LaSota fusion (F) cleavage motif-mediated fusion activity is affected by other regions of the F protein from different genotype Newcastle disease virus in a chimeric virus: implication for virulence attenuation

Abstract

The cleavage site sequence of the fusion (F) protein contributes to a wide range of virulence of Newcastle disease virus (NDV). In this study, we identified other important amino acid sequences of the F protein that affect cleavage and modulation of fusion. We generated chimeric Beaudette C (BC) viruses containing the cleavage site sequence of avirulent strain LaSota (Las-Fc) together with various regions of the F protein of another virulent strain AKO. We found that the F1 subunit is important for cleavage inhibition. Further dissection of the F1 subunit showed that replacement of four amino acids in the BC/Las-Fc protein with their AKO counterparts (T341S, M384I, T385A and I386L) resulted in an increase in fusion and replication in vitro. In contrast, the mutation N403D greatly reduced cleavage and viral replication, and affected protein conformation. These findings will be useful in developing improved live NDV vaccines and vaccine vectors.Virulent strains of Newcastle disease virus (NDV) cause a devastating disease in chickens leading to major economic losses in the poultry industry worldwide (Alexander, 1989). NDV is a member of the genus Avulavirus in the family Paramyxoviridae. The virion is enveloped, with a non-segmented, negative-sense ssRNA genome (Samal, 2011). The genome encodes a nucleocapsid protein (N), a phosphoprotein (P), a matrix protein (M), a fusion protein (F), a haemagglutinin-neuraminidase protein (HN) and a large polymerase protein (L).NDV isolates vary greatly in their pathogenicity for chickens and are categorized into three main pathotypes: lentogenic (avirulent), mesogenic (moderately virulent) and velogenic (highly virulent) (Alexander, 1989). The amino acid sequence at the F protein cleavage site has been identified as the primary determinant of virulence (Panda et al., 2004;Peeters et al., 1999). Virulent NDV strains have multibasic cleavage sequences that contain the preferred cleavage site of the intracellular protease furin (Arg-X-Arg/Lys-Arg # ), available in most cell types. In contrast, avirulent NDV strains typically contain one or two basic residues at the F protein cleavage site and are delivered to the plasma membrane in an uncleaved form for cleavage by extracellular proteases. Modification of the cleavage site sequence in the F protein has been shown to greatly alter the replication and pathogenicity of NDV (de Leeuw et al., 2005;Hu et al., 2011;Panda et al., 2004;Peeters et al., 1999; Xiao et al., 2012). In our previous study, we modified the F protein cleavage site sequence (RRQKR # F) of a highly virulent NDV strain Ban010 (genotype VII) to that of avirulent NDV strain LaSota (GRQGR # L), resulting in an avirulent derivative rBan/AF (Xiao et al., 2012). The parental virulent Ban010 virus produced extensive syncytia and plaque in cell culture in the absence of added protease, whereas the avirulent rBan/AF derivative caused only single-cell infections without syncytia or plaque formation in the presence or absence of extracellular protease. This result was unexpecte...

Two mutations in the HR2 region of Newcastle disease virus fusion protein with a cleavage motif “RRQRRL” are critical for fusogenic activity

“…However, the fusion level caused by a substitution construct La-F*/F48(118–499) was similar to that of F48-F* (data not shown), suggesting that except for residues D479 and S486, other amino acids in the 118–499 region of F48-F* might act synergistically to achieve the high level of fusion induced by La-F*. A recent study reported that the mutation N403D in the F protein of mesogenic strain BC with the avirulent Fcs “GRQGR↓L” substantially reduced the fusion activity [ 17 ]. Another report showed that the single mutation T458D or G459D in the HR2 region of the F protein in the virulent G7 strain enhanced cell fusion [ 24 ].…”

“…Alteration of the Fcs affects the fusion activity and virulence of NDV [ 6 , 11 – 14 ]. However, some strains and modified viruses containing an identical Fcs motif present varied fusogenic activity [ 15 – 17 ]. In our study, we observed that mutation of the F proteins of the virulent strain F48E9 and the avirulent strain LaSota to an identical motif, “RRQRR↓L”, altered membrane fusion in transfected cells, suggesting that other regions of the F protein are involved in inducing fusion in addition to the Fcs.…”

Roles of the Polymerase-Associated Protein Genes in Newcastle Disease Virus Virulence

“…The outer surface glycoproteins have been shown to play an important role in the virulence of NDV ( Panda et al, 2004 ; Wakamatsu et al, 2006 ; Vigerust and Shepherd, 2007 ; Kim et al, 2011 , 2016 ; Hu et al, 2012 ). We first confirmed the roles of the F and HN envelope glycoproteins in NDV virulence by reciprocally exchanging them in combination between the velogenic genotype VII strain SG10 and the lentogenic genotype II strain LaSota.…”

Efficacy of a Recombinant Genotype VII Vaccine against Challenge with Velogenic Newcastle Disease Virus

“…However, there is a potential risk for the vaccine virus regaining its virulence during massive vaccination campaign in the field [12]. Also, virus shedding of the live vaccines was constantly reported [13]. Therefore, development of inactivated vaccine is necessary if it could provide good protection as live vaccine does but reduce virus shedding.…”