Contribution of ebola virus glycoprotein, nucleoprotein, and VP24 to budding of VP40 virus-like particles

The VP40 matrix protein of Ebola virus buds from cells in the form of virus-like particles (VLPs) and plays a central role in virus assembly and budding. In this study, we utilized a functional budding assay and cotransfection experiments to examine the contributions of the glycoprotein (GP), nucleoprotein (NP), and VP24 of Ebola virus in facilitating release of VP40 VLPs. We demonstrate that VP24 alone does not affect VP40 VLP release, whereas NP and GP enhance release of VP40 VLPs, individually and to a greater degree in concert. We demonstrate further the following: (i). VP40 L domains are not required for GP-mediated enhancement of budding; (ii). the membrane-bound form of GP is necessary for enhancement of VP40 VLP release; (iii). NP appears to physically interact with VP40 as judged by detection of NP in VP40-containing VLPs; and (iv). the C-terminal 50 amino acids of NP may be important for interacting with and enhancing release of VP40 VLPs. These findings provide a more complete understanding of the role of VP40 and additional Ebola virus proteins during budding.     

Binding of virus-like particles of Norwalk virus to romaine lettuce veins

Noroviruses (NoV) annually cause millions of cases of gastrointestinal disease in the United States. NoV are associated with raw shellfish outbreaks, particularly oysters, which are thought to bioaccumulate NoV particles during the filter-feeding process. NoV outbreaks, however, have also been known to occur from other common-source food-borne vehicles, such as lettuce, frozen raspberries, and salad. In this study, we evaluated romaine lettuce as a potential vehicle for NoV transmission by testing the binding and distribution of NoV to the surface of romaine. Recombinant Norwalk virus-like particles (rNVLP) applied to the surface of romaine lettuce localized as large clusters primarily on the leaf veins. An extract of romaine lettuce leaves in phosphate-buffered saline (PBS) (romaine extract [RE]) bound rNVLP in a dose-dependent manner. RE did not bind rNVLP by histo-blood group antigens (HBGA), nor was RE competitive with rNVLP binding to porcine gastric mucin. These results suggested that non-HBGA molecules in RE bind rNVLP by a binding site(s) that is different from the defined binding pocket on the virion. Extracts of cilantro, iceberg lettuce, spinach, and celery also bound rNVLP. Samples of each of the vegetables spiked with rNVLP and tested with anti-NVLP antibody revealed by confocal microscopy the presence of rNVLP not only on the veins of cilantro but also throughout the surface of iceberg lettuce.     

Mutilation of RNA phage Qbeta virus-like particles: from icosahedrons to rods

Icosahedral virus-like particles (VLPs) of RNA phage Qbeta are stabilized by four disulfide bonds of cysteine residues 74 and 80 within the loop between beta-strands F and G (FG loop) of the monomeric subunits, which determine the five-fold and quasi-six-fold symmetry contacts of the VLPs. In order to reduce the stability of Qbeta VLPs, we mutationally converted the amino acid stretch 76-ANGSCD-81 within the FG loop into the 76-VGGVEL-81 sequence. It led to production in Escherichia coli cells of aberrant rod-like Qbeta VLPs, along with normal icosahedral capsids. The length of the rod-like particles exceeded 4-30 times the diameter of icosahedral Qbeta VLPs.     

Vesicular stomatitis virus as a vector to deliver virus-like particles of human norovirus: a new vaccine candidate against an important noncultivable virus

Human norovirus (HuNoV) is a major causative agent of food-borne gastroenteritis worldwide. Currently, there are no vaccines or effective therapeutic interventions for this virus. Development of an attenuated vaccine for HuNoV has been hampered by the inability to grow the virus in cell culture. Thus, a vector-based vaccine may be ideal. In this study, we constructed a recombinant vesicular stomatitis virus (rVSV-VP1) expressing VP1, the major capsid protein of HuNoV. Expression of the capsid protein by VSV resulted in the formation of HuNoV virus-like particles (VLPs) that are morphologically and antigenically similar to native virions. Recombinant rVSV-VP1 was attenuated in cultured mammalian cells as well as in mice. Mice inoculated with a single dose of rVSV-VP1 through intranasal and oral routes stimulated a significantly stronger humoral and cellular immune response than baculovirus-expressed VLP vaccination. Moreover, we demonstrated that mice inoculated with rVSV-VP1 triggered a comparable level of fecal and vaginal IgA antibody. Taken together, the VSV recombinant system not only provides a new approach to generate HuNoV VLPs in vitro but also a new avenue for the development of vectored vaccines against norovirus and other noncultivable viruses.     

Use of rotavirus virus-like particles as surrogates to evaluate virus persistence in shellfish

Rotavirus virus-like particles (VLPs) and MS2 bacteriophages were bioaccumulated in bivalve mollusks to evaluate viral persistence in shellfish during depuration and relaying under natural conditions. Using this nonpathogenic surrogate virus, we were able to demonstrate that about 1 log10 of VLPs was depurated after 1 week in warm seawater (22 degrees C). Phage MS2 was depurated more rapidly (about 2 log10 in 1 week) than were VLPs, as determined using a single-compartment model and linear regression analysis. After being relayed in the estuary under the influence of the tides, VLPs were detected in oysters for up to 82 days following seeding with high levels of VLPs (concentration range between 10(10) and 10(9) particles per g of pancreatic tissue) and for 37 days for lower contamination levels (10(5) particles per g of pancreatic tissue). These data suggest that viral particles may persist in shellfish tissues for several weeks.     

Production of FMDV virus-like particles by a SUMO fusion protein approach in Escherichia coli

Virus-like particles (VLPs) are formed by the self-assembly of envelope and/or capsid proteins from many viruses. Some VLPs have been proven successful as vaccines, and others have recently found applications as carriers for foreign antigens or as scaffolds in nanoparticle biotechnology. However, production of VLP was usually impeded due to low water-solubility of recombinant virus capsid proteins. Previous studies revealed that virus capsid and envelope proteins were often posttranslationally modified by SUMO in vivo, leading into a hypothesis that SUMO modification might be a common mechanism for virus proteins to retain water-solubility or prevent improper self-aggregation before virus assembly. We then propose a simple approach to produce VLPs of viruses, e.g., foot-and-mouth disease virus (FMDV). An improved SUMO fusion protein system we developed recently was applied to the simultaneous expression of three capsid proteins of FMDV in E. coli. The three SUMO fusion proteins formed a stable heterotrimeric complex. Proteolytic removal of SUMO moieties from the ternary complexes resulted in VLPs with size and shape resembling the authentic FMDV. The method described here can also apply to produce capsid/envelope protein complexes or VLPs of other disease-causing viruses.     

Incorporation of tick-borne encephalitis virus replicons into virus-like particles by a packaging cell line

RNA replicons derived from flavivirus genomes show considerable potential as gene transfer and immunization vectors. A convenient and efficient encapsidation system is an important prerequisite for the practical application of such vectors. In this work, tick-borne encephalitis (TBE) virus replicons and an appropriate packaging cell line were constructed and characterized. A stable CHO cell line constitutively expressing the two surface proteins prM/M and E (named CHO-ME cells) was generated and shown to efficiently export mature recombinant subviral particles (RSPs). When replicon NdDeltaME lacking the prM/M and E genes was introduced into CHO-ME cells, virus-like particles (VLPs) capable of initiating a single round of infection were released, yielding titers of up to 5 x 10(7)/ml in the supernatant of these cells. Another replicon (NdDeltaCME) lacking the region encoding most of the capsid protein C in addition to proteins prM/M and E was not packaged by CHO-ME cells. As observed with other flavivirus replicons, both TBE virus replicons appeared to exert no cytopathic effect on their host cells. Sedimentation analysis revealed that the NdDeltaME-containing VLPs were physically distinct from RSPs and similar to infectious virions. VLPs could be repeatedly passaged in CHO-ME cells but maintained the property of being able to initiate only a single round of infection in other cells during these passages. CHO-ME cells can thus be used both as a source for mature TBE virus RSPs and as a safe and convenient replicon packaging cell line, providing the TBE virus surface proteins prM/M and E in trans.     

利用反向遗传操作技术产生ZJI株鹅源新城疫病毒

利用反向遗传操作技术,将ZJI株鹅源新城疫病毒全基因组cNDA克隆(NDV3GM122)和含该毒株NP、P及L基因的3个表达载体(pCI-NP、pCI-P与pCI-L)共转染BSR-T7/5细胞;同时,将NDV3GM122与含新城疫病毒La Sota毒株NP、P及L基因的3个表达载体(pCIneoNP、pCIneoP与pCIneoL)进行共转染。通过间接免疫荧光实验(Indiectimmunofluorescence assay,IFA)以及接种鸡胚后进行血凝(Hemagglutinin,HA)与血凝抑制(Hemagglutinininhibition,HI)试验、RT-PCR扩增和电镜观察,结果均证实全基因组cDNA克隆NDV3GM122与La Sota毒株表达载体共转染组产生了有血凝性的鹅源新城疫病毒,而NDV3GM122与ZJI株表达载体共转染组暂未检测到有血凝性的病毒。ZJI株鹅源新城疫病毒的拯救成功为对该病毒进行功能基因组研究和疫苗的研制等后续工作打下了基础。     

Formation of virus-like particles by HIV-1 Gag proteins, expressed by a recombinant vaccinia virus

Monkey kidney cells CV-1 were infected with recombinant vaccinia virus carrying HIV-1 gag gene with a deletion of 230 nucleotide pairs from the 3'-terminus. The main gene product detected in the lysates of infected cells was the gag precursor rp50. The protein was accumulated on the cell membranes suggesting that it had a myristylated N-terminus, and was cleaved by a recombinant virus specific protease with the formation of two proteins, p17 and p24 corresponding in molecular masses to mature gag proteins. Virus-like particles similar to immature HIV virions were budding from the surface of infected cells. They look like the ring of optically dense material covered with a lipid bilayer, of the same size (100-120 nm) and of the same density in a sucrose gradient (1.16-1.18 g/ml) as HIV-1 virions. The particles contained rp50 and cellular heterogeneous RNA. Thus, the unprocessed gag precursor with deleted 77 amino acid residues from the C-terminus is able to form virus-like particles in the absence of env proteins and virus-specific RNA, and these particles are budding from the cell surface. The question about the use of extracellular Gag-particles for AIDS diagnostic work and construction of vaccines is discussed.     

Production of human papillomavirus type 16 L1 virus-like particles by recombinant Lactobacillus casei cells

Infections with human papillomavirus type 16 (HPV-16) are closely associated with the development of human cervical carcinoma, which is one of the most common causes of cancer death in women worldwide. At present, the most promising vaccine against HPV-16 infection is based on the L1 major capsid protein, which self-assembles in virus-like particles (VLPs). In this work, we used a lactose-inducible system based on the Lactobacillus casei lactose operon promoter (plac) for expression of the HPV-16 L1 protein in L. casei. Expression was confirmed by Western blotting, and an electron microscopy analysis of L. casei expressing L1 showed that the protein was able to self-assemble into VLPs intracellularly. The presence of conformational epitopes on the L. casei-produced VLPs was confirmed by immunofluorescence using the anti-HPV-16 VLP conformational antibody H16.V5. Moreover, sera from mice that were subcutaneously immunized with L. casei expressing L1 reacted with Spodoptera frugiperda-produced HPV-16 L1 VLPs, as determined by an enzyme-linked immunosorbent assay. The production of L1 VLPs by Lactobacillus opens the possibility for development of new live mucosal prophylactic vaccines.     

Generation of hepatitis C virus-like particles by use of a recombinant vesicular stomatitis virus vector

Hepatitis C virus (HCV), a major etiologic agent of hepatocellular carcinoma, presently infects approximately 400 million people worldwide, making the development of protective measures against HCV infection a key objective. Here we have generated a recombinant vesicular stomatitis virus (VSV), which expresses the HCV structural proteins, by inserting the contiguous Core, E1, and E2 coding region of HCV into the VSV genome. Recombinant VSV expressing HCV Core, E1, and E2 (VSV-HCV-C/E1/E2) grew to high titers in vitro and efficiently expressed the incorporated HCV gene product, which became fully processed into the individual HCV structural proteins. Biochemical and biophysical analysis indicated that the HCV Core, E1, and E2 proteins assembled to form HCV-like particles (HCV-LPs) possessing properties similar to the ultrastructural properties of HCV virions. Mice immunized with VSV-HCV-C/E1/E2 generated cell-mediated immune responses to all of the HCV structural proteins, and humoral responses, particularly to E2, were also readily evident. Our data collectively indicate that engineered VSVs expressing HCV Core, E1, and E2 and/or HCV-LPs represent useful tools in vaccine and immunotherapeutic strategies designed to address HCV infection.     

Formation of virus-like particles from O-type foot-and-mouth disease virus in insect cells using codon-optimized synthetic genes

A recombinant baculovirus was constructed to simultaneously express codon-optimized virus-like particles (VLP), A VP1-2A-VP3 and VP0 of serotype O foot-and-mouth disease virus (FMDV), from individual promoters. The target proteins were expressed in insect cells at high level, as shown by indirect sandwich ELISA; and the expressed VP1-2A-VP3 could autocatalytically be cleaved into the individual proteins, VP1-2A and VP3, as shown by Western-blot analyses. In addition, in the insect cells, the structural proteins, VP0, VP3 and VP1-2A, self-assembled into virus-like particles resembling the authentic FMDV particles. This information should prove useful for the development of more efficient VLP assembly using shorter genes.