Search Results (20)

Background/Objectives: Pathogenic mammarenaviruses cause severe hemorrhagic and neurologic disease in humans. Machupo virus (MACV), a New World (NW) mammarenavirus, causes Bolivian hemorrhagic fever in humans, and there are no approved vaccines. Methods: Here, we describe and compare the immunogenicity of three vaccines expressing the MACV glycoprotein complex (GPC) in C57BL/6 mice: a recombinant vesicular stomatitis virus (rVSV) and two different lipid nanoparticle (LNP)-encapsulated nucleoside-modified mRNA (mRNA-LNP) vaccines. The first mRNA-LNP vaccine, designated MACV mRNA, expresses the full-length MACV GPC. The second mRNA-LNP vaccine, called MACV VLP mRNA, encodes MACV GPC with appended sequences that induce the budding of virus-like particles (VLPs) with MACV GPC on the surface. This is the first description of any mRNA-LNP vaccine for MACV and the first comparison of mRNA and rVSVs as vaccine candidates for MACV. Results: We find that two doses of either MACV mRNA or MACV VLP mRNA are required for the induction of robust humoral and cellular immune responses including total MACV GPC IgG, neutralizing antibodies, cross-reactive antibodies that bind the related Junín virus GPC, and MACV-specific T-cell responses. To further investigate vaccination strategies for MACV, we also evaluated a heterologous prime-boost regimen involving the MACV mRNA vaccine coupled with the rVSV-based MACV vaccine. We find that the highest levels of MACV GPC-specific IgG and neutralizing titers were achieved when heterologous mRNA and rVSV prime-boost regimens were employed. Conclusions: These results elucidate differences in the immune response to different vaccine platforms for MACV and can inform future vaccine development for NW arenaviruses. Full article

Severe acute respiratory syndrome coronavirus (SARS-CoV) assembles and buds from the Golgi apparatus or the ER membrane, but the specific membrane microdomains utilized during this process remain underexplored. Here, we show that co-expression of the SARS-CoV structural proteins S, M, and N in HEK-293T cells is sufficient to generate genome-free SARS-CoV-like virus-like particles (VLPs), which preferentially bud from glycolipid-enriched membrane lipid raft microdomains. Immunofluorescence microscopy using raft-selective dyes (DiIC16) and spike-specific antibodies revealed strong co-localization of VLPs with lipid rafts. Detergent-resistant membrane analysis and sucrose gradient centrifugation further confirmed the presence of S protein in buoyant, raft-associated fractions alongside the raft marker CD44. Importantly, pharmacological disruption of rafts with methyl-β-cyclodextrin reduced VLP budding and S protein partitioning into raft domains, underscoring the requirement for intact lipid rafts in assembly. Additionally, our data support lipid raft-associated proteins’ (e.g., FNRA, VIM, CD59, RHOA) roles in modulating cellular responses conducive to viral replication and assembly. These findings highlight lipid rafts as crucial platforms for SARS-CoV morphogenesis and suggest new avenues for vaccine and antiviral development using VLPs and raft-targeting therapeutics. Full article

Ebola virus (EBOV) causes severe hemorrhagic fevers in humans, and effective countermeasures remain limited. The EBOV-encoded major matrix protein VP40 is essential for viral assembly, budding, and particle release, making it a promising target for antiviral drug development. However, no approved drugs currently target the viral particle formation process. In this study, we established a simple and highly sensitive screening system to evaluate VP40-mediated virus-like particle (VLP) formation under biosafety level −2 conditions. The system uses the HiBiT luminescence-based reporter fused to VP40, allowing for the detection of VP40 release. Our results demonstrate that the HiBiT sequence fused at the N-terminus [HiBiT-VP40 (N)] retains VP40′s ability to form VLPs, supporting its use as a functional reporter. Furthermore, we validated the system by assessing the role of Rab11-dependent trafficking in VP40-mediated budding and by evaluating the effect of nocodazole, a microtubule depolymerizer, on VLP release. This novel screening system provides a convenient and reliable platform for screening potential inhibitors targeting the late stages of EBOV infection, including viral particle formation and release. Additionally, its potential adaptability to other filoviruses suggests wide applicability in the discovery and development of additional novel therapeutic agents. Full article

Background/Objectives: N-Myristoyltransferase inhibitors (NMTi) represent a novel antiviral strategy against mammarenaviruses such as Lassa and Junin viruses. The Z matrix protein inhibits viral ribonucleoprotein (vRNP) activity in a dose-dependent manner. Here, we investigated whether Z-mediated vRNP inhibition depends on Z myristoylation or oligomerization. Methods: We used HEK293T cells transfected with wild-type (WT) or G2A-mutated Z constructs in LCMV minigenome (MG) assays. Cells were treated with the NMTi IMP-1088 and the proteasome inhibitor MG132. Z protein expression, vRNP activity, and VLP production were analyzed by immunofluorescence, western blotting, and colocalization analyses. Results: IMP-1088 treatment led to proteasome-mediated degradation of Z, reducing its inhibition of vRNP activity, which was restored by MG132. The non-myristoylated Z G2A mutant retained vRNP inhibitory activity but showed impaired oligomerization and budding capacity. These findings demonstrate that Z-mediated vRNP inhibition is independent of myristoylation and oligomerization. Conclusions: Z myristoylation and oligomerization are not required for its inhibitory vRNP activity. Targeting Z myristoylation with NMTi impairs virus assembly and budding without affecting Z-mediated inhibition of vRNP activity, supporting the development of NMTi as a promising broad-spectrum antiviral strategy against mammarenaviruses. Full article

HIV-1 budding as well as many other cellular processes require the Endosomal Sorting Complex Required for Transport (ESCRT) machinery. Understanding the architecture of the native ESCRT-III complex at HIV-1 budding sites is limited due to spatial resolution and transient ESCRT-III recruitment. Here, we developed a drug-inducible transient HIV-1 budding inhibitory tool to enhance the ESCRT-III lifetime at budding sites. We generated autocleavable CHMP2A, CHMP3, and CHMP4B fusion proteins with the hepatitis C virus NS3 protease. We characterized the CHMP-NS3 fusion proteins in the absence and presence of protease inhibitor Glecaprevir with regard to expression, stability, localization, and HIV-1 Gag VLP budding. Immunoblotting experiments revealed rapid and stable accumulation of CHMP-NS3 fusion proteins. Notably, upon drug administration, CHMP2A-NS3 and CHMP4B-NS3 fusion proteins substantially decrease VLP release while CHMP3-NS3 exerted no effect but synergized with CHMP2A-NS3. Localization studies demonstrated the relocalization of CHMP-NS3 fusion proteins to the plasma membrane, endosomes, and Gag VLP budding sites. Through the combined use of transmission electron microscopy and video-microscopy, we unveiled drug-dependent accumulation of CHMP2A-NS3 and CHMP4B-NS3, causing a delay in HIV-1 Gag-VLP release. Our findings provide novel insight into the functional consequences of inhibiting ESCRT-III during HIV-1 budding and establish new tools to decipher the role of ESCRT-III at HIV-1 budding sites and other ESCRT-catalyzed cellular processes. Full article

Human T-cell tropic virus type 1 (HTLV-1) is known to be mainly transmitted by cell-to-cell contact due to the lower infectivity of the cell-free virion. However, the reasons why cell-free HTLV-1 infection is poor remain unknown. In this study, we found that the retrovirus pseudotyped with HTLV-1 viral envelope glycoprotein (Env) was infectious when human immunodeficiency virus type 1 (HIV-1) was used to produce the virus. We found that the incorporation of HTLV-1 Env into virus-like particles (VLPs) was low when HTLV-1 Gag was used to produce VLPs, whereas VLPs produced using HIV-1 Gag efficiently incorporated HTLV-1 Env. The production of VLPs using Gag chimeras between HTLV-1 and HIV-1 Gag and deletion mutants of HIV-1 Gag showed that the p6 domain of HIV-1 Gag was responsible for the efficient incorporation of HTLV-1 Env into the VLPs. Further mutagenic analyses of the p6 domain of HIV-1 Gag revealed that the PTAP motif in the p6 domain of HIV-1 Gag facilitates the incorporation of HTLV-1 Env into VLPs. Since the PTAP motif is known to interact with tumor susceptibility gene 101 (TSG101) during the budding process, we evaluated the effect of TSG101 knockdown on the incorporation of HTLV-1 Env into VLPs. We found that TSG101 knockdown suppressed the incorporation of HTLV-1 Env into VLPs and decreased the infectivity of cell-free HIV-1 pseudotyped with HTLV-1 Env. Our results suggest that the interaction of TSG101 with the PTAP motif of the retroviral L domain is involved not only in the budding process but also in the efficient incorporation of HTLV-1 Env into the cell-free virus. Full article

The manufacture and downstream processing of virus-like particles (VLPs) using the baculovirus expression vector system (BEVS) is complicated by the presence of large concentrations of baculovirus particles, which are similar in size and density to VLPs, and consequently are difficult to separate. To reduce the burden of downstream processing, CRISPR-Cas9 technology was used to introduce insertion-deletion (indel) mutations within the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) gp64 open reading frame, which encodes the major envelope protein of AcMNPV. After comfirming the site-specific targeting of gp64 leading to reduced budded virus (BV) release, the gag gene of human immunodeficiency virus type 1 was expressed to produce Gag VLPs. This approach was effective for producing VLPs using the BEVS whilst simultaneously obstructing BV release. Full article

Ebolavirus (EBOV) is a negative-sense RNA virus that causes severe hemorrhagic fever in humans. The matrix protein VP40 facilitates viral budding by binding to lipids in the host cell plasma membrane and driving the formation of filamentous, pleomorphic virus particles. The C-terminal domain of VP40 contains two highly-conserved cysteine residues at positions 311 and 314, but their role in the viral life cycle is unknown. We therefore investigated the properties of VP40 mutants in which the conserved cysteine residues were replaced with alanine. The C311A mutation significantly increased the affinity of VP40 for membranes containing phosphatidylserine (PS), resulting in the assembly of longer virus-like particles (VLPs) compared to wild-type VP40. The C314A mutation also increased the affinity of VP40 for membranes containing PS, albeit to a lesser degree than C311A. The double mutant behaved in a similar manner to the individual mutants. Computer modeling revealed that both cysteine residues restrain a loop segment containing lysine residues that interact with the plasma membrane, but Cys³¹¹ has the dominant role. Accordingly, the C311A mutation increases the flexibility of this membrane-binding loop, changes the profile of hydrogen bonding within VP40 and therefore binds to PS with greater affinity. This is the first evidence that mutations in VP40 can increase its affinity for biological membranes and modify the length of Ebola VLPs. The Cys³¹¹ and Cys³¹⁴ residues therefore play an important role in dynamic interactions at the plasma membrane by modulating the ability of VP40 to bind PS. Full article

Foot and mouth disease is a livestock acute disease, causing economic losses in affected areas. Currently, control of this disease is performed by mandatory vaccination campaigns using inactivated viral vaccines. In this work, we describe the development of a chimeric VLP-based vaccine candidate for foot-and-mouth disease virus (FMDV), based on the co-expression of the HIV-1 Gag protein and a novel fusion rabies glycoprotein (RVG), which carries in its N-term the FMDV main antigen: the G-H loop. It is demonstrated by confocal microscopy that both Gag-GFP polyprotein and the G-H loop colocalize at the cell membrane and, that the Gag polyprotein of the HIV virus acts as a scaffold for enveloped VLPs that during the budding process acquires the proteins that are being expressed in the cell membrane. The obtained VLPs were spherical particles of 130 ± 40 nm in diameter (analyzed by TEM, Cryo-TEM and NTA) carrying an envelope membrane that efficiently display the GH-RVG on its surface (analyzed by gold immunolabeling). Immunostainings with a FMDV hyperimmune serum showed that the heterologous antigenic site, genetically fused to RVG, is recognized by specific G-H loop antibodies. Additionally, the cVLPs produced expose the G-H loop to the liquid surrounding (analyzed by specific ELISA). Finally, we confirmed that these FMD cVLPs are able to induce a specific humoral immune response, based on antibodies directed to the G-H loop in experimental animals. Full article

Filoviruses Ebola (EBOV) and Marburg (MARV) are devastating high-priority pathogens capable of causing explosive outbreaks with high human mortality rates. The matrix proteins of EBOV and MARV, as well as eVP40 and mVP40, respectively, are the key viral proteins that drive virus assembly and egress and can bud independently from cells in the form of virus-like particles (VLPs). The matrix proteins utilize proline-rich Late (L) domain motifs (e.g., PPxY) to hijack specific host proteins that contain WW domains, such as the HECT family E3 ligases, to facilitate the last step of virus–cell separation. We identified E3 ubiquitin ligase Smad Ubiquitin Regulatory Factor 2 (SMURF2) as a novel interactor with VP40 that positively regulates VP40 VLP release. Our results show that eVP40 and mVP40 interact with the three WW domains of SMURF2 via their PPxY motifs. We provide evidence that the eVP40–SMURF2 interaction is functional as the expression of SMURF2 positively regulates VLP egress, while siRNA knockdown of endogenous SMURF2 decreases VLP budding compared to controls. In sum, our identification of novel interactor SMURF2 adds to the growing list of identified host proteins that can regulate PPxY-mediated egress of VP40 VLPs. A more comprehensive understanding of the modular interplay between filovirus VP40 and host proteins may lead to the development of new therapies to combat these deadly infections. Full article

Most filoviruses cause severe disease in humans. For example, Ebola virus (EBOV) is responsible for the two most extensive outbreaks of filovirus disease to date, with case fatality rates of 66% and 40%, respectively. In contrast, Reston virus (RESTV) is apparently apathogenic in humans, and while transmission of RESTV from domestic pigs to people results in seroconversion, no signs of disease have been reported in such cases. The determinants leading to these differences in pathogenicity are not well understood, but such information is needed in order to better evaluate the risks posed by the repeated spillover of RESTV into the human population and to perform risk assessments for newly emerging filoviruses with unknown pathogenic potential. Interestingly, RESTV and EBOV already show marked differences in their growth in vitro, with RESTV growing slower and reaching lower end titers. In order to understand the basis for this in vitro attenuation of RESTV, we used various life cycle modeling systems mimicking different aspects of the virus life cycle. Our results showed that viral RNA synthesis was markedly slower when using the ribonucleoprotein (RNP) components from RESTV, rather than those for EBOV. In contrast, the kinetics of budding and entry were indistinguishable between these two viruses. These data contribute to our understanding of the molecular basis for filovirus pathogenicity by showing that it is primarily differences in the robustness of RNA synthesis by the viral RNP complex that are responsible for the impaired growth of RESTV in tissue culture. Full article

Ebola virus (EBOV) interacts with cells using multiple categories of cell-surface receptors, including C-type lectins and phosphatidylserine (PS) receptors. PS receptors typically bind to apoptotic cell membrane PS and orchestrate the uptake and clearance of apoptotic bodies. Many viruses coated with PS-containing lipid envelopes, acquired during budding from host cells, can also exploit these receptors for internalization. PS is restricted to the inner leaflet of the plasma membrane in homeostatic cells, an orientation that would be unfavorable for PS receptor-mediated uptake if conserved on the viral envelope. Therefore, it is theorized that viral infection induces host-cell PS externalization to the outer leaflet during replication. Cells have several membrane scramblase enzymes that enrich outer leaflet PS when activated. Here, we investigate the role of two scramblases, TMEM16F and XKR8, as possible mediators of cellular and viral envelope surface PS levels during recombinant vesicular stomatitis virus (VSV) in which the VSV glycoprotein was replaced with the Ebola glycoprotein (rVSV/EBOV-GP) replication and EBOV virus-like particle (VLP) production. We find that rVSV/EBOV-GP and EBOV VLPs produced in XKR8 knockout cells contain two- to threefold less PS in their outer leaflets. Consequently, rVSV/EBOV-GP produced in deltaXKR8 is 70% less efficient at infecting cells through apoptotic mimicry as compared to the viruses produced by parental cells. In addition, the budding efficiency of both recombinant VSV particles and VLPs was significantly reduced in cells lacking XKR8. Our data suggest that virion surface PS acquisition requires XKR8 activity, whereas the deletion of TMEM16F did not affect EBOV-GP-mediated entry of VLP production. Unexpectedly, we observed an additional role of XKR8 in rVSV/G, rVSV/EBOV-GP, and EBOV VLP budding. Full article

For highly pathogenic viruses, reporter assays that can be rapidly performed are critically needed to identify potentially functional mutations for further study under maximal containment (e.g., biosafety level 4 [BSL-4]). The Ebola virus nucleoprotein (NP) plays multiple essential roles during the viral life cycle, yet few tools exist to study the protein under BSL-2 or equivalent containment. Therefore, we adapted reporter assays to measure NP oligomerization and virion-like particle (VLP) production in live cells and further measured transcription and replication using established minigenome assays. As a proof-of-concept, we examined the NP-R111C substitution, which emerged during the 2013–2016 Western African Ebola virus disease epidemic and rose to high frequency. NP-R111C slightly increased NP oligomerization and VLP budding but slightly decreased transcription and replication. By contrast, a synthetic charge-reversal mutant, NP-R111E, greatly increased oligomerization but abrogated transcription and replication. These results are intriguing in light of recent structures of NP oligomers, which reveal that the neighboring residue, K110, forms a salt bridge with E349 on adjacent NP molecules. By developing and utilizing multiple reporter assays, we find that the NP-111 position mediates a complex interplay between NP’s roles in protein structure, virion budding, and transcription and replication. Full article

A prophylactic vaccine eliciting both broad neutralizing antibodies (bNAbs) to the HIV-1 envelope glycoprotein (Env) and strong T cell responses would be optimal for preventing HIV-1 transmissions. Replication incompetent HIV-1 virus-like particles (VLPs) offer the opportunity to present authentic-structured, virion-associated Env to elicit bNAbs, and also stimulate T cell responses. Here, we optimize our DNA vaccine plasmids as VLP expression vectors for efficient Env incorporation and budding. The original vector that was used in human trials inefficiently produced VLPs, but maximized safety by inactivating RNA genome packaging, enzyme functions that are required for integration into the host genome, and deleting accessory proteins Vif, Vpr, and Nef. These original DNA vaccine vectors generated VLPs with incomplete protease-mediated cleavage of Gag and were irregularly sized. Mutations to restore function within the defective genes revealed that several of the reverse transcriptase (RT) deletions mediated this immature phenotype. Here, we made efficient budding, protease-processed, and mature-form VLPs that resembled infectious virions by introducing alternative mutations that completely removed the RT domain, but preserved most other safety mutations. These VLPs, either expressed from DNA vectors in vivo or purified after expression in vitro, are potentially useful immunogens that can be used to elicit antibody responses that target Env on fully infectious HIV-1 virions. Full article

Human epidermal growth factor receptor-2 (HER2) is upregulated in 20% to 30% of breast cancers and is a marker of a poor outcome. Due to the development of resistance to passive immunotherapy with Trastuzumab, active anti-HER2 vaccination strategies that could potentially trigger durable tumor-specific immune responses have become an attractive research area. Recently, we have shown that budded virus-like particles (VLPs) produced in Sf9 insect cells are an ideal platform for the expression of complex membrane proteins. To assess the efficacy of antigen-displaying VLPs as active cancer vaccines, BALB/c mice were immunized with insect cell glycosylated and mammalian-like glycosylated HER2-displaying VLPs in combination with two different adjuvants and were challenged with HER2-positive tumors. Higher HER2-specific antibody titers and effector functions were induced in mice vaccinated with insect cell glycosylated HER2 VLPs compared to mammalian-like glycosylated counterparts. Moreover, insect cell glycosylated HER2 VLPs elicited a protective effect in mice grafted with HER2-positive mammary carcinoma cells. Interestingly, no protection was observed in mice that were adjuvanted with Poly (I:C). Here, we show that antigen-displaying VLPs produced in Sf9 insect cells were able to induce robust and durable immune responses in vivo and have the potential to be utilized as active cancer vaccines. Full article