Budding Assay Research Articles

The universal suppressor mutation restores membrane budding defects in the HSV-1 nuclear egress complex by stabilizing the oligomeric lattice.

Nuclear egress is an essential process in herpesvirus replication whereby nascent capsids translocate from the nucleus to the cytoplasm. This initial step of nuclear egress-budding at the inner nuclear membrane-is coordinated by the nuclear egress complex (NEC). Composed of the viral proteins UL31 and UL34, NEC deforms the membrane around the capsid as the latter buds into the perinuclear space. NEC oligomerization into a hexagonal membrane-bound lattice is essential for budding because NEC mutants designed to perturb lattice interfaces reduce its budding ability. Previously, we identified an NEC suppressor mutation capable of restoring budding to a mutant with a weakened hexagonal lattice. Using an established in-vitro budding assay and HSV-1 infected cell experiments, we show that the suppressor mutation can restore budding to a broad range of budding-deficient NEC mutants thereby acting as a universal suppressor. Cryogenic electron tomography of the suppressor NEC mutant lattice revealed a hexagonal lattice reminiscent of wild-type NEC lattice instead of an alternative lattice. Further investigation using x-ray crystallography showed that the suppressor mutation promoted the formation of new contacts between the NEC hexamers that, ostensibly, stabilized the hexagonal lattice. This stabilization strategy is powerful enough to override the otherwise deleterious effects of mutations that destabilize the NEC lattice by different mechanisms, resulting in a functional NEC hexagonal lattice and restoration of membrane budding.

α-Tocopherol reduces VLDL secretion through modulation of intracellular ER-to-Golgi transport of VLDL.

Avoiding hepatic steatosis is crucial for preventing liver dysfunction, and one mechanism by which this is accomplished is through synchronization of the rate of very low density lipoprotein (VLDL) synthesis with its secretion. Endoplasmic reticulum (ER)-to-Golgi transport of nascent VLDL is the rate-limiting step in its secretion and is mediated by the VLDL transport vesicle (VTV). Recent in vivo studies have indicated that α-tocopherol (α-T) supplementation can reverse steatosis in nonalcoholic fatty liver disease, but its effects on hepatic lipoprotein metabolism are poorly understood. Here, we investigated the impact of α-T on hepatic VLDL synthesis, secretion, and intracellular ER-to-Golgi VLDL trafficking using an in vitro model. Pulse-chase assays using [3H]-oleic acid and 100 µmol/L α-T demonstrated a disruption of early VLDL synthesis, resulting in enhanced apolipoprotein B-100 expression, decreased expression in markers for VTV budding, ER-to-Golgi VLDL transport, and reduced VLDL secretion. Additionally, an in vitro VTV budding assay indicated a significant decrease in VTV production and VTV-Golgi fusion. Confocal imaging of lipid droplet (LD) localization revealed a decrease in overall LD retention, diminished presence of ER-associated LDs, and an increase in Golgi-level LD retention. We conclude that α-T disrupts ER-to-Golgi VLDL transport by modulating the expression of specific proteins and thus reduces VLDL secretion.

Phosphatidylserine clustering by the Ebola virus matrix protein is a critical step in viral budding

Phosphatidylserine (PS) is a critical lipid factor in the assembly and spread of numerous lipid‐enveloped viruses. Here, we describe the ability of the Ebola virus (EBOV) matrix protein eVP40 to induce clustering of PS and promote viral budding in vitro, as well as the ability of an FDA‐approved drug, fendiline, to reduce PS clustering and subsequent virus budding and entry. To gain mechanistic insight into fendiline inhibition of EBOV replication, multiple in vitro assays were run including imaging, viral budding and viral entry assays. Fendiline lowers PS content in mammalian cells and PS in the plasma membrane, where the ability of VP40 to form new virus particles is greatly lower. Further, particles that form from fendiline‐treated cells have altered particle morphology and cannot significantly infect/enter cells. These complementary studies reveal the mechanism by which EBOV matrix protein clusters PS to enhance viral assembly, budding, and spread from the host cell while also laying the groundwork for fundamental drug targeting strategies.

Host Regulation of Ebola Virus Egress and Spread: Role of Cytoskeletal Filamin Proteins

Ebola virus (EBOV) causes severe hemorrhagic fever associated with high mortality rates in humans. EBOV and other filoviruses are emerging BSL‐4 pathogens for which the development of novel and effective therapeutics is urgently needed. Egress and spread of EBOV is mediated by the VP40 matrix protein (eVP40), whereby expression of eVP40 alone directs the production and egress of virus‐like particles (VLPs) that accurately mimics the budding process of live infectious virus. We have shown that eVP40 hijacks or recruits select host proteins to facilitate the budding process. On the other hand, we have also identified several host proteins that interact with eVP40 to negatively regulated eVP40‐mediated budding. Thus, a better understanding of the eVP40‐host interactome will provide important insights into the mechanisms of virus egress and spread, as well as inform about potential targets for the development of novel antiviral therapeutics. Here, we investigated the possible role for host filamin A and B proteins (actin crosslinking proteins that function in cell morphology and migration at the plasma membrane) in regulating both entry and egress of EBOV. Toward this end, we employed filamin A (FLNaKD) and filamin B knockdown cells (FLNbKD) to assess EBOV entry by using live EBOV, as well as recombinant viruses and/or pseudotypes expressing the EBOV surface glycoprotein (GP). We also used the KD cells to assess virus egress by using our well‐established eVP40 VLP budding assay. In sum, our results indicate that expression of host filamin proteins appears to be important for EBOV to efficiently complete different stages of its lifecycle.

Red Light and Glucose Enhance Cytokinin-Mediated Bud Initial Formation in Physcomitrium patens.

Growth and development of Physcomitrium patens is endogenously regulated by phytohormones such as auxin and cytokinin. Auxin induces the transition of chloronema to caulonema. This transition is also regulated by additional factors such as quantity and quality of light, carbon supply, and other phytohormones such as strigolactones and precursors of gibberrelic acid. On the other hand, cytokinins induce the formation of bud initials following caulonema differentiation. However, the influence of external factors such as light or nutrient supply on cytokinin-mediated bud initial formation has not been demonstrated in Physcomitrium patens. This study deals with the effect of light quality and nutrient supply on cytokinin-mediated bud initial formation. Bud initial formation has been observed in wild type plants in different light conditions such as white, red, and blue light in response to exogenously supplied cytokinin as well as glucose. In addition, budding assay has been demonstrated in the cry1a mutant of Physcomitrium in different light conditions. The results indicate that carbon supply and red light enhance the cytokinin response, while blue light inhibits this process in Physcomitrium.

Exosomal EPHA2 derived from highly metastatic breast cancer cells promotes angiogenesis by activating the AMPK signaling pathway through Ephrin A1-EPHA2 forward signaling.

Rationale: Angiogenesis is a fundamental process of tumorigenesis, growth, invasion and metastatic spread. Extracellular vesicles, especially exosomes, released by primary tumors promote angiogenesis and cancer progression. However, the mechanism underlying the pro-angiogenic potency of cancer cell-derived exosomes remains poorly understood.Methods: Exosomes were isolated from breast cancer cells with high metastatic potential (HM) and low metastatic potential (LM). The pro-angiogenic effects of these exosomes were evaluated by in vitro tube formation assays, wound healing assays, rat arterial ring budding assays and in vivo Matrigel plug assays. Subsequently, RNA sequencing, shRNA-mediated gene knockdown, overexpression of different EPHA2 mutants, and small-molecule inhibitors were used to analyze the angiogenesis-promoting effect of exosomal EPHA2 and its potential downstream mechanism. Finally, xenograft tumor models were established using tumor cells expressing different levels of EPHA2 to mimic the secretion of exosomes by tumor cells in vivo, and the metastasis of cancer cells were monitored using the IVIS Spectrum imaging system and Computed Tomography.Results: Herein, we demonstrated that exosomes produced by HM breast cancer cells can promote angiogenesis and metastasis. EPHA2 was rich in HM-derived exosomes and conferred the pro-angiogenic effect. Exosomal EPHA2 can be transferred from HM breast cancer cells to endothelial cells. Moreover, it can stimulate the migration and tube-forming abilities of endothelial cells in vitro and promote angiogenesis and tumor metastasis in vivo. Mechanistically, exosomal EPHA2 activates the AMPK signaling via the ligand Ephrin A1-dependent canonical forward signaling pathway. Moreover, inhibition of the AMPK signaling impairs exosomal EPHA2-mediated pro-angiogenic effects.Conclusion: Our findings identify a novel mechanism of exosomal EPHA2-mediated intercellular communication from breast cancer cells to endothelial cells in the tumor microenvironment to provoke angiogenesis and metastasis. Targeting the exosomal EPHA2-AMPK signaling may serve as a potential strategy for breast cancer therapy.

SEC24 isoform specificity regulates the assembly of γ‐secretase from dimeric subcomplexes

AbstractBackgroundγ‐Secretase is a diaspartyl protease with presenilin (PSEN) as the catalytic subunit. It targets a plethora of type I transmembrane proteins and, as such, affects a broad range of physiological processes. Because of this, aberrant functioning of γ‐secretase is linked to several diseases including cancer and Alzheimer’s disease (AD). Herein, mutations in the PSEN genes are associated with rare cases of familial AD and shift the production to longer, more aggregation prone amyloid β peptides. γ‐Secretase consists, besides the catalytic component PSEN, of nicastrin, APH1 and PEN‐2 that are co‐translationally translocated in the endoplasmic reticulum (ER). As γ‐secretase has a stoichiometry of 1:1:1:1, the assembly process is spatially and temporally regulated. Although it is generally agreed that this occurs in early biosynthetic compartments, it remains far from understood whether full assembly occurs already in the ER and to what extent Golgi‐to‐ER recycling is involved.MethodWe used advanced cell fractionation protocols and an in vitro reconstituted ER budding assay to explore in detail the stepwise assembly process of γ‐secretase in early biosynthetic compartments.ResultCell fractionation combined with blue native electrophoresis shows that subcomplexes are mainly present in ER‐enriched fractions while full complexes appear in the ER‐Golgi intermediate compartment, indicating that final assembly occurs beyond the ER. In vitro reconstitution of ER export reveals that none of the γ‐secretase subunits is required for ER‐exit of the others. However, knock‐out of any subunit induces the accumulation of preceding subcomplexes in de novo formed COPII vesicles. Mutating a motif in the cytosolic loop domain of PSEN1, which is lost in the familial AD‐associated PSEN1∆E9 mutant, abrogates ER‐exit of PSEN1 as well as PEN‐2, but not of nicastrin. This is explained molecularly by the selectivity of Sec24A/B and Sec24C/D in packaging PSEN1 and nicastrin, respectively, and further argues against full complex assembly prior to ER‐exit.ConclusionIn conclusion, our data support a model wherein individual subunits and dimeric subcomplexes preferentially exit the ER. Full assembly likely occurs in intermediate compartment/cis‐Golgi allowing complexes to escape active Golgi‐to‐ER recycling as part of a secondary quality control mechanism in controlling cellular γ‐secretase levels and activity.

Proteomic Profiling of Mitochondrial-Derived Vesicles in Brain Reveals Enrichment of Respiratory Complex Sub-assemblies and Small TIM Chaperones.

The generation of mitochondrial-derived vesicles (MDVs) is implicated in a plethora of vital cell functions, from mitochondrial quality control to peroxisomal biogenesis. The discovery of distinct subtypes of MDVs has revealed the selective inclusion of mitochondrial cargo in response to varying stimuli. However, the true scope and variety of MDVs is currently unclear, and unbiased approaches have yet to be used to understand their biology. Furthermore, as mitochondrial dysfunction has been implicated in many neurodegenerative diseases, it is essential to understand MDV pathways in the nervous system. To address this, we sought to identify the cargo in brain MDVs. We used an in vitro budding assay and proteomic approach to identify proteins selectively enriched in MDVs. 72 proteins were identified as MDV-enriched, of which 31% were OXPHOS proteins. Interestingly, the OXPHOS proteins localized to specific modules of the respiratory complexes, hinting at the inclusion of sub-assemblies in MDVs. Small TIM chaperones were also highly enriched in MDVs, linking mitochondrial chaperone-mediated protein transport to MDV formation. As the two Parkinson's disease genes PINK1 and Parkin have been previously implicated in MDV biogenesis in response to oxidative stress, we compared the MDV proteomes from the brains of wild-type mice with those of PINK1-/- and Parkin-/- mice. No significant difference was found, suggesting that PINK1- and Parkin-dependent MDVs make up a small proportion of all MDVs in the brain. Our findings demonstrate a previously uncovered landscape of MDV complexity and provide a foundation from which further novel MDV functions can be discovered. Data are available via ProteomeXchange with identifier PXD020197.

Amino Acid Deletions in p6Gag Domain of HIV-1 CRF07_BC Ameliorate Galectin-3 Mediated Enhancement in Viral Budding.

HIV-1 CRF07_BC is a recombinant virus with amino acid (a.a.) deletions in p6Gag, which are overlapped with the Alix-binding domain. Galectin-3 (Gal3), a β-galactose binding lectin, has been reported to interact with Alix and regulate HIV-1 subtype B budding. This study aims to evaluate the role of Gal3 in HIV-1 CRF07_BC infection and the potential effect of a.a. deletions on Gal3-mediated regulation. A total of 38 HIV-1+ injecting drug users (IDUs) were enrolled in the study. Viral characterization and correlation of Gal3 were validated. CRF07_BC containing 7 a.a. deletions and wild-type in the p6Gag (CRF07_BC-7d and -wt) were isolated and infectious clones were generated. Viral growth kinetic and budding assays using Jurkat-CCR5/Jurkat-CCR5-Gal3 cells infected with CRF07_BC were performed. Results indicate that 69.4% (25/38) of the recruited patients were identified as CRF07_BC, and CRF07_BC-7d was predominant. Slow disease progression and significantly higher plasma Gal3 were noted in CRF07_BC patients (p < 0.01). Results revealed that CRF07_BC infection resulted in Gal3 expression, which was induced by Tat. Growth dynamic and budding assays indicated that Gal3 expression in Jurkat-CCR5 cells significantly enhanced CRF07_BC-wt replication and budding (p < 0.05), while the promoting effect was ameliorated in CRF07_BC-7d. Co-immunoprecipitation found that deletions in the p6Gag reduced Gal-3-mediated enhancement of the Alix–Gag interaction.

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 IC50of 6.17 ± 0.24 μMby 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 IC50of ∼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.

Both Svp26 and Mnn6 are required for the efficient ER exit of Mnn4 in Saccharomyces cerevisiae.

Incorporation of membrane and secretory proteins into COPII vesicles are facilitated either by the direct interaction of cargo proteins with COPII coat proteins, or by ER exit adaptor proteins which mediate the interaction of cargo proteins with COPII coat proteins. Svp26 is one of the ER exit adaptor proteins in the yeast Saccharomyces cerevisiae. The ER exit of several type II membrane proteins have been reported to be facilitated by Svp26. We demonstrate here that the efficient incorporation of Mnn4, a type II membrane protein required for mannosyl phosphate transfer to glycoprotein-linked oligosaccharides, into COPII vesicles is also dependent on the function of Svp26. We show that Mnn4 is localized to the Golgi. In addition to Mnn4, Mnn6 is known to be also required for the transfer of mannosyl phosphate to the glycans. We show, by indirect immunofluorescence, that Mnn6 localizes to the ER. As in the case with Svp26, deletion of the MNN6 gene results in the accumulation of Mnn4 in ER. In vitro COPII vesicle budding assays show that Svp26 and Mnn6 facilitate the incorporation of Mnn4 into COPII vesicles. In contrast to Svp26, which is itself efficiently captured into the COPII vesicles, Mnn6 was not incorporated into the COPII vesicles. Mnn4 and Mnn6 have the DXD motif which is often found in the many glycosyltransferases and functions to coordinate a divalent cation essential for the reaction. Alcian blue dye binding assay shows that substitution of the first D in this motif present in Mnn4 by A impairs the Mnn4 function. In contrast, amino acid substitutions in DXD motifs present in Mnn6 did not affect the function of Mnn6. These results suggest that Mnn4 may be directly involved in the mannosyl phosphate transfer reaction.

Host Protein BAG3 is a Negative Regulator of Lassa VLP Egress.

Lassa fever virus (LFV) belongs to the Arenaviridae family and can cause acute hemorrhagic fever in humans. The LFV Z protein plays a central role in virion assembly and egress, such that independent expression of LFV Z leads to the production of virus-like particles (VLPs) that mimic egress of infectious virus. LFV Z contains both PTAP and PPPY L-domain motifs that are known to recruit host proteins that are important for mediating efficient virus egress and spread. The viral PPPY motif is known to interact with specific host WW-domain bearing proteins. Here we identified host WW-domain bearing protein BCL2 Associated Athanogene 3 (BAG3) as a LFV Z PPPY interactor using our proline-rich reading array of WW-domain containing mammalian proteins. BAG3 is a stress-induced molecular co-chaperone that functions to regulate cellular protein homeostasis and cell survival via Chaperone-Assisted Selective Autophagy (CASA). Similar to our previously published findings for the VP40 proteins of Ebola and Marburg viruses, our results using VLP budding assays, BAG3 knockout cells, and confocal microscopy indicate that BAG3 is a WW-domain interactor that negatively regulates egress of LFV Z VLPs, rather than promoting VLP release. Our results suggest that CASA and specifically BAG3 may represent a novel host defense mechanism, whereby BAG3 may dampen egress of several hemorrhagic fever viruses by interacting and interfering with the budding function of viral PPxY-containing matrix proteins.

Lysophospholipids Facilitate COPII Vesicle Formation

SummaryCoat protein complex II (COPII) proteins form vesicles from the endoplasmic reticulum to export cargo molecules to the Golgi apparatus. Among the many proteins involved in this process, Sec12 is a key regulator, functioning as the guanosine diphosphate (GDP) exchange factor for Sar1p, the small guanosine triphosphatase (GTPase) that initiates COPII assembly. Here we show that overexpression of phospholipase B3 in the thermosensitive sec12-4 mutant partially restores growth and protein transport at non-permissive temperatures. Lipidomics analyses of these cells show a higher content of lysophosphatidylinositol (lysoPI), consistent with the lipid specificity of PLB3. Furthermore, we show that lysoPI is specifically enriched in COPII vesicles isolated from in vitro budding assays. As these results suggested that lysophospholipids could facilitate budding under conditions of defective COPII coat dynamics, we reconstituted COPII binding onto giant liposomes with purified proteins and showed that lysoPI decreases membrane rigidity and enhances COPII recruitment to liposomes. Our results support a mechanical facilitation of COPII budding by lysophospholipids.

Repurposing Fendiline as a novel anti‐viral therapeutic

The most prolonged and fatal outbreak of the Ebola virus (EBOV) occurred in 2014, claiming the lives of thousands. Although more than 40 years have passed since the initial discovery of EBOV, there are still no available treatments with FDA approval. Clinical case studies from human survivors highlighted that a hallmark of survival is a measurable immune response against specific viral proteins, such as VP40. VP40, is the canonical mediator of EBOV budding. Therefore, slowing down viral propagation through inhibiting replication, trafficking or budding could be the key to developing an efficacious treatment.VP40 interacts with host proteins and lipids to oligomerize at the plasma membrane (PM) to form the filamentous structure of the virus. The initial association between VP40 and the PM is through electrostatic and stereospecific interactions with phosphatidyl serine (PS). Following PM docking, VP40 filamentous oligomerization is also dependent upon the presence of PS at the PM. These filaments are the building block for nascent viral particles, which will ultimately bud from the cell to propagate the virus within the host. Therefore, PS within the inner leaflet of the PM is essential for viral budding and propagation. Moreover, VP40's ability to produce virus like particles (VLPs) when expressed in mammalian cells, even in the absence of the other viral proteins, allows EBOV budding to be studied in a BSL‐2 setting.Fendiline, an FDA approved drug for coronary heart disease, has been shown to significantly reduce PS from the PM. Therefore, this project focuses on repurposing Fendiline as a novel anti‐viral therapy to inhibit EBOV budding. We hypothesized that treating HEK 293 with Fendiline, would reduce VP40's ability to bind to the PM, oligomerize, and ultimately inhibit VLP production.To monitor Fendiline's effect on PS in HEK293 cells, live cell confocal imaging experiments were performed on HEK 293 cells expressing the high affinity PS probe, GFP‐LactC2. After 48 hours of treatment, 5 uM Fendiline significantly reduced GFP‐LactC2 localization at the PM by ~25% (p=0.03). Lipidomic analysis of HEK 293 cells also revealed a significant reduction in total cellular levels of PS after 48 hours of 5 uM treatment (~30%, p=0.03). To assess the effect of Fendiline on VP40 binding to the PM, HEK 293 cells expressing GFP‐VP40 were treated with Fendiline and monitored for 48 hours. Again, after 48 hours of 5 uM treatment, there was a significant reduction of GFP‐VP40 localization to the PM (~15%, p=0.0001). Functional budding assays were performed to assess the effect of Fendiline on VLP production. After 48 hours, 5 uM Fendiline reduced VLP production by ~60% (p=0.025). Scanning electron microscopy experiments revealed a significant reduction in filamentous particle length and density after 48 hours of 1 uM or 5 uM Fendiline treatment compared to untreated cells expressing FLAG‐VP40(p&lt;0.01).Our project has highlighted that repurposing drugs such as Fendiline and targeting lipid metabolism are promising avenues for developing anti‐viral therapies. The next stop for this project is to extend the studies into live virus models in a BSL‐4 setting.Support or Funding InformationThis work has been supported by NIH training grant T32GM075762 and NIH AI081077This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

ESCRT-III is required for scissioning new peroxisomes from the endoplasmic reticulum.

Dynamic control of peroxisome proliferation is integral to the peroxisome's many functions. The endoplasmic reticulum (ER) serves as a source of preperoxisomal vesicles (PPVs) that mature into peroxisomes during de novo peroxisome biogenesis and support growth and division of existing peroxisomes. However, the mechanism of PPV formation and release from the ER remains poorly understood. In this study, we show that endosomal sorting complexes required for transport (ESCRT)-III are required to release PPVs budding from the ER into the cytosol. Absence of ESCRT-III proteins impedes de novo peroxisome formation and results in an aberrant peroxisome population in vivo. Using a cell-free PPV budding assay, we show that ESCRT-III proteins Vps20 and Snf7 are necessary to release PPVs from the ER. ESCRT-III is therefore a positive effector of membrane scission for vesicles budding both away from and toward the cytosol. These findings have important implications for the evolutionary timing of emergence of peroxisomes and the rest of the internal membrane architecture of the eukaryotic cell.

Hemorrhagic Fever Virus Budding Studies.

Independent expression of the VP40 or Z matrix proteins of filoviruses (marburgviruses and ebolaviruses) and arenaviruses (Lassa fever and Junín), respectively, gives rise to the production and release of virus-like particles (VLPs) that are morphologically identical to infectious virions. We can detect and quantify VLP production and egress in mammalian cells by transient transfection, SDS-PAGE, Western blotting, and live cell imaging techniques such as total internal reflection fluorescence (TIRF) microscopy. Since the VLP budding assay accurately mimics budding of infectious virus, this BSL-2 assay is safe and useful for the interrogation of both viral and host determinants required for budding and can be used as an initial screen to identify and validate small molecule inhibitors of virus release and spread.

GTPase Sar1 regulates the trafficking and secretion of the virulence factor gp63 in Leishmania

Metalloprotease gp63 (Leishmania donovani gp63 (Ldgp63)) is a critical virulence factor secreted by Leishmania However, how newly synthesized Ldgp63 exits the endoplasmic reticulum (ER) and is secreted by this parasite is unknown. Here, we cloned, expressed, and characterized the GTPase LdSar1 and other COPII components like LdSec23, LdSec24, LdSec13, and LdSec31 from Leishmania to understand their role in ER exit of Ldgp63. Using dominant-positive (LdSar1:H74L) and dominant-negative (LdSar1:T34N) mutants of LdSar1, we found that GTP-bound LdSar1 specifically binds to LdSec23, which binds, in turn, with LdSec24(1-702) to form a prebudding complex. Moreover, LdSec13 specifically interacted with His6-LdSec31(1-603), and LdSec31 bound the prebudding complex via LdSec23. Interestingly, dileucine 594/595 and valine 597 residues present in the Ldgp63 C-terminal domain were critical for binding with LdSec24(703-966), and GFP-Ldgp63L594A/L595A or GFP-Ldgp63V597S mutants failed to exit from the ER. Moreover, Ldgp63-containing COPII vesicle budding from the ER was inhibited by LdSar1:T34N in an in vitro budding assay, indicating that GTP-bound LdSar1 is required for budding of Ldgp63-containing COPII vesicles. To directly demonstrate the function of LdSar1 in Ldgp63 trafficking, we coexpressed RFP-Ldgp63 along with LdSar1:WT-GFP or LdSar1:T34N-GFP and found that LdSar1:T34N overexpression blocks Ldgp63 trafficking and secretion in Leishmania Finally, we noted significantly compromised survival of LdSar1:T34N-GFP-overexpressing transgenic parasites in macrophages. Taken together, these results indicated that Ldgp63 interacts with the COPII complex via LdSec24 for Ldgp63 ER exit and subsequent secretion.

Interdomain salt-bridges in the Ebola virus protein VP40 and their role in domain association and plasma membrane localization.

The Ebola virus protein VP40 is a transformer protein that possesses an extraordinary ability to accomplish multiple functions by transforming into various oligomeric conformations. The disengagement of the C-terminal domain (CTD) from the N-terminal domain (NTD) is a crucial step in the conformational transformations of VP40 from the dimeric form to the hexameric form or octameric ring structure. Here, we use various molecular dynamics (MD) simulations to investigate the dynamics of the VP40 protein and the roles of interdomain interactions that are important for the domain-domain association and dissociation, and report on experimental results of the behavior of mutant variants of VP40. The MD studies find that various salt-bridge interactions modulate the VP40 domain dynamics by providing conformational specificity through interdomain interactions. The MD simulations reveal a novel salt-bridge between D45-K326 when the CTD participates in a latch-like interaction with the NTD. The D45-K326 salt-bridge interaction is proposed to help domain-domain association, whereas the E76-K291 interaction is important for stabilizing the closed-form structure. The effects of the removal of important VP40 salt-bridges on plasma membrane (PM) localization, VP40 oligomerization, and virus like particle (VLP) budding assays were investigated experimentally by live cell imaging using an EGFP-tagged VP40 system. It is found that the mutations K291E and D45K show enhanced PM localization but D45K significantly reduced VLP formation.

Quinoxaline-based inhibitors of Ebola and Marburg VP40 egress

We prepared a series of quinoxalin-2-mercapto-acetyl-urea analogs and evaluated them for their ability to inhibit viral egress in our Marburg and Ebola VP40 VLP budding assays in HEK293T cells. We also evaluated selected compounds in our bimolecular complementation assay (BiMC) to detect and visualize a Marburg mVP40–Nedd4 interaction in live mammalian cells. Antiviral activity was assessed for selected compounds using a live recombinant vesicular stomatitis virus (VSV) (M40 virus) that expresses the EBOV VP40 PPxY L-domain. Finally selected compounds were evaluated in several ADME assays to have an early assessment of their drug properties. Our compounds had low nM potency in these assays (e.g., compounds 21, 24, 26, 39), and had good human liver microsome stability, as well as little or no inhibition of P450 3A4.

Suppression of production of baboon endogenous virus by dominant negative mutants of cellular factors involved in multivesicular body sorting pathway

Baboon endogenous virus (BaEV) is an infectious endogenous gammaretrovirus isolated from a baboon placenta. BaEV-related sequences have been identified in both Old World monkeys and African apes, but not in humans or Asian apes. Recently, it was reported that BaEV-like particles were produced from Vero cells derived from African green monkeys by chemical induction, and thus BaEV-like particles may contaminate biological products manufactured using Vero cells. In this study, we constructed an infectious molecular clone of BaEV strain M7. We found two putative L-domain motifs, PPPY and PSAP, in the pp15 region of Gag. To examine the function of the L-domain motifs, we conducted virus budding assay using L-domain motif mutants. We revealed that the PPPY motif, but not the PSAP motif, plays a major role as the L-domain in BaEV budding. We also demonstrated that Vps4A/B are involved in BaEV budding. These data suggest that BaEV Gag recruits the cellular endosomal sorting complex required for transport (ESCRT) machinery through the interaction of the PPPY L-domain with cellular factors. These data will be useful for controlling contamination of BaEV-like particles in biological products in the future.