A luciferase-based budding assay for Ebola virus

The VP40 matrix protein of Ebola virus (EBOV) is capable of budding from mammalian cells as a virus-like particle (VLP) and is the major protein involved in virus egress. A functional budding assay has been developed based upon this characteristic of VP40 to assess the contributions of VP40 sequences as well as host proteins to the budding process. This well-defined assay has been modified for potential use in a high-throughput format in which the detection and quantification of firefly luciferase protein in VLPs represents a direct measure of VP40 budding efficiency. Luciferase was found to be incorporated into budding VP40 VLPs. Furthermore, co-expression of EBOV glycoprotein (GP) enhances release of VLPs containing VP40 and luciferase. In contrast, when luciferase is co-expressed with a budding deficient mutant of VP40, luciferase levels in the VLP fraction decrease significantly. This assay represents a promising high-throughput approach to identify inhibitors of EBOV budding.     

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.     

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.     

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.     

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.     

Calmodulin antagonists suppress gap junction coupling in isolated Hensen cells of the guinea pig cochlea

The effect of calmodulin (CaM) antagonists W7, trifluoperazine (TFP) and a calmodulin inhibitory peptide on gap junction coupling in isolated Hensen cells of the organ of Corti was analysed by the double whole-cell patch-clamp technique. Addition of the conventional antagonists W7 and TFP in the micromolar range caused a rapid decrease of gap junction conductance after a delay of a few minutes in a dose-dependent manner. Fluorescence spectroscopy of cytoplasmic free calcium concentration ([Ca(2+)](i)) by Fura-2 showed no significant change of [Ca(2+)](i) by W7. Chelation of [Ca(2+)](i) by 10 mM BAPTA or use of nominally Ca(2+)-free external bath did not suppress the W7-induced gap junction uncoupling. The results suggest that W7 and TFP induce gap junction uncoupling at unchanged global [Ca(2+)](i) in Hensen cells. To obtain additional evidence for an involvement of CaM in regulating gap junction conductance a calmodulin inhibitory peptide, the MLCK peptide (250 nM), was added to the standard pipette solution. Again gap junction uncoupling was observed, but on a significantly slower time scale. This is the first study of an effect of calmodulin antagonists on gap junction coupling in isolated Hensen cells. The question whether the effect of calmodulin inhibitors is specific and involves CaM-dependent gating of gap junction coupling in Hensen cells is discussed.     

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.     

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.     

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.     

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     

Further analysis of the role of calcium in rotavirus morphogenesis

Previously we reported that calcium plays an important role in the maturation of bovine rotavirus (M. S. Shahrabadi and P. W. K. Lee, 1986. Virology 152, 298-307). We now demonstrate that the formation of mature double-shelled (L) particles was strictly dependent on the concentration of calcium present in the growth medium. The formation of single-shelled (D) particles did not appear to be a calcium-mediated process. Subsequent labeling studies using 45Ca revealed that calcium was incorporated into the L particles but not the D particles. The previously noted decreased level of the outer capsid protein VP7 (42K) in calcium-deprived cultures was now found to be due to the preferential degradation, and not to the impaired synthesis, of this protein in the absence of calcium. It was further demonstrated that calcium had a stabilizing effect on VP7 and that VP7 synthesized in the presence of calcium was not degraded upon subsequent calcium deprivation. Protein degradation during calcium deprivation was apparently limited to the mature form of VP7 since the unglycosylated precursor (pVP7), formed in the presence of tunicamycin, was found to be stable under this condition. Electron microscopic examination of infected cells revealed that in the presence of calcium, virus maturation took place by the budding of viral cores through the endoplasmic reticulum (ER). No such budding was observed in calcium-deprived cells. In these cells mature virions were absent and membrane fragments could be found associated with viral cores or single-shelled particles.     

Inhibition of measles virus budding by phenothiazines

HeLa cells infected with measles virus show an accumulation of virus-specific strands at the plasma membrane after addition of the anticalmodulin drugs trifluoperazine (TFP) and chlorpromazine (CPZ), whereas spherical virus particles are almost completely absent. At low drug concentrations (10-15 microM TFP; 30-40 microM CPZ) the inhibitory effect is dependent on the presence of extracellular calcium. The strands complete the budding process after removal of the drugs. Restoration of virus budding is not sensitive to cycloheximide and immunoprecipitation experiments give evidence that the viral protein synthesis is not qualitatively altered in the presence of TFP. The data indicate that both drugs arrest the budding process at an intermediate stage at the plasma membrane. The inability of the strands to comigrate with cytochalasin B-induced actin patches suggests that the inhibition of budding is probably the result of an impaired interaction of viral structures with the cytoskeleton.     

钙调素拮抗剂与钙通道阻滞剂对离体大鼠心脏钙反常损伤心肌保护作用的研究

目的:观察钙调素拮抗剂三氟拉嗪(TFP)及钙通道阻滞剂异博定(VP)对缺钙-复钙损伤心肌的保护作用。方法:应用TFP与VP对离体大鼠心脏进行灌流。检测心肌钙含量、灌流液中乳酸脱氢酶(LDH)及心肌蛋白漏出量等指标。结果:TFP显著减少损伤心肌的钙含量、LDH及蛋白漏出量,与对照组相比差异显著(P＜0.01)。VP灌注组与对照组相比各项指标差异无显著(P＞0.05)。TFP与VP联合应用各项检测指标值明显低于单独应用TFP(P＜0.01)。结论:TFP有效减轻钙反常心肌损伤的程度;VP对钙反常心肌无保护作用。联合应用TFP与VP的心肌保护效果更佳。钙反常时Ca²⁺大量内流主要通过钙调素介导,但慢钙通道在钙超载形成过程中也可能起一定作用。     

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。     

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.     

Intra-peritoneal and intra-rectal immunogenicity induced by rotavirus virus like particles 2/6/7 in mice

We previously developed virus like particles of rotavirus (RV) with VP2, VP6, and VP7 proteins (VLP2/6/7) using stable High-five cell line. To evaluate the immunogenicity of our construct, we assessed the humoral and cytokine responses induced by VLP2/6/7 in BALB/c mice immunized intra-peritoneally and intra-rectally. Enzyme-linked immunosorbent assay (ELISA) and Relative quantitative (RQ) Real-time PCR were used to evaluate the antibody (IgG and IgA) levels in serum and mRNA levels of IL-6, IL-10 and IFN-γ in spleen cells, respectively. Our results showed that VLP2/6/7 is capable of intra-peritoneal (I.P.) and intra-rectal (I.R.) induction of serum IgG and IgA responses. IgA was detected in fecal samples of immunization groups by I.P. and I.R. routes. Interestingly, I.R. route induced higher IgA titer compared with I.P. route which was statistically significant. Moreover, mRNA levels of IL-6 and IFN-γ were significantly elevated in mice immunized intra-peritoneally with VLP2/6/7 compared to control group. As such, the mean change was 7.4 (P < 0.05) and 14.8 (P < 0.001) for IFN-γ and IL-6, respectively. Likewise, the same pattern was found when mice were immunized intra-rectally. Although elevated, the difference in the mean change for IL-10 was not statistically significant when compared to control group. Our findings indicated that VLPs constructed via a stable insect cell line are able to induce both humoral and cellular responses, a similar pattern as observed after immunization with live RVs.     

Monoclonal antibodies to Ebola virus: isolation, characteristics, and study of cross reactivity with Marburg virus

Thirteen hybridoma strains producing monoclonal antibodies (Mabs) to Ebola virus were prepared by fusion of NS-O mouse myeloma cells with splenocytes of BALB/c mice immunized with purified and inactivated Ebola virus (Mayinga strain). Mabs directed against viral proteins were selected and tested by ELISA. Protein specificity of 13 Mabs was determined by immunoblotting with SDS-PAGE proteins of Ebola virus. Of these, 11 hybridoma Mabs reacted with 116 kDa protein (NP) and 2 with Ebola virus VP35. Antigenic cross-reactivity between Ebola and Marburg viruses was examined in ELISA and immunoblotting with polyclonal and monoclonal antibodies. In ELISA, polyclonal antibodies of immune sera to Ebola or Marburg viruses reacted with heterologous filoviruses, and two anti-NP Ebola antibodies (Mabs 7E1 and 6G8) cross-reacted with both viruses. Target proteins for cross-reactivity, Ebola NP (116 kDa) and Marburg NP (96 kDa), and VP35 of both filoviruses were detected by immunoblotting with polyclonal and monoclonal antibodies (6G8) to Ebola virus.     

Identification and mapping of single nucleotide polymorphisms in the varicella-zoster virus genome

We have analysed the expression and cellular localisation of the matrix protein VP40 from Ebola virus. Full-length VP40 and an N-terminal truncated construct missing the first 31 residues [VP40(31-326)] both locate to the plasma membrane of 293T cells when expressed transiently, while a C-terminal truncation of residues 213 to 326 [VP40(31-212)] shows only expression in the cytoplasm, when analysed by indirect immunofluorescence and plasma membrane preparations. In addition, we find that full-length VP40 [VP40(1-326)] and VP40(31-326) are both released into the cell culture supernatant and float up in sucrose gradients. The efficiency of their release, however, is dependent on the presence of the N-terminal 31 residues. VP40 that is released into the supernatant is resistant to trypsin digestion, a finding that is consistent with the formation of viruslike particles detected by electron microscopy. Together, these results provide strong evidence that Ebola virus VP40 is sufficient for virus assembly and budding from the plasma membrane.     

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.