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Newcastle disease virus (NDV) is the pathogen of a zoonosis that is primarily transmitted by poultry and has severe infectivity and a high fatality rate. Many studies have focused on the role of the NDV fusion (F) protein in the cell-cell membrane fusion process. However, little attention has been given to the heptad repeat region, HR4, which is located in the NDV F2 subunit. Here, site-directed mutants were constructed to study the function of the NDV F protein HR4 region and identify the key amino acids in this region. Nine conserved amino acids were substituted with alanine or the corresponding amino acid of other aligned paramyxoviruses. The desired mutants were examined for changes in fusogenic activity through three kinds of membrane fusion assays and expression and proteolysis through IFA, FACS and WB. The results showed that when conserved amino acids (L81, Y84, L88, L91, L92, P94, L95 and I99) were replaced with alanine, the fusogenic activity of the F protein was abolished, possibly because of failed protein expression not only on the cell surface but also inside cells. These data indicated that the conserved amino acids above in NDV F HR4 are critical for normal protein synthesis and expression, possibly for the stability of the F protein monomer, formation of trimer and conformational changes., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Zhiyu Wang reports financial support was provided by National Natural Science Foundation of China. Yaqing Liu reports financial support was provided by Doctoral Research Foundation of Affiliated Hospital of Qingdao University. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Newcastle disease, designated a class A disease of poultry by the Office international des epizooties (OIE), is an acute infection caused by Newcastle disease virus (NDV). The merging of the envelope of NDV with the membrane of a target host cell is the key step in the infection pathway, which is driven by the concerted action of two glycoproteins: haemagglutinin-neuraminidase (HN) and fusion (F) protein. When the HN protein binds to the host cell surface receptor, the F protein is activated to mediate fusion. The three-dimensional structure of the F protein has been reported to have low electron density between the DIII domain and the HRB domain, and this electron-poor region is defined as the HRB linker. To clarify the contributing role of the HRB linker in the NDV F protein-mediated fusion process, 6 single amino acid mutants were obtained by site-directed mutagenesis of the HRB linker. The expression of the mutants and their abilities to mediate fusion were analysed, and the key amino acids in the HRB linker were identified as L436, E439, I450, and S453, as they can modulate the fusion ability or expression of the active form to a certain extent. The data shed light on the crucial role of the F protein HRB linker in the acquisition of a normal fusogenic phenotype.

Newcastle disease (ND), caused by the Newcastle disease virus (NDV), is transmitted by poultry with severe infectivity and a high fatality rate. The fusion (F) protein on the NDV envelope facilitates the merger of the viral and host cell membranes with the help of the homologous hemagglutinin-neuraminidase protein (HN). The transmembrane (TM) domains of viral fusion proteins are typically required for fusion, but the key amino acids in NDV F TM domains have not been identified. Site-directed mutagenesis was utilized to change the conserved amino acids at 500, 501, 502, 505, 510, 513, 516, 519, and 520 to alanine. It was found that mutants L519 and V520 had an interrupted protein expression, decreased to below 10%, and mutants A500, I505, V513, and V516 had a hypoactive impact on fusion activity, decreased to 85.38%, 67.05%, 55.38% and 51.13% of wt F, respectively. The results indicated that the TM domain plays a vital part in the fusion activity of the NDV F protein.

To gain insights into the role of the head-stalk linker region in the fusion triggering, we constructed mutants by deleting or substituting the linker region (115-NGAANNSG-122) of Newcastle disease virus (NDV) haemagglutinin-neuraminidase (HN) with the corresponding sequences of other paramyxoviruses. The results showed that these HN mutants exhibited different levels of fusion-triggering activity, but most of them maintained comparable levels with wide-type HN in both receptor recognition and neuraminidase activity. We tried to figure out reasons for fusion alteration through assessing the expression and the oligomeric state of HN mutants. Moreover, four mutants with significant fusion changes were introduced into the headless HN stem (HN1-123) to intensively investigate the role of the linker region in fusion triggering. Consequently, the stability of HN oligomers and the structural integrity of the 4 helical-bundle of stalk have complicated influences on the alteration of fusion-triggering activities for different mutants. These data suggested that the head-stalk linker could regulate the fusion triggering at both full-length and headless HN levels.

Background: The paramyxovirus haemagglutinin-neuraminidase (HN) is a multifunctional protein that is responsible for attachment to receptors, removal of receptors from infected cells to prevent viral self-aggregation (neuraminidase, NA) and fusion promotion. It is commonly accepted that there are two receptor binding sites in the globular head of HN, and the second receptor binding site is only involved in the function of receptor binding and fusion promotion., Methods: 10 conserved residues in the second receptor binding site of Newcastle disease virus (NDV) HN were chosen and substituted to alanine (A). The desired mutants were examined to detect the functional change in hemadsorption (HAD) ability, NA activity and fusion promotion ability., Results: The HAD and fusion promotion ability of mutants C172A, R174A, C196A, D198A, Y526A and E547A were abolished. Compared with wild-type (wt) HN, the HAD of mutants T167A, S202A and R516A decreased to 55.81, 44.53, 69.02%, respectively, and the fusion promotion ability of these three mutants decreased to 54.74, 49.46, 65.26%, respectively; however, mutant G171A still maintained fusion promotion ability comparable with wt HN but had impaired HAD ability. All the site-directed mutations altered the NA activity of NDV HN without affecting protein cell surface expression., Conclusions: The data suggest that mutants C172A, R174A, C196A, D198A, Y526A and E547A do not allow the conformational change that is required for fusion promotion ability and HAD activity, while the other mutants only affect the conformational change to a limited extent, except mutant G171A with intact fusion promotion ability. Overall, the conserved amino acids in the second receptor binding site, especially residues C172, R174, C196, D198, Y526 and E547, are crucial to normal NDV HN protein function.

The avian infectious disease, Newcastle disease (ND), caused by Newcastle disease virus (NDV) can cause severe economic losses to poultry whether vaccinated or not in many countries. In this study, a strain of NDV isolated from an outbreak in China was subjected to biological, phylogenetic and genetic characterization. The results showed that the mean death time (MDT) was 52.4 h and the intracerebral pathogenicity indices (ICPI) value was 1.95. In addition, amino acid sequencing result showed that it had a sequence 112 R-R-Q-R-R ↓ F 117 at fusion protein cleaving site (FPCS) indicating a velogenic strain. And its genome length is 15,192 nucleotide (nt) with the conserved complementary 3' leader and 5' trailer regions encoding six genes, 3'-NP-P-M-F-HN-L-5'. Based on phylogenetic analyses for hyper-variable region and complete genome of F gene, the strain studied here can be clustered into genotype IX, Class II, which has little evolution distance with strains of genotype III, being considered as a transitional strain in the evolution history of NDV. The rescue of infectious cDNA is proceeded in 9-day-old embryonated SPF chicken eggs. Despite the death of the first generation, the allantoic fluid harvested from the first generation lost its pathogenicity after passage. And we found the phenomenon happened due to the antibody appearing in the allantoic fluid. These findings offer our understanding of circulating strains of NDV in China and lay scientific foundations for making more efficient vaccines for Newcastle disease., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Newcastle disease virus (NDV), an avian paramyxovirus, causes Newcastle disease (ND) which is a highly contagious and fatal viral disease affecting poultry and most species of birds. The fusion (F) protein of NDV mediates membrane fusion, which is essential to the processes of viral entry, replication, and dissemination. Although several domains of NDV F are known to have important effects on regulating the membrane fusion activity, the role of the region around domain III (DIII) and domain I (DI) still remains ill-defined. Site-directed mutagenesis was utilized to change the conserved amino acids at 269, 274, 277, 286, 287, 290, 295, and 297 to alanine in order to investigate the effects of these conserved amino acids around the DIII and DI linker region of the NDV F protein on fusion activity. It was found that five of these substitutions almost abolished fusion activity except for mutants I269A, Q286A, and N297A, which showed 57.1%, 161.1%, and 97.7% of the wt F level, respectively. Four (I274A, D277A, V287A, and P290A) of these five mutants likely result in interfering with folding or transporting of the molecule since these proteins were minimally expressed at the cell surface, formed aggregates, or not proteolytically cleaved. However, mutant L295A almost abolished fusion activity even with a similar level of cell surface expression. These data indicated that conserved amino acids around the DIII-DI linker region are critical for the folding of the F protein and have an important influence on fusion activity.

Newcastle disease (ND), which is caused by Newcastle disease virus (NDV), is a highly contagious disease in chickens and is a great threat to the poultry industry. Fusion of the viral and target cell membranes is a prerequisite for NDV's entry into host cells. This process is directly mediated by the fusion (F) protein. Although several domains of F are known to regulate membrane fusion activity, the roles of the DI-DII linker (residues 376-381) of the NDV F protein in membrane fusion still remain unclear. To investigate the roles of this linker in NDV F-induced cell-cell fusion, mutations were engineered into this linker by site-directed mutagenesis. These mutants were analysed with respect to cell surface expression and membrane fusion activity. Each of the mutated F proteins in this linker was expressed at the cell surface at a similar level to wild-type (WT) F. However, most of them resulted in significant alterations in fusion activity. In particular, the mutants G377S, A378D, L379A and T380P were able to independently mediate cell fusion in the absence of HN protein in BHK-21 cells. Taken together, the results indicated that the DI-DII linker region has an important effect on the fusion activity of NDV F and mutants in this region could alter the requirement for HN for the promotion of membrane fusion.

Newcastle disease virus (NDV), an avain paramyxovirus, has been assigned to the genus Avulavirus within the family Paramyxoviridae. It causes Newcastle disease (ND) that is a highly contagious and fatal viral disease affecting poultry and most species of birds. The hemagglutinin-neuraminidase (HN) protein of NDV has multiple functions including mediating hemadsorption (HAD), neuraminidase (NA), and fusion promotion activities affecting the process of viral attachment, entry, replication and dissemination. Fusion ability of the NDV was highly correlated to its virulence. Mutations in the HN globular head and headless HN of NDV were constructed to determinate the impact of highly conserved amino acids in the globular head of paramyxovirus HN proteins and the roles of the stalk region of HN in the fusion process. It was found that the interaction between F and HN mutants E401A, G402A, G468A, V469A, Y526A, and T527A was equal to that in F and wt HN. The mutations of G402A, G468A, V469A, and T527A had various effects on cell fusion promotion, receptor binding ability, and NA activity, but the membrane merging rate was comparable to wt HN. The elimination of hemadsorption ability and NA activity of E401A and Y526A resulted in the loss of the fusion promotion function of HN. The conclusion was that receptor binding and NA had a common active site and E401 and Y526 amino acids were essential for virus attachment, entry, and dissemination. In addition, G468A mutation made different contributions to HAD and NA, which indicated that G468 was one of the potential key amino acids in switching the two functions between receptor binding and sialic acid destruction of HN. It was also proven that the headless HN of NDV could promote the fusion event mediated by F. Thus, it revealed a novel mechanism in F activation of NDV.

To explore the effects of amino acids Gln and Asn within the specific fusion domain of fusion (F) protein on the specific membrane fusion in Newcastle disease virus (NDV), the mutants Q204E-Q205E and N245D were constructed in the specific fusion domain of F protein. The mutant genes were co-expressed with homologous or heterologous hemagglutinin-neuraminidase (HN) in BHK21 cells. Cell fusion functions of mutants were analyzed with Giemsa staining and reporter gene methods. Cell surface expression efficiency was analyzed with immunofluorescence assay and fluorescence-activated cell sorter analysis. Co-immunoprecipitation was performed to analyze the interaction of mutant F proteins with the homotypic HN protein. Both Q204E-Q205E and N245D mutations caused increased cell-cell fusion activity when they were co-expressed with homotypic HN protein. The mutant F proteins had slight changes in cell surface expression compared with that of wild-type F protein. The interactions of Q204E-Q205E or N245D with their homotypic HN increased significantly (P < 0.01) compared with the wild-type F protein. Neither Q204-Q205E nor N245D caused cell fusion in the presence of heterologous HN protein. Our data suggested that the residues Q204, Q205, and N245 play a critical role in the regulation of cell fusion. They may decrease the interaction of wild-type NDV F and NDV HN to suppress the fusion activity for survival of the infected host, which may enable a persistent virus infection and long-term virus reproduction and spread.

Objectives: To determine the effects of heptad repeat regions (HRs) and N-linked carbohydrate sites of the Newcastle disease virus hemagglutinin-neuraminidase (HN) protein on fusion of HN and fusion (F) proteins and HN-F interaction., Methods: We mutated six 'a' residues in the HRs and four asparagines in N-linked carbohydrate sites to alanine in the HN protein. A vaccinia-T7 RNA polymerase expression system was used to express HN cDNAs in BHK-21 cells to determine the HN functions. Deglycosylation was treated with PGNase F digestion. The formation of HN-F protein complexes was determined by the coimmunoprecipitation assay., Results: Each HR-mutated protein interfered with fusion and the HN-F interaction. The G4-mutated protein not only impaired fusion and HN-F interaction but also decreased activities in cell fusion promotion, hemadsorption and neuraminidase., Conclusions: It is assumed that two different mechanisms for mutations in these two regions are responsible for the decreased fusion promotion activity and the reduced ability of interaction with F protein. Mutations in the HRs impair fusion and HN-F interaction by altering the transmission of a signal from the globular domain to the F-specific region in the stalk, but the G4 mutation modulates fusion and HN-F interaction by the misfolding of some important structures., (Copyright © 2012 S. Karger AG, Basel.)

Objectives: To explore the effects of ectodomain sequences between HR1 and HR2 of F1 protein on the specific interaction with its homologous hemagglutinin-neuraminidase (HN) in paramyxoviruses., Methods: Site-directed mutagenesis was used to obtain mutants containing new enzyme sites on the F genes of Newcastle disease virus (NDV) and human parainfluenza virus (hPIV), and four DNA segments located between the HR1 and HR2 (NDV F-1, hPIV F-1, NDV F-2 and hPIV F-2) were obtained by cutting mutant F genes with specific endonucleases. Gene recombination was used to get chimeric F proteins NDV-C1 and hPIV-C1 by exchanging NDV F-1 and hPIV F-1 each other, and NDV-C2 and hPIV-C2 were also obtained by the same way. All the mutants and chimeric F proteins were co-expressed with their homologous or heterologous HN proteins in eukaryocytes. The fusion functions were assayed with Giemsa staining and reporter gene method for qualitative and quantitative analyses, respectively. The cell surface expression of F proteins was assayed with fluorescence-activated cell sorter (FACS) for quantitative analysis., Results: All the mutants of F proteins had the same functions as their relevant wild types. Chimeric F proteins NDV-C1 and hPIV-C1 had 76.34 and 65.82% of fusion activities, and NDV-C2 and hPIV-C2 had 96.25 and 93.78% of fusion activities, respectively, as compared with their relevant wild types. The analysis of FACS indicated that all the mutants and chimeric F proteins had almost the same expression efficiencies as their relevant wild types., Conclusions: The segments of NDV F-1 and hPIV F-1 were important for their specific membrane fusion, but NDV F-2 and hPIV F-2 were not., (2007 S. Karger AG, Basel)

Objectives: To determine the effects of heptad repeat regions (HRs) and N-linked carbohydrate sites of the Newcastle disease virus hemagglutinin-neuraminidase (HN) protein on fusion of HN and fusion (F) proteins and HN-F interaction. Methods: We mutated six 'a' residues in the HRs and four asparagines in N-linked carbohydrate sites to alanine in the HN protein. A vaccinia-T7 RNA polymerase expression system was used to express HN cDNAs in BHK-21 cells to determine the HN functions. Deglycosylation was treated with PGNase F digestion. The formation of HN-F protein complexes was determined by the coimmunoprecipitation assay. Results: Each HR-mutated protein interfered with fusion and the HN-F interaction. The G4-mutated protein not only impaired fusion and HN-F interaction but also decreased activities in cell fusion promotion, hemadsorption and neuraminidase. Conclusions: It is assumed that two different mechanisms for mutations in these two regions are responsible for the decreased fusion promotion activity and the reduced ability of interaction with F protein. Mutations in the HRs impair fusion and HN-F interaction by altering the transmission of a signal from the globular domain to the F-specific region in the stalk, but the G4 mutation modulates fusion and HN-F interaction by the misfolding of some important structures. Copyright © 2012 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]