Construction Of Newcastle Disease Virus-like Particles And Mechanism Of Budding

Virus-like particle (VLP) is a highly effective type of subunit vaccine that mimics the structure of virus particle without infectious genetic material. Indeed, many VLP completely lack the DNA or RNA genome of the virus and thus have the authentic conformation of viral capsid proteins, without any of the drawbacks such as, reversion, recombination and re-assortment. VLP is prepared based on the fact that expression of the capsid proteins of viruses leads to the spontaneous assembly of particles that are structurally similar to authentic viruses.VLP has been used as an efficient method to study the characteristics, polymorphism, assembly, buddiong and vaccine efficacy for many viruses. For example, VLP were widely used in the research on the polymorphism, assembly, budding and vaccine development of many viruses, including HIV, HPV, HBV, HCV, HEV and rovirus.VLP play more and more important role in research on polymorphism, assembly, budding and vaccine development.Newcastle disease virus (NDV) is an important avian pathogen causing severe economic losses in the poultry industry worldwide. Most researches about NDV were focused on the structure and function of single gene and its expression product. Almost no direct information was obtained about the mechanism of the interactions between six proteins during RNA transcription and synthesis, during the virus assembly, propagation and budding. In order to clarify the mechanism and factors of NDV budding, improve the immune response of NDV subunit vaccine and evaluate the immunogenicity and immunoreactivity of NDV-like particles generally, the following research using VLP technique will be carried out. The purpose of this study was to assemble VLP of NDV using the mammalian cells and baculoviruse expression systems and use the VLP to investigate the mechanism of the assembly of structual proteins, formation of VLP and budding of NDV.Two expression systems were adopted to study the budding and assembly of NDV-like particles in this study. One is the mammalian cells expression system to express the M, NP, F and HN proteins to study the mechanism of interaction of NDV and cells. Another is baculoviruses expression system to express large quartity of forgein proteins in order to purify the expressed proteins and assemble NDV-like particles in vitro.In mammalian cell expression system, four recombinant expression plasmids, pCAGG-M, pCAGG-NP, pCAGG-F and pCAGG-HN that harbour M, NP, F and HN gene of F₄₈E₉ strain NDV, respectively, were constructed. These recombinant expression plasmids were separately transinfected into HeLa cells with liposome to express proteins. The expressed proteins were detected with antisera against NDV whole molecule by indirect immunofluorescence assay. To confirm these result, the expression of M and NP protein in HeLa cells was confirmed by polyclone antisera against M and NP proteins expressed in E.coli cells. In addition, HeLa cells co-transfected with four recombinant expression plasmids showed syncytium formation. F protein is a fusion protein that can induce cell fusion with the presence of HN protein, which was evidenced by the fact that pCAGG-F and pCAGG-HN co-transfected HeLa cells can express F and HN proteins with the biological functions. The co-expression of M, NP, F and HN proteins in the supernatant culture medium were confirmed bywestern blot.Based on these results, HeLa cells were transfected with individual or co-transfected with recombinant expression plasmids to discove which structural protein is essential to promote budding of NDV in virus-free system. The result showed that expression of M protein of NDV in HeLa cells could drive the budding of VLP observed by electron microscope. VLP containing M protein resemble NDV with the size of lOOnm, much smaller than NDV 250nm. However, after transfection of HeLa cells with pCAGG-M+pCAGG-NP+pCAGG-F or pCAGG-M+pCAGG-NP+pCAGG-HN, much bigger-size VLP around 150nm were observed by EM. When the HeLa cells were co-transfected with four recombinant expression plasmids, the VLP were about 200nm in diameter, similar as NDV.In baculovirus expression system, baculovirus transfer vector pAcAB4 that harbour four promoters, two plO promoters and two polyhedrin promoters was used to constructe the recombinant baculovirus. For this purpose, Stu I site in F gene and Xba I site in NP gene of F4gE9 strain NDV were first mutated by PCR methods. Genes encoding F, NP, M and HN proteins were amplified and inserted into pMDl 8-T vector and were sequenced. Then F, NP, M and HN genes were subcloned into baculovirus transfer vector pAcAB4 in turn. F and M gene were under the control of the promoter plO and NP and HN gene were downstream of the promoter polyhedrin. Recombinant baculovirus transfer vector pAcAB4-F-NP-M-HN were constructed successfully and confirmed by the sequence analyses. Sf9 insect cells were co-transfected with pAcAB4-F-NP-M-HN and linearized baculovirus DNA. Recombinant baculovirus rBac-F-NP-M-HN subsequently acquired was confirmed by PCR and then plaque purified and amplified. Both culture supernatant and rBac-F-NP-M-HN infected Sf9 cells were collected at 72h post-infection. The simultaneous expression of F, NP, M and HN proteins in culture supernatant were identified by western blot. In contrast, only HN proteins was detected in infected cells. These results suggested that VLP can self-assemble under the condition of co-expression of M, NP, F and HN proteins leave the cells via the budding mechanism.To further analyze the budding mechanism of VLP, four baculovirus transfer vectors that harbour M, NP, F and HN genes, respectively, were constructed and co-transfected with linearized baculovirus DNA into the insect cells. The recombinant baculoviruses were identified by PCR and named rBac-M, rBac-NP, rBac-F and rBac-HN. These recombinant baculoviruses were further plaque purified and amplified. Expression of M, NP, F and HN proteins were also identified by indirect immunofluorescence assay. Then, the insect cells were either infected with one recombinant baculovirus or with two or more baculovirus. The ultrathin section of infected insect cells was made and VLP were observed by electron microscope. The empty particles with 50nm in diameter were observed in the insect cells infected with rBac-M whereas they were not observed in insect cells infected by wild type baculovirus. Infection by rBac-M or with the help of rBac-NP leaded to the self-assembly of VLP and budding out of the insect cells. When insect cells were infected by rBac-M or co-infected with rBac-NP, rBac-F or additional rBac-HN, different size of empty particles were observed in ultrathin section samples. The diameter of particles was 50-l00nm, much smaller than the NDV. Similar empty particles were also observed in the insect Sf9 cells infected with rBac-F-NP-M-HN. The culture supernate ofinsect cells infected by recombinant baculovirus were subjected to negative staining. VLP that resemble the authentic virion were observed in culture supernate of insect cells infected with rBac-M or rBac-F-NP-M-HN. But the size of VLP coming from rBac-M in supernatant was smaller than that of the authentic NDV particles. VLP comprising of exprssion of rBac-F-NP-M-HN resemble close the NDV in size.lt suggested that M, NP, F and HN structural proteins assemble outside cellular nucleus. The interaction and assembly among the structural proteins did not complete until the particles budded from the infected cells, just like budding of authentic virion. Some endochylema proteins in host cells may be necessary to complete the assembly. The studies in baculovius expression system reach the similar conclusions as that of mammalian cell expression system. The expression of M protein lead to assembly of VLP and budding out of the cells. F or HN glycoprotein doesn't play key roles in assembly of VLP and just lead to the differences in the size of VLP.This was the first study to construct NDV-like particles in mammalian cell and baculovirus expression systems. It concluded that M protein is the major force to drive the virus budding. The expression of M protein alone could drive the VLP budding and release from the cells. M protein of NDV, at the presence of other structural proteins could assemble into VLP and resemble the authentic virion in size and shape.Further studies are required to address the following questions: which domains or motifs in M protein are essential in VLP budding? What roles other structral proteins especially F and HN glycoproteins except M protein play in the assembly and budding of NDV? Whether nonstructral proteins of NDV and host cells are important in virus assembly and budding?...