Calcium-Dependent Nonspecific Permeability of the Inner Mitochondrial Membrane Is Not Induced in Mitochondria of the Yeast Endomyces magnusii

Mitochondria of the yeast Endomyces magnusii were examined for the presence of a Ca2+- and phosphate-induced permeability of the inner mitochondrial membrane (pore). For this purpose, coupled mitochondria were incubated under conditions known to induce the permeability transition pore in animal mitochondria, i.e., in the presence of high concentrations of Ca2+ and P(i), prooxidants (t-butylhydroperoxide), oxaloacetate, atractyloside (an inhibitor of ADP/ATP translocator), SH-reagents, by depletion of adenine nucleotide pools, and deenergization of the mitochondria. Large amplitude swelling, collapse of the membrane potential, and efflux of the accumulated Ca2+ were used as parameters for demonstrating pore induction. E. magnusii mitochondria were highly resistant to the above-mentioned substances. Deenergization of mitochondria or depletion of adenine nucleotide pools have no effect on low-amplitude swelling or the other parameters. Cyclosporin A, a specific inhibitor of the nonspecific permeability transition in animal mitochondria, did not affect the parameters measured. It is thus evident that E. magnusii mitochondria lack a functional Ca2+-dependent pore, or possess a pore differently regulated as compared to that of mammalian mitochondria.     

Characterization of Chikungunya Virus-Like Particles

Chikungunya virus (CHIKV) is becoming a global concern due to the increasing number of outbreaks throughout the world and the absence of any CHIKV-specific vaccine or treatment. Virus-like particles (VLPs) are multistructured proteins that mimic the organization and conformation of native viruses but lack the viral genome. They are noninfectious and potentially safer vaccine candidates. Recent studies demonstrated that the yield of CHIKV VLPs varies depending on the strains, despite the 95% amino acid similarity of the strains. This might be due to the codon usage, since protein expression is differently controlled by different organisms. We optimized the region encoding CHIKV structural proteins, C-E3-E2-6k-E1, inserted it into a mammalian expression vector, and used the resulting construct to transfect 293 cells. We detected 50-kDa proteins corresponding to E1 and/or E2 in the cell lysate and the supernatant. Transmission electron microscopy revealed spherical particles with a 50- to 60-nm diameter in the supernatant that resembled the native CHIKV virions. The buoyant density of the VLPs was 1.23 g/mL, and the yield was 20 µg purified VLPs per 10⁸ cells. The VLPs aggregated when mixed with convalescent sera from chikungunya patients, indicating that their antigenicity is similar to that of native CHIKV. Antibodies elicited with the VLPs were capable of detecting native CHIKV, demonstrating that the VLPs retain immunogenicity similar to that of the native virion. These results indicated that CHIKV VLPs are morphologically, antigenically, and immunologically similar to the native CHIKV, suggesting that they have potential for use in chikungunya vaccines.     

Effects of the number of amino acid residues in the signal segment upstream or downstream of the NS2B-3 cleavage site on production and secretion of prM/M-E virus-like particles of West Nile virus

Expression of genes for precursor M (prM) and envelope (E) proteins of West Nile virus (WNV) leads to the production of small, capsidless, and non-infectious virus-like particles (VLPs) possessing the E antigen which is responsible for viral entry and immune protection. It has been reported that processing of the secretion signal affects viral release. We examined the secretion efficiency of VLPs into the culture medium from RK13 or 293 T cells transfected with expression vectors for prM and E proteins of WNV which were constructed to comprise different lengths of signal peptides upstream of the prM-E domain. The number of amino acid residues present in the segment markedly affected the production, processing, and secretion of VLPs. Secreted VLPs possessed both the processed M protein and the glycosylated E protein. In addition, immunization with VLPs induced neutralizing antibodies in C3H/HeN mice. These results indicate that the number of amino acid residues comprising the N-terminus of the signal segment controls the efficiency of assembly, maturation, and release of VLPs in the absence of viral protease, which in turn indicates the potential of VLPs as a candidate for an effective WNV subunit vaccine.     

Mosaic RNA Phage VLPs Carrying Domain III of the West Nile Virus E Protein

The virus-neutralising domain III (DIII) of the West Nile virus glycoprotein E was exposed on the surface of RNA phage AP205 virus-like particles (VLPs) in mosaic form. For this purpose, a 111 amino acid sequence of DIII was added via amber or opal termination codons to the C-terminus of the AP205 coat protein, and mosaic AP205-DIII VLPs were generated by cultivation in amber- or opal-suppressing Escherichia coli strains. After extensive purification to 95 % homogeneity, mosaic AP205-DIII VLPs retained up to 11–16 % monomers carrying DIII domains. The DIII domains appeared on the VLP surface because they were fully accessible to anti-DIII antibodies. Immunisation of BALB/c mice with AP205-DIII VLPs resulted in the induction of specific anti-DIII antibodies, of which the level was comparable to that of the anti-AP205 antibodies generated against the VLP carrier. The AP205-DIII-induced anti-DIII response was represented by a significant fraction of IgG2 isotype antibodies, in contrast to parallel immunisation with the DIII oligopeptide, which failed to induce IgG2 isotype antibodies. Formulation of AP-205-DIII VLPs in alum adjuvant stimulated the level of the anti-DIII response, but did not alter the fraction of IgG2 isotype antibodies. Mosaic AP205-DIII VLPs could be regarded as a promising prototype of a putative West Nile vaccine.     

A specific domain of the Chikungunya virus E2 protein regulates particle formation in human cells: implications for alphavirus vaccine design

Virus-like particles (VLPs) can be generated from Chikungunya virus (CHIKV), but different strains yield variable quantities of particles. Here, we define the genetic basis for these differences and show that amino acid 234 in E2 substantially affects VLP production. This site is located within the acid-sensitive region (ASR) known to initiate a major conformational change in E1/E2. Selected other mutations in the ASR, or changes in pH, also increased VLP yield. These results demonstrate that the ASR of E2 plays an important role in regulating particle generation.     

Detection of multiple forms of polyphosphate phosphohydrolase from Endomyces magnusii]

Labile polyphosphate phosphohydrolase from Endomyces magnusii is 27-fold purified by means of fractionation with ammonium sulphate, gel filtration on Sephadex G-75 and Biogel P-60 and chromatography on DEAE cellulose. Chromatography on DEAE Sephadex A-50, isoelelctric focusing and polyacrylamide gel electrophoresis of the enzyme preparation revealed 3 different fractions with polyphosphate phosphohydrolase activity (PPPH1, PPPH2 and PPPH3). Relative content of these fractions in E. magnusii cells is 30%, 55% and 15% respectively. Isoelectric points are: PPPH1--pH 5.1--5.2; PPPH2--pH 6.0--6.1; PPPH3--pH 6.3--6.4. PPPH1 and PPPH2 are found to be the most labile. PPPH3 is more stable under isolation procedure and storage. The fractions have similar molecular weight (48 000 +/- 3000).     

Analysis of antigenicity and topology of E2 glycoprotein present on recombinant hepatitis C virus-like particles

Purification of hepatitis C virus (HCV) from sera of infected patients has proven elusive, hampering efforts to perform structure-function analysis of the viral components. Recombinant forms of the viral glycoproteins have been used instead for functional studies, but uncertainty exists as to whether they closely mimic the virion proteins. Here, we used HCV virus-like particles (VLPs) generated in insect cells infected with a recombinant baculovirus expressing viral structural proteins. Electron microscopic analysis revealed a population of pleomorphic VLPs that were at least partially enveloped with bilayer membranes and had viral glycoprotein spikes protruding from the surface. Immunogold labeling using specific monoclonal antibodies (MAbs) demonstrated these protrusions to be the E1 and E2 glycoproteins. A panel of anti-E2 MAbs was used to probe the surface topology of E2 on the VLPs and to compare the antigenicity of the VLPs with that of truncated E2 (E2(660)) or the full-length (FL) E1E2 complex expressed in mammalian cells. While most MAbs bound to all forms of antigen, a number of others showed striking differences in their abilities to recognize the various E2 forms. All MAbs directed against hypervariable region 1 (HVR-1) recognized both native and denatured E2(660) with comparable affinities, but most bound either weakly or not at all to the FL E1E2 complex or to VLPs. HVR-1 on VLPs was accessible to these MAbs only after denaturation. Importantly, a subset of MAbs specific for amino acids 464 to 475 and 524 to 535 recognized E2(660) but not VLPs or FL E1E2 complex. The antigenic differences between E2(660,) FL E1E2, and VLPs strongly point to the existence of structural differences, which may have functional relevance. Trypsin treatment of VLPs removed the N-terminal part of E2, resulting in a 42-kDa fragment. In the presence of detergent, this was further reduced to a trypsin-resistant 25-kDa fragment, which could be useful for structural studies.     

The M, E, and N structural proteins of the severe acute respiratory syndrome coronavirus are required for efficient assembly, trafficking, and release of virus-like particles

The production of virus-like particles (VLPs) constitutes a relevant and safe model to study molecular determinants of virion egress. The minimal requirement for the assembly of VLPs for the coronavirus responsible for severe acute respiratory syndrome in humans (SARS-CoV) is still controversial. Recent studies have shown that SARS-CoV VLP formation depends on either M and E proteins or M and N proteins. Here we show that both E and N proteins must be coexpressed with M protein for the efficient production and release of VLPs by transfected Vero E6 cells. This suggests that the mechanism of SARS-CoV assembly differs from that of other studied coronaviruses, which only require M and E proteins for VLP formation. When coexpressed, the native envelope trimeric S glycoprotein is incorporated onto VLPs. Interestingly, when a fluorescent protein tag is added to the C-terminal end of N or S protein, but not M protein, the chimeric viral proteins can be assembled within VLPs and allow visualization of VLP production and trafficking in living cells by state-of-the-art imaging technologies. Fluorescent VLPs will be used further to investigate the role of cellular machineries during SARS-CoV egress.     

Formation of L-malic acid by yeasts of the genus Dipodascus

The yeast strains of the genus Dipodascus were used for the bioconversion of fumaric acid to L-malic acid. Under nongrowth conditions, the fumarase activity in the intact cells or in the cell-free extract of Dipodascus was 10 times higher than that of Saccharomyces cerevisiae cells. Pretreatment of the Dipodascus with malonate was not necessary because succinate was not detected as a by-product. The fumarase activity in Dipodascus magnusii CCM 8235 was increased approximately 100% when Triton X-305 (0.1%) was added to the reaction mixture.     

Effect of the DnaK chaperone on the conformational quality of JCV VP1 virus‐like particles produced in Escherichia coli

Protein nanoparticles such as virus‐like particles (VLPs) can be obtained by recombinant protein production of viral capsid proteins and spontaneous self‐assembling in cell factories. Contrarily to infective viral particles, VLPs lack infective viral genome while retaining important viral properties like cellular tropism and intracellular delivery of internalized molecules. These properties make VLPs promising and fully biocompatible nanovehicles for drug delivery. VLPs of human JC virus (hJCV) VP1 capsid protein produced in Escherichia coli elicit variable hemagglutination properties when incubated at different NaCl concentrations and pH conditions, being optimal at 200 mM NaCl and at pH range between 5.8 and 7.5. In addition, the presence or absence of chaperone DnaK in E. coli cells influence the solubility of recombinant VP1 and the conformational quality of this protein in the VLPs. The hemagglutination ability of hJCV VP1 VLPs contained in E. coli cell extracts can be modulated by buffer composition in the hemagglutination assay. It has been also determined that the production of recombinant hJCV VP1 in E. coli is favored by the absence of chaperone DnaK as observed by Western Blot analysis in different E. coli genetic backgrounds, indicating a proteolysis targeting role for DnaK. However, solubility is highly compromised in a DnaK^? E. coli strain suggesting an important role of this chaperone in reduction of protein aggregates. Finally, hemagglutination efficiency of recombinant VP1 is directly related to the presence of DnaK in the producing cells. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:744–748, 2014     

Expression and characterization of virus-like particles containing rubella virus structural proteins.

Rubella virus (RV) virions contain two envelope glycoproteins (E1 and E2) and a capsid protein (C). Noninfectious RV-like particles (VLPs) containing three structural proteins were expressed in a BHK cell line (BHK-24S) by using an inducible promoter. These VLPs were found to resemble RV virons in terms of their size, their morphology, and some biological activities. In immunoblotting studies, VLPs were found to bind similarly to native RV virions with 10 of a panel of 12 RV-specific murine monoclonal antibodies. Immunization of mice with VLPs induced specific antibody responses against RV structural proteins as well as virus-neutralizing and hemagglutination-inhibiting antibodies. After immunization of mice with VLPs, in vitro challenge of isolated lymphocytes with inactivated RV and individual RV structural proteins stimulated proliferation. Our data suggest the possibility of using VLPs as immunogens for serodiagnostic assays and RV vaccines.     

Ca2+-release pathways from mitochondria of the yeast Endomyces magnusii

Ca2+-release pathways from Ca2+-preloaded mitochondria of the yeast Endomyces magnusii were studied. In the presence of phosphate as a permeant anion, Ca2+ was released from respiring mitochondria only after massive cation loading at the onset of anaerobiosis. Intensive aeration of the mitochondrial suspension rapidly inhibited the efflux of Ca2+ and induced its reuptake. The Ca2+ release was not affected by cyclosporin A, an inhibitor of the nonselective permeability transition of mammalian mitochondria. With acetate as the permeant anion, a spontaneous net Ca2+ efflux began after uptake of about 75% of the added cation. The rate of this efflux was insensitive to cyclosporin A, aeration, and Na+ and was proportional to the Ca2+ load. The Ca2+ release was inhibited by La3+, Mn2+, Mg2+, TPP+, and nigericin (in the presence of KCl) and activated by spermine and hypotonicity. We conclude that Ca2+ efflux from preloaded E. magnusii mitochondria is very similar to the Na+-independent specific pathway for Ca2+ release operative in mitochondria from nonexcitable mammalian tissues.     

Ca(2+) efflux in mitochondria from the yeast Endomyces magnusii.

Calcium release pathways in Ca(2+)-preloaded mitochondria from the yeast Endomyces magnusii were studied. In the presence of phosphate as a permeant anion, Ca(2+) was released from respiring mitochondria only after massive cation loading at the onset of anaerobiosis. Ca(2+) release was not affected by cyclosporin A, an inhibitor of the mitochondrial permeability transition. Aeration of the mitochondrial suspension inhibited the efflux of Ca(2+) and induced its re-uptake. With acetate as the permeant anion, a spontaneous net Ca(2+) efflux set in after uptake of approximately 150 nmol of Ca(2+)/mg of protein. The rate of this efflux was proportional to the Ca(2+) load and insensitive to aeration, protonophorous uncouplers, and Na(+) ions. Ca(2+) efflux was inhibited by La(3+), Mn(2+), Mg(2+), tetraphenylphosphonium, inorganic phosphate, and nigericin and stimulated by hypotonicity, spermine, and valinomycin in the presence of 4 mm KCl. Atractyloside and t-butyl hydroperoxide were without effect. Ca(2+) efflux was associated with contraction, but not with mitochondrial swelling. We conclude that the permeability transition pore is not involved in Ca(2+) efflux in preloaded E. magnusii mitochondria. The efflux occurs via an Na(+)-independent pathway, in many ways similar to the one in mammalian mitochondria.     

Norovirus GII.4 strain antigenic variation

Noroviruses are the principal cause of epidemic gastroenteritis worldwide. Multiple reports have concluded that the major capsid proteins of GII.4 strains, which cause 80% of norovirus infections worldwide, are evolving rapidly, resulting in new epidemic strains. Surrogate neutralization assays using sera from outbreaks and from immunized mice suggest that, as with influenza virus, antigenic variation maintains GII.4 persistence in the face of human population herd immunity. To test this hypothesis, mice were hyperimmunized with virus-like particles (VLPs) representing an early (GII.4-1987) and a contemporary (GII.4-2006) GII.4 strain. Anti-GII.4-1987 IgG monoclonal antibodies (MAbs) strongly reacted with GII.4 VLPs derived between only 1987 and 2002. Ligand binding blockade was more efficient with GII.4-1987 and GII.4-1997 VLPs than with GII.4-2002. Anti-GII.4-2006 IgG MAbs recognized either a broad panel of GII.4 VLPs (1987 to 2006) or a subset of contemporary (2004 to 2006) VLPs. Most 2006 antibodies did not recognize or only poorly recognized GII.4 VLPs of 2007 or 2008, documenting rapid antigenic evolution of GII.4 capsids. Generally, 2006 MAbs blocked homotypic VLP-ligand binding but were unable to block VLPs representing strains primarily circulating during or earlier than 2002. These analyses demonstrate that both subtle and significant evolutionary change has occurred within antibody epitopes between epidemic strains, providing direct evidence that the GII.4 noroviruses are undergoing antigenic variation, likely in response to herd immunity. As with influenza virus, HIV, and hepatitis C virus, norovirus antigenic variation will significantly influence the design of efficacious vaccines and immunotherapeutics against these important human pathogens.     

戊型肝炎病毒样颗粒在重组汉逊酵母中的发酵表达、纯化及其免疫原性分析

为了研制戊型肝炎新型基因工程疫苗,利用汉逊酵母表达系统表达重组戊型肝炎病毒样颗粒,成功构建了重组戊型肝炎疫苗工程菌株HP/HEV2.3,对该菌株的发酵条件和纯化工艺进行了研究。先将工作种子批进行发酵培养,收集发酵后的细胞培养物;对其先后进行细胞破碎、澄清和超滤、硅胶吸附和解吸附、超滤浓缩换液、色谱纯化及除菌过滤,制得重组汉逊酵母戊型肝炎病毒样颗粒,纯化收率为33%,纯度达99%;电镜观察显示该重组汉逊酵母戊型肝炎病毒样颗粒与天然戊型肝炎病毒颗粒理论大小一致,为32 nm;基因序列与理论一致;SDS-PAGE分析结果表明其表达的外源蛋白质分子量与预期的目的蛋白质分子量大小一致,均为56 k Da,表达量占细胞总蛋白的26%,表达水平为1.0 g/L发酵液;Western blotting、ELISA活性检测及小鼠免疫接种效力试验ED_(50)结果表明,此重组汉逊酵母戊型肝炎病毒样颗粒具有良好的抗原性和免疫原性,可用于制造戊型肝炎新型基因工程疫苗。     

Zika Virus Baculovirus-Expressed Virus-Like Particles Induce Neutralizing Antibodies in Mice

The newly emerged mosquito-borne Zika virus (ZIKV) strains pose a global challenge owing to its ability to cause microcephaly and neurological disorders. Several ZIKV vaccine candidates have been proposed, including inactivated and live attenuated virus vaccines, vector-based vaccines, DNA and RNA vaccines. These have been shown to be efficacious in preclinical studies in mice and nonhuman primates, but their use will potentially be a threat to immunocompromised individuals and pregnant women. Virus-like particles (VLPs) are empty particles composed merely of viral proteins, which can serve as a safe and valuable tool for clinical prevention and treatment strategies. In this study, we used a new strategy to produce ZIKV VLPs based on the baculovirus expression system and demonstrated the feasibility of their use as a vaccine candidate. The pre-membrane (prM) and envelope (E) proteins were co-expressed in insect cells and self-assembled into particles similar to ZIKV. We found that the ZIKV VLPs could be quickly and easily prepared in large quantities using this system. The VLPs were shown to have good immunogenicity in immunized mice, as they stimulated high levels of virus neutralizing antibody titers, ZIKV-specific IgG titers and potent memory T cell responses. Thus, the baculovirus-based ZIKV VLP vaccine is a safe, effective and economical vaccine candidate for use against ZIKV.     

Antigenicity and immunogenicity of novel chimeric hepatitis B surface antigen particles with exposed hepatitis C virus epitopes

The small envelope protein of hepatitis B virus (HBsAg-S) can self-assemble into highly organized virus like particles (VLPs) and induce an effective immune response. In this study, a restriction enzyme site was engineered into the cDNA of HBsAg-S at a position corresponding to the exposed site within the hydrophilic a determinant region (amino acid [aa] 127-128) to create a novel HBsAg vaccine vector allowing surface orientation of the inserted sequence. We inserted sequences of various lengths from hypervariable region 1 (HVR1) of the hepatitis C virus (HCV) E2 protein containing immunodominant epitopes and demonstrated secretion of the recombinant HBsAg VLPs from transfected mammalian cells. A number of different recombinant proteins were synthesized, and HBsAg VLPs containing inserts up to 36 aa were secreted with an efficiency similar to that of wild-type HBsAg. The HVR1 region exposed on the particles retained an antigenic structure similar to that recognized immunologically during natural infection. VLPs containing epitopes from either HCV-1a or -1b strains were produced that induced strain-specific antibody responses in immunized mice. Injection of a combination of these VLPs induced antibodies against both HVR1 epitopes that resulted in higher titers than were achieved by vaccination with the individual VLPs, suggesting a synergistic effect. This may lead to the development of recombinant particles which are able to induce a broad anti-HCV immune response against the HCV quasispecies or other quasispecies-like infectious agents.     

Efficient assembly and release of SARS coronavirus-like particles by a heterologous expression system

Virus-like particles (VLPs) produced by recombinant expression of the major viral structural proteins could be an attractive method for severe acute respiratory syndrome (SARS) control. In this study, using the baculovirus system, we generated recombinant viruses that expressed S, E, M and N structural proteins of SARS-CoV either individually or simultaneously. The expression level, size and authenticity of each recombinant SARS-CoV protein were determined. In addition, immunofluorescence and FACS analysis confirmed the cell surface expression of the S protein. Co-infections of insect cells with two recombinant viruses demonstrated that M and E could assemble readily to form smooth surfaced VLPs. On the other hand, simultaneous high level expression of S, E and M by a single recombinant virus allowed the very efficient assembly and release of VLPs. These data demonstrate that the VLPs are morphological mimics of virion particles. The high level expression of VLPs with correct S protein conformation by a single recombinant baculovirus offers a potential candidate vaccine for SARS.     

Three different M1 RNA-containing viruslike particle types in Saccharomyces cerevisiae: in vitro M1 double-stranded RNA synthesis.

Killer strains of Saccharomyces cerevisiae bear at least two different double-stranded RNAs (dsRNAs) encapsidated in 39-nm viruslike particles (VLPs) of which the major coat protein is coded by the larger RNA (L-A dsRNA). The smaller dsRNA (M1 or M2) encodes an extracellular protein toxin (K1 or K2 toxin). Based on their densities on CsCl gradients, L-A- and M1-containing particles can be separated. Using this method, we detected a new type of M1 dsRNA-containing VLP (M1-H VLP, for heavy) that has a higher density than those previously reported (M1-L VLP, for light). M1-H and M1-L VLPs are present together in the same strains and in all those we tested. M1-H, M1-L, and L-A VLPs all have the same types of proteins in the same approximate proportions, but whereas L-A VLPs and M1-L VLPs have one dsRNA molecule per particle, M1-H VLPs contain two M1 dsRNA molecules per particle. Their RNA polymerase produces mainly plus single strands that are all extruded in the case of M1-H particles but are partially retained inside the M1-L particles to be used later for dsRNA synthesis. We show that M1-H VLPs are formed in vitro from the M1-L VLPs. We also show that the peak of M1 dsRNA synthesis is in fractions lighter than M1-L VLPs, presumably those carrying only a single plus M1 strand. We suggest that VLPs carrying two M1 dsRNAs (each 1.8 kilobases) can exist because the particle is designed to carry one L-A dsRNA (4.5 kilobases).     

The function of the spike protein of mouse hepatitis virus strain A59 can be studied on virus-like particles: cleavage is not required for infectivity.

The spike protein (S) of the murine coronavirus mouse hepatitis virus strain A59 (MHV-A59) induces both virus-to-cell fusion during infection and syncytium formation. Thus far, only syncytium formation could be studied after transient expression of S. We have recently described a system in which viral infectivity is mimicked by using virus-like particles (VLPs) and reporter defective-interfering (DI) RNAs (E. C. W. Bos, W. Luytjes, H. Van der Meulen, H. K. Koerten, and W. J. M. Spaan, Virology 218:52-60, 1996). Production of VLPs of MHV-A59 was shown to be dependent on the expression of M and E. We now show in several ways that the infectivity of VLPs is dependent on S. Infectivity was lost when spikeless VLPs were produced. Infectivity was blocked upon treatment of the VLPs with MHV-A59-neutralizing anti-S monoclonal antibody (MAb) A2.3 but not with nonneutralizing anti-S MAb A1.4. When the target cells were incubated with antireceptor MAb CC1, which blocks MHV-A59 infection, VLPs did not infect the target cells. Thus, S-mediated VLP infectivity resembles MHV-A59 infectivity. The system can be used to identify domains in S that are essential for infectivity. As a first application, we investigated the requirements of cleavage of S for the infectivity of MHV-A59. We inserted three mutant S proteins that were previously shown to be uncleaved (E. C. W. Bos, L. Heijnen, W. Luytjes, and W. J. M. Spaan, Virology 214:453-463, 1995) into the VLPs. Here we show that cleavage of the spike protein of MHV-A59 is not required for infectivity.