| Paramyxoviruses are a group of important viruses that are pathogenic to human beings and animals. They can be epidemic all over the globe. The family of paramyxoviridae is divided into two subfamilies: paramyxovirinae and pneumovirinae. Paramyxovirinae is divided into three genuses: paramyxovirus, morbillivirus and rubulavirus; pneumovirinae only contains pneumovirus. Paramyxovirus includes human parainfluenza virus (hPIV), Newcastle disease virus (NDV) and Sendia virus (SV).hPIVs mainly cause diseases of respiratory and reproduction systems, including bronchitis, bronchiolitis, pneumonia, conjunctivitis, orchitis and ovaritis etc. Sometimes, it can cause death. Also in this family there are many animal pathogens, including NDV, an avian virus that is a serious agricultural problem in many regions of the world at present. NDV can cause avian and cattle diseases and usually disastrous and wide epidemic. Paramyxoviruses can also cause diseases of horses and swine, such as pneumonia, necrotic bronchitis, enchephalitis, etc. Some viruses are emerging ones, including Nipah virus, Hendra virus, Salem virus, avian pneumovirus and human metapneumovirus belonging to paramyxoviridae. They can cause severe human and animal diseases, infecting many organs or tissues, especially central nervous system. Clinically, fever and headach are common, and the fatality is very high.NDV is of other clinical significances. For example, it can induce interferon and can be used to treat tumors. NDV is a typical member of paramyxoviridae,therefore it is of significance to study NDV in theory and practice.Besides paramyxoviridae, many other human pathogenic viruses have enveloped glycoproteins that can cause cell fusion, such as herpes simplex virus, human immunodeficiency virus, etc. Cell fusion is a critical step in viral multiplification and pathogenesis. Thus, studies on paramyxoviruses F protein possess wide significances in viral theory and practice.Paramyxoviruses are enveloped, negative-stranded, non-segmented RNA viruses. Cell fusion is a critical step in viral information transduction, multiplification and pathogenesis. The NDV and hPIV membrane contain two types of glycoproteins that facilitate fusion process, the hemagglutinin-neuraminidase (HN) and fusion (F) glycoproteins. The fusion glycoprotein is divided into head, stalk, transmembrane region and tail, and believed to disrupt the target cell membrane and induce membrane fusion. The fusion protein monomer is synthesized as a precursor designated Fo and cleaved during transport through the Golgi membranes into two disulfide-linked subunits, Fi and F2. This processing exposes the hydrophobic domain at the amino terminus of the Fi subunit and is essential for the biological activities of the mature protein. This hydrophobic domain, called the fusion peptide, is highly conserved among paramyxovirus F proteins and considered to be involved directly in mediating membrane fusion.The HN protein is a multifunctional molecule with three distinct activities: receptor recognition activity (binding to cellular receptors), neuraminidase activity (NA) and fusion-promotion activity (FP).Although the F protein is directly responsible for fusion of the viral and cellular membranes, co-expression of HN with the F protein is generally required for most paramyxoviruses. Furthermore, the requirement for the HN protein in fusion is virus specific, which means that F protein can only induce cell fusion when co-expressed with homogenous HN protein. For example, cell fusion will take place when NDV F and NDV HN or hPIV F and hPIV HN are co-expressed. Otherwise, there will be no cell fusion observed. This phenomenon proves that there is a kind of signal or information exchange or transmission between the two proteins. The specific membrane fusion has been interpreted as evidence for a necessary interaction between the two proteins, which, in some way, activates the fusion activity of the F protein. Experiments showed that after HN binds toreceptor, conformation of HN changes and induces that of F also changes. Then hydrophobic fusion peptides are released into cell membranes, so cell fusion is triggered.The interaction between F and HN is one of the focuses on enveloped glycoproteins of paramyxoviridae in the world. In order to localize an active domain that interacts with homogenous HN on F protein of paramyxoviruses and to understand the molecular mechanism of cell fusion, site-directed mutagenesis and gene recombination were used to get chimeric F proteins. The results showed that the specific interaction domain with HN on F protein is in the whole stalk and part of head, i.e. in Fi except transmembrane and tail parts.In order to identify active domain that interacts with homogenous HN on F protein of paramyxoviruses, on the basis of previous experiments, site-directed mutagenesis was used to get mutants with a new enzyme site and gene recombination was used to get chimeric F proteins. Chimeric F proteins were expressed with homogenous HN or with heterogenous HN in eukaryocytes, and their fusion functions were assayed by qualitative analysis of cell fusion and fluorescence-activated cell sorter (FACS) analysis.1. Effects of fusion peptide on the specific membrane fusionThe chimeric proteins NDV C-F and hPIV C-F were obtained by exchanging fusion peptide (FP) of NDV F and hPIV F each other. They were co-expressed with homogenous HN or with heterogenous HN in eukaryocytes. No cell fusion was observed when NDV C-F and hPIV HN were co-expressed or hPIV C-F and NDV HN were co-expressed; the fusion activity had almost the same function as that of NDV F wild type when NDV C-F and NDV HN were co-expressed; as well as the fusion activity of hPIV C-F and hPIV HN co-expressed.The analysis of FACS indicated that the expression efficiencies of both NDV C-F and hPIV C-F were the same as those of their relevant wild types.2. Effects of HR1 on the specific membrane fusionThe chimeric proteins NDV C-HR1 and hPIV C-HR1 were obtained byexchanging HR1 of NDV F and hPIV F each other. They were co-expressed with homogenous HN or with heterogenous HN in eukaryocytes. No cell fusion was observed when NDV C-HRl and hPIV HN were co-expressed or hPIV C-HRl and NDV HN were co-expressed; the fusion activity had 53.91 % compared with NDV F wild type when NDV C-HRl and NDV HN were co-expressed; the fusion activity had 83.15% compared with hPIV F wild type when hPIV C-HRl and hPIV HN were co-expressed.The analysis of FACS indicated that the expression efficiencies of both NDV C-HRl and hPIV C-HRl were lower than those of their relevant wild types.3. Effects of HR2 on the specific membrane fusionThe chimeric proteins NDV C-HR2 and hPIV C-HR2 were obtained by exchanging HR2 of NDV F and hPIV F each other. They were co-expressed with homogenous HN or with heterogenous HN in eukaryocytes. No cell fusion was observed when NDV C-HR2 and hPIV HN were co-expressed or hPIV C-HR2 and NDV HN were co-expressed; the fusion activity had 107.23 % compared with NDV F wild type when NDV C-HR2 and NDV HN were co-expressed; the fusion activity had 12.01 % compared with hPIV F wild type when hPIV C-HR2 and hPIV HN were co-expressed.The analysis of FACS indicated that the expression efficiencies of NDV C-HR2 were the same as those of NDV F wild type; the expression efficiencies of hPIV C-HR2 were lower than those of hPIV wild type.4. Effects of ectodomain non-conservative on the specific membrane fusionThe chimeric proteins NDV C-M and hPIV C-M were obtained by exchanging ectodomain non-conservative of NDV F and hPIV F each other. They were co-expressed with homogenous HN or with heterogenous HN in eukaryocytes. No cell fusion was observed when NDV C-M and hPIV HN were co-expressed or hPIV C-M and NDV HN were co-expressed; the fusion activity had 78.73 % compared with NDV F wild type when NDV C-M and NDV HN were co-expressed; the fusionactivity had 75.14% compared with hPIV F wild type when hPIV C-M and hPIV HN were co-expressed.The analysis of FACS indicated that the expression efficiencies of both NDV C-M and hPIV C-M almost had the same as their relevant wild types.5. Effects of the segment adjacent to the fusion peptide of HR1 on the specific membrane fusionThe chimeric proteins NDV C-HR1-N and hPIV C-HR1-N were obtained by exchanging the segment adjacent to the fusion peptide of HR1 of NDV F and hPIV F. They were co-expressed with homogenous HN or with heterogenous HN in eukaryocytes. No cell fusion was observed when NDV C-HR1-N and hPIV HN were co-expressed or hPIV C-HR1-N and NDV HN were co-expressed; the fusion activity had 56.23% compared with NDV F wild type when NDV C-HR1-N and NDV HN were co-expressed; the fusion activity had 85.76% compared with hPIV F wild type when hPIV C-HR1-N and hPIV HN were co-expressed.The analysis of FACS indicated that the expression efficiencies of both NDV C-HR1-N and hPIV C-HR1-N were lower than those of their relevant wild types.6. Effects of the segment adjacent to the TM of HR2 on the specific membrane fusionThe chimeric proteins NDV C-HR2-C and hPIV C-HR2-C were obtained by exchanging the segment adjacent to the TM of HR2 of NDV F and hPIV F each other. They were co-expressed with homogenous HN or with heterogenous HN in eukaryocytes. No cell fusion was observed when NDV C-HR2-C and hPIV HN were co-expressed or hPIV C-HR2-C and NDV HN were co-expressed; the fusion activity had 98.25 % compared with NDV F wild type when NDV C-HR2-C and NDV HN were co-expressed; the fusion activity had 8.43 % compared with hPIV F wild type when hPIV C-HR2-C and hPIV HN were co-expressed.The analysis of FACS indicated that the expression efficiencies of NDV C-HR2-C almost had the same as NDV F wild type; the expression efficiencies of hPIV C-HR2-C were lower than those of hPIV wild type.7. Effects of two segments of ectodomain non-conservative on the specific membrane fusionEctodomain non-conservative of both NDV F and hPIV F were divided into two segments Ml (N terminus of non-conservative) and M2 (C terminus of non-conservative). The chimeric proteins NDV C-Ml and hPIV C-Ml were obtained by exchanging Ml of NDV F and hPIV F; NDV C-M2 and hPIV C-M2 were obtained by exchanging M2 of NDV F and hPIV F. All of them were co-expressed with homogenous HN or with heterogenous HN in eukaryocytes. No cell fusion was observed when NDV C-Ml and hPIV FIN were co-expressed or NDV C-M2 and hPIV HN were co-expressed; the fusion activity had 76.34% compared with NDV F wild type when NDV C-Ml and NDV HN were co-expressed; the fusion activity had 96.25% compared with NDV F wild type when NDV C-M2 and NDV HN were co-expressed; no cell fusion was observed when hPIV C-Ml and NDV HN were co-expressed or hPIV C-M2 and NDV HN were co-expressed; the fusion activity had 65.82 % compared with hPIV F wild type when hPIV C-Ml and hPIV HN were co-expressed; the fusion activity had 95.78% compared with hPIV F wild type when hPIV C-M2 and hPIV HN were co-expressed.The analysis of FACS indicated that the expression efficiencies of all the four chimeric proteins almost had the same as their relevant wild types.The results suggested as folio wings:The FP was not important for the specific membrane fusion, because its chimeric proteins do not affect their relevant membrane fusion activities.HRls of both NDV F and hPIV F were important for their specific membrane fusion. The results of exchanging the N terminus of HR1 are the same as those of exchanging the whole HR1. Therefore, the specific membrane fusion region of HR1 mainly lies in the N terminus of HR1, i.e., peptides of HR1 adjacent to the fusion peptide.The HR2 of NDV F was not important for its specific membrane fusion because it can be replaced by HR2 of hPIV F. HR2 of hPIV F was very important for itsspecific membrane fusion. The results of exchanging the C terminus of HR2 are the same as those of exchanging the whole HR2. Thus, the specific membrane fusion region of HR2 mainly lies in the C terminus of HR2, i.e., peptides of HR2 adjacent to the transmembrane region.Besides HRl and HR2, the non-conservitive sequences between HRl and HR2 of NDV F and hPIV F were also important for their specific membrane fusion. Furthermore, the specific region of non-conservitive sequences mainly lies in Ml fragment, i.e., the N terminus of non-conservitive sequences.
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