Expression Of HN And F Proteins Of NDV And The Syncytium Formation In HHCC Cells

Newcastle disease virus (NDV),a non-segmented and negative-stranded RNA virus, belongs to the Rubulavirus genus of the Paramyxoviridae family. Due to the properties of natural tumor-selective viral replication and non-pathogen in human, NDV was used in the treatment of cancer in recent years. The ways that NDV applied clinically in cancer therapy were focused on two fields: i) Single autologous tumor cells were infected with NDV ex vivo to produce tumour vaccine which expected to stimulate active immunity in patients after the surgery. ii) Oncolytic NDV virons were directly injected into cancer patients for oncolytic virotherapy or NDV was genetically manipulated as a virus vector to carry foreign genes in cancer gene therapy.The full length of RNA genome of NDV is about 15kb, comprising with six genes, in which the two genes encode hemagglutinin-neuraminidase (HN) and fusion protein (F), expressing on the virus envelope. The functions of HN protein were listed as followings: i) Receptor recognition: to recognize sialic acid-containing glycoproteins on host cell surfaces to mediate viral particles binding to cell membrane, finally switching on infection. ii) Neuraminidase activity: the catalytic function of the enzyme cleaves sialic acid residues off of newly formed glycoproteins inside the host cells, to ensure proper budding and non-aggregation of virus particles. iii) Promoting fusion: HN interacting with F protein can cause cell fusion, then make the membranes fusion of NDV and target cell, finally lead to NDV entering into the cytoplasm and then replicating. Except causing membrane fusion, the amino acid sequence of F protein plays an important role in viral virulence.The objectives of the study: The functions of HN and F proteins of an oncolytic strain NDV-Italien were studied respectively, and the interaction of the two proteins after cotransfection to stimulate the membrane fusion in hepatocellular carcinoma cells was investigated. The study suggests a new way for hepatoma therapy.Our study included three parts. Firstly, we constructed an eukaryotic expression plasmid containing HN gene of NDV-Italien and identified HN expression by western blot and immunofluorescence, further detected the function of erythroagglutination and neuramidinase of HN protein. Secondly, we constructed an eukaryotic expression plasmid containing F gene of NDV-Italien and identified F expression by immunofluorescence. We cotransfected the eukaryotic expression plasmids of HN and F into COS-7 cells, and detected the function of membrane fusion by hematoxylin staining. Thirdly, we cotransfected the eukaryotic expression plasmids of HN and F into HHCC cells, and identified the effect of membrane fusion on the promotion of syncytium in HHCC cells by hematoxylin staining.HN and F cDNA were synthesized from viral RNA by RT-PCR. The amplified fragments were respectively cloned in T vector, sequenced and recloned in expression vector pCDNA3.1. HN and F gene was cloned correctly into expression vector pcDNA3.1 by PCR and restriction analysis. The recombination vectors were named respectively pcDNA3.1-HN and pcDNA3.1-F. The recombination vectors pcDNA3.1-HN and pcDNA3.1-F were respectively transfected into COS-7 cell. The results of western blot analysis and immunofluorescence showed the HN and F proteins were expressed transiently in COS-7 cells. Function results showed HN protein has the erythroagglutination and neuramidinase activity and interaction between HN and F protein can cause membrane fusion. Further, the expression vectors pcDNA3.1-HN and pcDNA3.1-F were cotransfected into HHCC cells, the results showed interaction between HN and F protein can cause the effect of membrane fusion on the promotion of syncytium in HHCC cells.