag (Pr55Gag) protein. MA binds to membranes and RNAs, helps transport Pr55Gag proteins to virus assembly sites at the plasma membranes of infected cells, and facilitates the incorporation of HIV-1 envelope (Env) proteins into virions by virtue of an interaction with the Env protein cytoplasmic tails (CTs). MA has been shown to crystallize as a trimer and to organize on membranes in hexamer lattices. MA mutations that localize to residues near the ends of trimer spokes have been observed to impair Env protein assembly into virus particles, and several of these are suppressed by the 62QR mutation at the hubs of trimer interfaces. We have examined the binding activities of wild-type (WT) MA and 62QR MA variants and found that the 62QR mutation stabilized MA trimers but did not alter the way MA proteins organized on membranes. Relative to WT MA, the 62QR protein showed small effects on membrane and RNA binding. However, 62QR proteins bound significantly better to Env CTs than their WT counterparts, and CT binding efficiencies correlated with trimerization efficiencies. Our data suggest a model in which multivalent binding of trimeric HIV-1 Env proteins to MA trimers contributes to the process of Env virion incorporation. IMPORTANCE The HIV-1 Env proteins assemble as trimers, and incorporation of the proteins into virus particles requires an interaction of Env CT domains with the MA domains of the viral precursor Gag proteins. Despite this knowledge, little is known about the mechanisms by which MA facilitates the virion incorporation of Env proteins. To help elucidate this process, we examined the binding activities of an MA mutant that stabilizes MA trimers. We found that the mutant proteins organized similarly to WT proteins on membranes, and that mutant and WT proteins revealed only slight differences in their binding to RNAs or lipids. However, the mutant proteins showed better binding to Env CTs than the WT proteins, and CT binding correlated with MA trimerization. Our results suggest that multivalent binding of trimeric HIV-1 Env proteins to MA trimers contributes to the process of Env virion incorporation.