Recovery And Biological Properties Analysis Of Genotype ⅦNDV Bearing Mutations In HN Intervening Region

The HN protein is a type Ⅱ homotetrameric glycoprotein that exists on the surface of virions and infected cells. The ectodomain of HN has a globular head perched on top of a membrane-anchored stalk domain. Whereas the globular head is responsible for attachment to sialic acid-containing receptor(s), functions of neuraminidase activity (NA) and contains all of the known antigen sites, while the stalk interacts with the F to promote membrane fusion. Analysis of the HN stalk identifies a partially conserved segment which harbors two conserved heptad repeats (HR1and HR2) and a linear intervening region (IR) between the HRs. The HRs form a a-coiled-coil structure which has great contributions to HN structure and function. Some amino acid substitutions occurred in HN stalk can influence the functions of HN especially the fusion promotion activity in some extent, which could result in a modification of virus biological properties. While most studies at present focused on the alterations of HN protein functions caused by amino acid mutations without enough attention to the modifications of virus biological behaviors and the limited results also need to be explored and verified further. Therefore, we brought adjacent single mutation into the IR of HN stalk and rescued the mutated viruses; we studied the biological effects of amino acid substitutions in IR from protein level to viral level systematically.In protein level, mutations influenced the HN functions in different degree. When incubation in4℃, the HAd value of HN-A89Q, HN-L90A and HN-E91A were significantly higher than HNwt with an increase of47.3%,33%,45.8%respectively; While the HN-S92A, HN-P93A and HN-L94A were opposite, their values were marked decreased to68%,74.2%and64.5%of the wt level separately. Except the HN-L94A mutant, the A89Q, L90A, E91A, S92A and P93A mutations were impaired the NA activity of HN protein in some extent, especially the E91A and P93A mutants, only held29.7%and20.7%of wt level. And the fusion promotion of HNs containing A89Q, L90A, S92A and L94A substitution separately were notably lower than HNwt, the fusion index of them were44%,60.4%,75%,41.3%of wt level, and the syncytia caused by them were less and smaller than the parent. While the E91A and P93A had no visible influence on fusion promotion activity of protein.The viral level, we successfully rescued5HN-mutated viruses:r-L90A, r-E91A, r-S92A, r-P93A and r-L94A, and we demonstrated that the r-E91A, r-S92A, r-P93A and r-L94A owned hereditary stability. The results also showed that mutations decreased the propagation of virus in BHK-21cells and attenuated the virulence, especially the L94A mutation which enormously weakened the proliferation activity of virus, the highest titer of r-L94A was reduced by10000times than the NDV97wild type, and it ultimately turned the virus to be an intermediately virulent strain from the velogenic parent. In addition, mutations reduced the infectivity of virus and had different influence on viral hemagglutinin activity, neuraminidase activity and tissue tropism.In summary, we concluded that:The domains that are responsible for HN functions are consisted of different key amino acids, mutations occurred in the IR had different influence on these domains and also HN functions, while these functions have close connections between them. The functional modifications of HN have great influence on the interactions of viral proteins and also interaction between virus and its host. It is of great value to study the protein functional changes occurred by amino acid substitutions for analyzing modifications of viral biological characters. The IR mutations had different performances to different genotype HN proteins, which indicated that the different of genes and structures had influences on performances of same mutations. There also existed diversities in viral recovery efficiency for different mutations and genotypes with same mutations, which suggested the distinctions of mutation research in different viral strains and genotypes. Some amino acids substitutions of IR attenuated the virus and IR should to be a candidate target for viral alteration and development of attenuated vaccine.This work provides a theory and accumulates data for development of novel NDV vaccines and small-molecule drugs.