Mutations in the Cytoplasmic Domain of the Newcastle Disease Virus Fusion Protein Confer Hyperfusogenic Phenotypes Modulating Viral Replication and Pathogenicity

Abstract

bThe Newcastle disease virus (NDV) fusion protein (F) mediates fusion of viral and host cell membranes and is a major determinant of NDV pathogenicity. In the present study, we demonstrate the effects of functional properties of F cytoplasmic tail (CT) amino acids on virus replication and pathogenesis. Out of a series of C-terminal deletions in the CT, we were able to rescue mutant viruses lacking two or four residues (r⌬2 and r⌬4). We further rescued viral mutants with individual amino acid substitutions at each of these four terminal residues (rM553A, rK552A, rT551A, and rT550A). In addition, the NDV F CT has two conserved tyrosine residues (Y524 and Y527) and a dileucine motif (LL536-537). In other paramyxoviruses, these residues were shown to affect fusion activity and are central elements in basolateral targeting. The deletion of 2 and 4 CT amino acids and single tyrosine substitution resulted in hyperfusogenic phenotypes and increased viral replication and pathogenesis. We further found that in rY524A and rY527A viruses, disruption of the targeting signals did not reduce the expression on the apical or basolateral surface in polarized Madin-Darby canine kidney cells, whereas in double tyrosine mutant, it was reduced on both the apical and basolateral surfaces. Interestingly, in rL536A and rL537A mutants, the F protein expression was more on the apical than on the basolateral surface, and this effect was more pronounced in the rL537A mutant. We conclude that these wild-type residues in the NDV F CT have an effect on regulating F protein biological functions and thus modulating viral replication and pathogenesis. N ewcastle disease virus (NDV) is a highly prevalent avian pathogen that infects essentially all species of birds and is of major economic importance to the poultry industry (1, 2). The disease varies in degree of severity, ranging from an inapparent infection to outbreaks of severe respiratory and neurologic disease that can have 100% mortality. Based on severity of the disease, NDV strains are grouped into lentogenic (nonpathogenic or mildly pathogenic), mesogenic (moderately pathogenic), and velogenic (highly pathogenic) pathotypes (3). NDV belongs to the genus Avulavirus within the family Paramyxoviridae, a family of enveloped, nonsegmented, negative-sense RNA viruses (4).The envelope of NDV contains two surface glycoproteins, HN and F (5,6). HN mediates viral attachment by binding to sialic acid cellular receptors, and F protein facilitates viral entry into the cells by fusing the viral envelope with the host cell membrane (7,8). The virus fusion process involves a series of major coordinated conformational changes in the F protein that bring together and merges the opposing membranes (5, 9).The NDV F protein ( Fig. 1) is synthesized as an inactive precursor, F 0 (66 kDa), that is cleaved posttranslationally by host cell proteases into two disulfide-linked subunits, N-terminal F 2 (12.5 kDa) and C-terminal F 1 (55 kDa) (8, 10). The F cleavage site is a major determinant of NDV tropism and viru...

Phylogeny, pathogenicity and transmissibility of a genotype XII Newcastle disease virus in chicken and goose

“…Aside from those at the cleavage site, it has been shown that mutations in other regions of the NDV F protein can also affect viral replication and pathogenicity (Ji et al, ; Samal, Khattar, Kumar, Collins, & Samal, ; Samal et al, ). We found that the F proteins of all subgenotype XIIb NDVs from China contained the substitutions N240S and T496I, unlike the other genotype XII NDVs from Peru and Vietnam and the genotype VII and IX NDVs.…”

The Epstein-Barr Virus (EBV) Glycoprotein B Cytoplasmic C-Terminal Tail Domain Regulates the Energy Requirement for EBV-Induced Membrane Fusion

“…Studies of the CTDs of paramyxovirus fusion proteins and the HSV-1 gB CTD, as well as our study of the EBV gB CTD, indicate that this domain has an important regulatory role in fusion activation. Both hyperfusogenic and hypofusogenic mutations can be found within the CTD (22,(43)(44)(45)(46)(47)(48)(49). A recent study with the HSV-1 gB CTD suggests that the hyperfusion phenotype is associated with diminished membrane interactions (29).…”

A Y527A mutation in the fusion protein of Newcastle disease virus strain LaSota leads to a hyperfusogenic virus with increased replication and immunogenicity

“…Several reports have also indicated a role for the cytoplasmic tail (CT) of F protein in virus entry, F protein cleavage, and fusogenicity (Morrison, 2003;Lamb & Parks, 2007;Chen et al, 2001). It has been found in other enveloped viruses that tyrosinecontaining signals, especially Y-X-X-aliphatic/aromatic consensus motifs, in the CT of viral membrane proteins are associated with the transport and delivery of proteins (Samal et al, 2013;Ball et al, 1997;Brewer & Roth,strains of NDV (Dolganiuc et al, 2003). We have previously shown that mutations in the two conserved tyrosine (Y524 and Y527) residues in the cytoplasmic domain of the F protein of moderately virulent NDV strain Beaudette C conferred a hyperfusogenic phenotype, increasing viral replication and pathogenicity (Samal et al, 2013).…”