Influence of calcium/calmodulin on budding of Ebola VLPs: implications for the involvement of the Ras/Raf/MEK/ERK pathway

The VP40 matrix protein of Ebola virus is able to bud from mammalian cells as a virus-like particle (VLP). Interactions between L-domain motifs of VP40 and host proteins such as Tsg101 and Nedd4 serve to facilitate budding of VP40 VLPs. Since intracellular levels of calcium are known to influence localization and function of host proteins involved in virus budding, we sought to determine, whether alterations of calcium or calmodulin levels in cells would affect budding of VP40 VLPs. VP40 VLP release was assessed in cells treated with BAPTA/AM, a calcium ion chelator, or with ionomycin, a calcium ionophore. In addition, VLP budding was assessed in cells treated with W7, W13, or TFP; all calmodulin antagonists. Results from these experiments indicated that: (i) budding of VP40 VLPs was reduced in a dose-dependent manner in the presence of BAPTA/AM, and slightly enhanced in the presence of ionomycin, (ii) VP40 VLP budding was reduced in a dose-dependent manner in the presence of W7, whereas VP40 VLP budding was unaffected in the presence of cyclosporine-A, (iii) budding of VSV-WT and a VSV recombinant (M40 virus) possessing the L-domains of Ebola VP40 was inhibited in the presence of W7, W13, or TFP, (iv) inhibition of virus budding by W7, W13, and TFP appears to be L-domain independent, and (v) the mechanism of calcium/calmodulin-mediated inhibition of Ebola VLP budding may involve the Ras/Raf/MEK/ERK signaling pathway.     

Ebola virus-like particles produced in insect cells exhibit dendritic cell stimulating activity and induce neutralizing antibodies

Recombinant baculoviruses (rBV) expressing Ebola virus VP40 (rBV-VP40) or GP (rBV-GP) proteins were generated. Infection of Sf9 insect cells by rBV-VP40 led to assembly and budding of filamentous particles from the cell surface as shown by electron microscopy. Ebola virus-like particles (VLPs) were produced by coinfection of Sf9 cells with rBV-VP40 and rBV-GP, and incorporation of Ebola GP into VLPs was demonstrated by SDS-PAGE and Western blot analysis. Recombinant baculovirus infection of insect cells yielded high levels of VLPs, which were shown to stimulate cytokine secretion from human dendritic cells similar to VLPs produced in mammalian cells. The immunogenicity of Ebola VLPs produced in insect cells was evaluated by immunization of mice. Analysis of antibody responses showed that most of the GP-specific antibodies were of the IgG2a subtype, while no significant level of IgG1 subtype antibodies specific for GP was induced, indicating the induction of a Th1-biased immune response. Furthermore, sera from Ebola VLP immunized mice were able to block infection by Ebola GP pseudotyped HIV virus in a single round infection assay, indicating that a neutralizing antibody against the Ebola GP protein was induced. These results show that production of Ebola VLPs in insect cells using recombinant baculoviruses represents a promising approach for vaccine development against Ebola virus infection.     

Filovirus budding

Family Filoviridae, which includes Ebola virus (EBOV) and Marburg virus (MARV), is a growing threat to human and non-human primate populations in central Africa. Although many facets of the filovirus life cycle remain to be deciphered, a great deal has been learned in recent years. In particular, a clearer understanding of the roles played by viral, as well as cellular, proteins in the assembly and budding processes has been achieved. This review will discuss the current state of filovirus budding research, with especial emphasis placed on the viral matrix protein VP40 and its relationship with the cellular vesicular sorting pathway. Possible budding functions of the viral glycoprotein (GP), as well as the membrane-associated viral protein 24 (VP24), will also be described, and a model for filovirus budding will be proposed.     

Paramyxovirus Sendai virus-like particle formation by expression of multiple viral proteins and acceleration of its release by C protein

Envelope viruses maturate by macromolecule assembly and budding. To investigate these steps, we generated virus-like particles (VLPs) by co-expression of structural proteins of Sendai virus (SeV), a prototype of the family Paramyxoviridae. Simultaneous expression of matrix (M), nucleo- (N), fusion (F), and hemagglutinin-neuraminidase (HN) proteins resulted in the generation of VLPs that had morphology and density similar to those of authentic virus particles, although the efficiency of release from cells was significantly lower than that of the virus. By using this VLP formation as a model of virus budding, roles of individual proteins in budding were investigated. The M protein was a driving force of budding, and the F protein facilitated and the HN protein suppressed VLP release. Either of the glycoproteins, F or HN, as well as the N protein, significantly shifted density of VLPs to that of virus particles, suggesting that viral proteins bring about integrity of VLPs by protein-protein interactions. We further found that co-expression of a nonstructural protein, C, but not V, enhanced VLP release to a level comparable to that of virus particles, demonstrating that the C protein plays a role in virus budding.     

A facile quantitative assay for viral particle genesis reveals cooperativity in virion assembly and saturation of an antiviral protein

Conventional assays of viral particle assembly and release are time consuming and laborious. We have developed an enzymatic virus-like particle (VLP) genesis assay that rapid and quantitative and is also versatile and applicable to diverse viruses including HIV-1 and Ebola virus. Using this assay, which has a dynamic range of several orders of magnitude, we show that the efficiency of VLP assembly and release, i.e., the fraction of the expressed protein that is assembled into extracellular particles, is dependent on the absolute level of expression of either HIV-1 Gag or Ebola virus VP40. We also demonstrate that the activity of the antiviral factor tetherin is dependent on the level of HIV-1 Gag expression and the numbers of VLPs generated, and appears to become saturated as these parameters are increased.     

Development,characterization and use of monoclonal VP40-antibodies for the detection of Ebola virus

Ebola virus (EBOV) causes uncommon but dramatic outbreaks in remote regions of Africa, where diagnostic facilities are limited. In order to develop diagnostic tests, which can be handled and distributed easily, monoclonal antibodies (mAbs) to EBOV, species Zaire, were produced from mice immunized with inactivated viral particles. Nine stable hybridoma cell lines were obtained producing specific mAbs directed against the viral structural protein VP40. These mAbs were characterized by enzyme-linked immunosorbent, immunoblot and immunofluorescence assays. Subsequently, an antigen capture enzyme-linked immunosorbent assay was established, which detects VP40 of all known species of EBOV. This assay could detect viral material in spiked human serum that has been sodium dodecylsulfate-inactivated. The established enzyme-linked immunosorbent assay therefore has the ability to become a very useful tool for obtaining an accurate diagnosis in the field, limiting the risk of laboratory infections.     

Virus-like particles of calicivirus as epitope carriers

Summary.  The VP60 of rabbit haemorrhagic disease virus (RHDV), when expressed in baculovirus, self-assembles into virus-like particles (VLP) which are antigenically and immunogenically indistinguishable from native virions. When the N-terminal 30 amino acid residues of VP60 were deleted and substituted by a well characterized six residue epitope from bluetongue virus capsid protein VP7 (Btag), the fusion protein retained its ability to self-assemble into VLPs. However, the size of these particles was only 27 nm, compared to 40 nm of VLPs derived from native VP60. The antigenicity of both VP60 and the Btag was retained as evident from ELISA and Western blot analyses. When Btag was fused at the C-terminus of VP60 without deletion, the fusion proteins formed VLPs of 40 nm in size and also retained their antigenicity, but the Btag antigenicity appeared weak at this fusion site. Received December 9, 1998/Accepted March 19, 1999     

Filovirus-like particles as vaccines and discovery tools

Ebola and Marburg viruses are members of the family Filoviridae, which cause severe hemorrhagic fevers in humans. Filovirus outbreaks have been sporadic, with mortality rates currently ranging from 30 to 90%. Unfortunately, there is no efficacious human therapy or vaccine available to treat disease caused by either Ebola or Marburg virus infection. Expression of the filovirus matrix protein, VP40, is sufficient to drive spontaneous production and release of virus-like particles (VLPs) that resemble the distinctively filamentous infectious virions. The addition of other filovirus proteins, including virion proteins (VP)24, 30 and 35 and glycoprotein, increases the efficiency of VLP production and results in particles containing multiple filovirus antigens. Vaccination with Ebola or Marburg VLPs containing glycoprotein and VP40 completely protects rodents from lethal challenge with the homologous virus. These candidate vaccines are currently being tested for immunogenicity and efficacy in nonhuman primates. Furthermore, the Ebola and Marburg VLPs are being used as a surrogate model to further understand the filovirus life cycle, with the goal of developing rationally designed vaccines and therapeutics. Thus, in addition to their use as a vaccine, VLPs are currently being used as tools to learn lessons about filovirus pathogenesis, immunology, replication and assembly requirements.     

Recruitment of Alix/AIP1 to the plasma membrane by Sendai virus C protein facilitates budding of virus-like particles

Sendai virus (SeV) is unique in that one of the viral accessory proteins, C, enhances budding of virus-like particles (VLPs) formed by SeV matrix protein M by physically interacting with Alix/AIP1. C protein itself does not have the ability to form VLPs, while M protein provides viral budding force, like other enveloped viruses. Here we show that SeV C protein recruits Alix/AIP1 to the plasma membrane (PM) to facilitate VLP budding. SeV M-VLP budding is sensitive to overexpression of a dominant-negative (DN) form of VPS4A only in the presence of the C proteins, which is able to recruit Alix/AIP1 to the PM. Our results indicate that SeV M and C proteins play separate roles in the budding process: M protein drives budding and C protein enhances the efficiency of the utilization of cellular MVB sorting machinery for efficient VLP budding.     

Ebola and Marburg virus-like particles activate human myeloid dendritic cells

The filoviruses, Ebola (EBOV) and Marburg (MARV), are potential global health threats, which cause deadly hemorrhagic fevers. Although both EBOV and MARV logarithmically replicate in dendritic cells (DCs), these viruses do not elicit DC cytokine secretion and fail to activate and mature infected DCs. Here, we employed virus-like particles (VLPs) of EBOV and MARV to investigate whether these genome-free particles maintain similar immune evasive properties as authentic filoviruses. Confocal microscopy indicated that human myeloid-derived DCs readily took up VLPs. However, unlike EBOV and MARV, VLPs induced maturation of DCs including upregulation of costimulatory molecules (CD40, CD80, CD86), major histocompatibility complex (MHC) class I and II surface antigens, and the late DC maturation marker CD83. The chemokine receptors CCR5 and CCR7 were also modulated on VLP-stimulated DCs, indicating that DC could migrate following VLP exposure. Furthermore, VLPs also elicited DC secretion of the pro-inflammatory cytokines TNF-alpha, IL-8, IL-6, and MIP-1alpha. Most significantly, in stark contrast to DC treated with intact EBOV or MARV, DC stimulated with EBOV or MARV VLPs showed enhanced ability to support human T-cell proliferation in an allogenic mixed lymphocyte response (MLR). Thus, our findings suggest that unlike EBOV and MARV, VLPs are effective stimulators of DCs and have potential in enhancing innate and adaptive immune responses.     

Productive replication of Ebola virus is regulated by the c-Abl1 tyrosine kinase

Ebola virus causes a fulminant infection in humans resulting in diffuse bleeding, vascular instability, hypotensive shock, and often death. Because of its high mortality and ease of transmission from human to human, Ebola virus remains a biological threat for which effective preventive and therapeutic interventions are needed. An understanding of the mechanisms of Ebola virus pathogenesis is critical for developing antiviral therapeutics. Here, we report that productive replication of Ebola virus is modulated by the c-Abl1 tyrosine kinase. Release of Ebola virus-like particles (VLPs) in a cell culture cotransfection system was inhibited by c-Abl1-specific small interfering RNA (siRNA) or by Abl-specific kinase inhibitors and required tyrosine phosphorylation of the Ebola matrix protein VP40. Expression of c-Abl1 stimulated an increase in phosphorylation of tyrosine 13 (Y(13)) of VP40, and mutation of Y(13) to alanine decreased the release of Ebola VLPs. Productive replication of the highly pathogenic Ebola virus Zaire strain was inhibited by c-Abl1-specific siRNAs or by the Abl-family inhibitor nilotinib by up to four orders of magnitude. These data indicate that c-Abl1 regulates budding or release of filoviruses through a mechanism involving phosphorylation of VP40. This step of the virus life cycle therefore may represent a target for antiviral therapy.     

Virus-Like Particle Vaccine Confers Protection against a Lethal Newcastle Disease Virus Challenge in Chickens and Allows a Strategy of Differentiating Infected from Vaccinated Animals

In this study, we developed Newcastle disease virus (NDV) virus-like particles (VLPs) expressing NDV fusion (F) protein along with influenza virus matrix 1 (M1) protein using the insect cell expression system. Specific-pathogen-free chickens were immunized with oil emulsion NDV VLP vaccines containing increasing dosages of VLPs (0.4, 2, 10, or 50 μg of VLPs/0.5-ml dose). Three weeks after immunization, the immunogenicity of the NDV VLP vaccines was determined using a commercial enzyme-linked immunosorbent assay (ELISA) kit, and a lethal challenge using a highly virulent NDV strain was performed to evaluate the protective efficacy of the NDV VLP vaccines. NDV VLP vaccines elicited anti-NDV antibodies and provided protection against a lethal challenge in a dose-dependent manner. Although the VLP vaccines containing 0.4 and 2 μg of VLPs failed to achieve high levels of protection, a single immunization with NDV VLP vaccine containing 10 or 50 μg could fully protect chickens from a lethal challenge and greatly reduced challenge virus shedding. Furthermore, we could easily differentiate infected from vaccinated animals (DIVA) using the hemagglutination inhibition (HI) test. These results strongly suggest that utilization of NDV VLP vaccine in poultry species may be a promising strategy for the better control of NDV.     

JC病毒样颗粒可直接转运进入细胞核

目的探讨JC病毒(JCV)病毒样颗粒(VLP)是否可以直接转运进入细胞核。方法心用JCV主要外壳蛋白VP1体外表达、重组VIP，在其表面标记异硫氰基荧光素(FTTC)，同时在其内部包裹荧光染料Cy3，感染培养的HeLa细胞和SVG细胞，荧光显微镜观察VLP入核转运。结果HeLa和SVG细胞感染包裹Cy3的FTTC-VLP时，FTTC与Cy3同时出现于细胞核内相同部位；而感染FTTC—VP1与Cy3混合物时，FTTC虽可在细胞核内检测到，但Cy3信号几乎消失。包裹Cy3的VIP用SDSPAGE展开，荧光显像后行考马斯亮蓝(CBB)染色，发现Cy3和VLP移行至不同部位，证明Cy3不能与VP1结合，提示VLP以完整的颗粒形式转运进入细胞核。应用包裹外源性DNA的VLP感染培养的HeLa和SVG细胞，发现包裹的DNA在细胞浆和细胞核内均可检测到，提示JCV入核过程与VIP相同。结论VLP可以不经裂解直接转运进入细胞核，JCV入核转运可能与VLP相同。     

Protective immune responses against foot-and-mouth disease virus by vaccination with a DNA vaccine expressing virus-like particles

To display antigenic protein on the surface of virus-like particles (VLPs) presents a potentially powerful strategy for vaccine development. We genetically engineered the major capsid protein VP1 of foot-and-mouth disease virus (FMDV) into the predominant epitope C of HBV core gene to yield a chimeric core-VP1 VLP. The VLP was successfully expressed in HeLa cells transfected with core-VP1 DNA construct. Compared with a regular VP1 DNA construct, immunization with core-VP1 DNA induced significantly higher levels of antigen-specific IgG production, T cell proliferation, cytotoxic T lymphocyte response, and cytokine production in mice. Most importantly, the level of neutralizing antibody elicited by core-VP1 immunization was significantly higher than that with VP1 DNA immunization, which correlated well with animal protection level from subsequent live FMDV challenge. Thus, immunization with chimeric VLP induces higher efficacy and provides an attractive DNA vaccine strategy for controlling FMDV infection in future.     

A novel intranasal virus-like particle (VLP) vaccine designed to protect against the pandemic 1918 influenza A virus (H1N1)

We have prepared a virus-like particle (VLP) vaccine bearing the surface glycoproteins HA and NA of the 1918 influenza A virus by infecting Sf9 cells with a quadruple recombinant baculovirus that expresses the four influenza proteins (HA, NA, M1, and M2) required for the assembly and budding of the VLPs. The presence of HA and M1 in the purified VLPs was confirmed by Western blot, and that of NA by a neuraminidase enzymatic assay. For in vivo studies, the 1918 VLP vaccine was formulated with or without an oligonucleotide containing two CpG motifs and administered in two doses 2 wk apart via the intranasal route. The antibody titers in mice immunized with VLP vaccines were higher than in mice vaccinated with an inactivated swine virus (H1N1) control, when CHO cells expressing 1918 HA were used as antigen. The opposite result was obtained when disrupted swine virus was the antigen for the ELISA test. Vaccine efficacy was evaluated by challenging immunized mice with the 1918 antigenically related influenza virus A/swine/Iowa/15/30 (H1N1) and measuring viral titers in the upper and lower respiratory tract. Mice immunized with VLP vaccine plus CpG demonstrated significantly lower viral titers in the nose and lungs than did the control on days 2 and 4 postchallenge and completely cleared the virus by day 6. Furthermore, they did not show symptoms of disease although there was a minor decrease in body weight. Mice vaccinated with VLP alone also demonstrated significantly lower viral titers in the nose and lungs than did the placebo group as well as the inactivated virus group on days 4 and 6 postchallenge. These results suggest that it is feasible to make a safe and immunogenic vaccine to protect against the extremely virulent 1918 virus, using a novel and safe cell-based technology.     

Induction of insert-specific immune response in mice by hamster polyomavirus VP1 derived virus-like particles carrying LCMV GP33 CTL epitope

Hamster polyomavirus (HaPyV) major capsid protein VP1 based chimeric virus-like particles (VLPs) carrying model GP33 CTL epitope derived from Lymphocytic choriomeningitis virus (LCMV) were generated in yeast and examined for their capability to induce CTL response in mice. Chimeric VP1-GP33 VLPs were effectively processed in antigen presenting cells in vitro and in vivo and induced antigen-specific CD8+ T cell proliferation. Mice immunized only once with VP1-GP33 VLPs without adjuvant developed an effective GP33-specific memory T cell response: 70% were fully and 30% partially protected from LCMV infection. Moreover, aggressive growth of tumors expressing GP33 was significantly delayed in these mice in vivo. Therefore, HaPyV VP1-derived VLP harboring CTL epitopes are attractive vaccine candidates for the induction of insert-specific CTL immune response.     

Functional characterization of Ebola virus L-domains using VSV recombinants

VSV recombinants containing the overlapping L-domain sequences from Ebola virus VP40 (PTAPPEY) were recovered by reverse-genetics. Replication kinetics of M40-WT, M40-P24L, and M40-Y30A were indistinguishable from VSV-WT in BHK-21 cells, whereas the double mutant (M40-P2728A) was defective in budding. Insertion of the Ebola L-domain region into VSV M protein was sufficient to alter the dependence on host proteins for efficient budding. Indeed, M40 recombinants containing a functional PTAP motif specifically incorporated endogenous tsg101 into budding virions and were dependent on tsg101 expression for efficient budding. Thus, VSV represents an excellent negative-sense RNA virus model for elucidating the functional aspects and diverse host interactions associated with the L-domains of Ebola virus.