Identification of a Mutation in Editing of Defective Newcastle Disease Virus Recombinants That Modulates P-Gene mRNA Editing and Restores Virus Replication and Pathogenicity in Chicken Embryos

Abstract

Editing of P-gene mRNA of Newcastle disease virus (NDV) enables the formation of two additional proteins (V and W) by inserting one or two nontemplated G residues at a conserved editing site (5-AAAAAGGG). The V protein of NDV plays an important role in virus replication and is also a virulence factor presumably due to its ability to counteract the antiviral effects of interferon. A recombinant virus possessing a nucleotide substitution within the A-stretch (5-AAgAAGGG) produced 20-fold-less V protein and, in consequence, was impaired in replication capacity and completely attenuated in pathogenicity for chicken embryos. However, in a total of seven serial passages, restoration of replication and pathogenic capacity in 9-to 11-day-old chicken embryos was noticed. Determining the sequence around the editing site of the virus at passage 7 revealed a C-to-U mutation at the second nucleotide immediately upstream of the 5-A 5 stretch (5-GuUAAgAAGGG). The V mRNA increased from an undetectable level at passage 5 to ca. 1 and 5% at passages 6 and 7, respectively. In addition, similar defects in another mutant possessing a different substitution mutation (5-AAAcAGGG) were restored in an identical manner within a total of seven serial passages. Introduction of the above C-to-U mutation into the parent virus (5-GuUAAAAAGGG) altered the frequency of P, V, and W mRNAs from 68, 28, and 4% to 15, 44, and 41%, respectively, demonstrating that the U at this position is a key determinant in modulating P-gene mRNA editing. The results indicate that this second-site mutation is required to compensate for the drop in edited mRNAs and consequently to restore the replication capacity, as well as the pathogenic potential, of editing-defective NDV recombinants. Newcastle disease virus (NDV) belongs to the new genusAvulavirus within the family Paramyxoviridae. NDV is the causative agent of one of the most devastating diseases of poultry worldwide. The negative-strand RNA virus genome of NDV encodes six genes (in the order 3Ј-NP-P-M-F-HN-L-5Ј). Except for the P gene mRNA, all express one major viral structural protein from a single open reading frame (ORF). The P gene mRNAs of the subfamily Paramyxovirinae express several proteins by the use of alternative reading frames and by a process of mRNA editing. The mRNA editing, which involves insertion of pseudotemplated G residue(s) occurs by a polymerase stuttering mechanism (10,11,24). Like in other members of paramyxovirinae, the P gene mRNA of NDV is edited by inserting one or two G residues into a G run within a conserved editing locus (5Ј-AAAAAGGG) (16, 21). As a result, three P gene derived mRNA species are produced which encode for the P, V, and W ORFs (unedited, with ϩ1 and ϩ2 frameshifts, respectively). The V and W proteins are colinear to the N-terminal half of the P protein but have different carboxy-terminal parts.The majority of the accessory proteins, including V and/or C proteins encoded by the P genes of different members of the subfamily Paramyxovirinae have been shown to be d...

Nipah Virus Edits Its P Gene at High Frequency To Express the V and W Proteins

“…By comparison, a recent report on four strains of measles virus shows a combined editing frequency of 42%, with 60% of transcripts encoding P, 35% of transcripts encoding V, and the remaining 5% encoding the hypothetical W protein (1). Similar numbers have been reported for Newcastle disease virus (15). The only other paramyxoviruses known to hyperedit their P genes are human and bovine parainfluenza virus 3 (PIV3) viruses, which predominantly insert 1 to 6 G residues at equal frequency, although up to 12 insertions have been documented (6,19).…”

Distinct and Overlapping Roles of Nipah Virus P Gene Products in Modulating the Human Endothelial Cell Antiviral Response

“…A potential explanation for this could be that the site in which the V ORF early stop codon was incorporated was just several nucleotides from the putative editing site, which may have negatively affected the RNA editing frequency. While mutagenesis studies of nucleotides 5′ to the editing site have been performed for several paramyxoviruses [63] , [64] , the incorporation of early stop codons for paramyxovirus V proteins however has not definitively been shown to alter RNA editing frequency [53] , [54] . On the other hand, another possibility could be that certain residues in the unique C-terminus of the NiV V protein itself has a role in facilitating RNA editing, as it has been shown for Sendai virus [65] .…”

Signaling through RIG-I and type I interferon receptor: Immune activation by Newcastle disease virus in man versus immune evasion by Ebola virus (Review)

“…The V protein is a frameshift variant of the NDV phosphoprotein P. and, while the P protein consists of 395 amino acids, the V protein consists of only 239 amino acids. The incorporation of two G nucleotides at the RNA editing site of the P protein results in the frameshift variant protein V (13). The V protein of NDV has been shown to inhibit the IFN response in birds in two ways: i) through the inhibition of IFN signaling by targeting STAT1 for degradation (14); and ii) through interaction with melanoma differentiation-associated gene 5 (MDA5), leading to the inhibition of interferon regulatory factor 3 (IRF-3) activation and IFN-β induction (15).…”