Conserved amino acids around the DIII-DI linker region of the Newcastle disease virus fusion protein are critical for protein folding and fusion activity

Abstract

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.

Genome-wide transposon mutagenesis of paramyxoviruses reveals constraints on genomic plasticity

“…F-ORF-5367 and F-ORF-5384, S5 Table). These insertants correspond to the hinge region between domains III and I in the fusion protein, which has been implicated in fusion regulation [17], Thus, we identified insertants that specifically compensated for the alteration of the highly conserved A138 residue in the F-protein fusion peptide.…”

Residues 315 and 369 in HN Protein Contribute to the Thermostability of Newcastle Disease Virus

“…The fusion activities of the NDVs were examined as previously described ( Jin et al, 2016 ; Chi et al, 2019 ). Briefly, 10 5 TCID 50 /mL of NDVs were heat-treated at 56°C for 10 min, and then infected with Vero cells for 2 h at 37°C.…”

“…The fusion activities of the NDVs were examined as previously described (Jin et al, 2016;Chi et al, 2019). Briefly, 10 5 TCID 50 /mL of NDVs were heat-treated at 56 • C for 10 min, and then infected with Vero cells for 2 h at 37 • C. Then the infected cells supernatant was discarded and kept in the DMEM (Gibco) supplemented with 2% FBS.…”

YLMY Tyrosine Residue within the Cytoplasmic Tail of Newcastle Disease Virus Fusion Protein Regulates Its Surface Expression to Modulate Viral Budding and Pathogenicity

“…Mutations G377S, A378D, and L379A in the DI-DII linker of the F protein induced much larger syncytia than the wild type, but with F protein expression at the cell surface comparable to that of the wild type. A possible explanation is that this mutant may have had an influence on the interaction between HN and F ( 6 , 39 ). Further experiments are needed to ascertain whether a specific mechanism exists through which the YLMY motifs regulate F-mediated fusion.…”

“…Before participating in fusion, the F protein is initially synthesized by NDV as the inactive precursor F 0 , which has to be cleaved to become the disulfide-bonded F 1 protein and F 2 active complexed form ( 5 ). The F 1 subunit contains two hydrophobic regions, the fusion peptide (FP), which resides at the new N-terminal after cleavage, and the transmembrane (TM) domain, which anchors the protein to the membrane of a virus or target cell, as well as two heptad repeat (HR) regions, HRA and HRB ( 6 ). All of these protein architectures are ectodomains, which are known to be important structural features of the F protein ectodomain.…”