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.

FIG. 1.

Recombinant NDV constructs. A schematic representation of the NDV gene order is shown in the negative-strand genomic RNA. The nucleotide sequences around the editing site (positions 2274 to 2291) are presented in a positive sense (5′ to 3′). The introduced nucleotide modifications (lowercase letters) and amino acid substitutions (one-letter code) in the P proteins of mutant viruses are shown in boldface.

FIG. 2.

Infectious titers of recombinant NDVs. Embryonated chicken eggs were inoculated at a dose of 4 log₁₀ EID₅₀ per egg and incubated for 4 days. Tenfold serial dilutions of the harvested allantoic fluid samples were prepared and inoculated into 9-to 11-day-old embryonated eggs (10 eggs/dilution). After 3 to 4 days of incubation, an HA test was carried out, and the calculated infectious titers in the log₁₀ EID₅₀/ml are shown on the top of the bars.

FIG. 3.

Pathogenicity of serially passaged NDV-P1 in SPF chicken embryos. Embryonated eggs were inoculated with the parent virus at passage 3 (rNDV) or the mutant NDV-P1 at passage 5 (P1-5), passage 6 (P1-6), or passage 7 (P1-7) and then incubated for 7 days or until the embryos had died. NDV-P1, which was completely safe for 11-day-old chicken embryos at passage 5, became nearly as virulent as the parent virus at passage 7.

FIG. 4.

Pattern of P-gene mRNA editing in cells infected with serially passaged NDV-P1. The sequences around the editing site from independent clones (Table 1) derived from rNDV or NDV-P1 at passage 5 (P1-5), passage 6 (P1-6), or passage 7 (P1-7) were determined. The percentages of unedited mRNAs representing P ORF (P) or edited mRNAs with insertion of one G residue coding for V ORF (V) or with insertions of two G residues coding for W ORF (W) are shown on the top of the bars.

FIG. 5.

(A) Infectious titers of recombinant NDV-PC4 at passages 3 and 7. Titers were determined as described under the legend for Fig. 2. (B) Pathogenicity of NDV-PC4 at passage 3 (PC4-3) and passage 7 (PC4-7) in SPF chicken embryos. Mortality was determined as described in the legend for Fig. 3. (C) Editing frequency of NDV-PC4 at passages 3 (PC4-3) and passage 7 (PC4-7). mRNA editing was determined from independent clones (Table 1) as described under the legend for Fig. 4.

FIG. 5.

(A) Infectious titers of recombinant NDV-PC4 at passages 3 and 7. Titers were determined as described under the legend for Fig. 2. (B) Pathogenicity of NDV-PC4 at passage 3 (PC4-3) and passage 7 (PC4-7) in SPF chicken embryos. Mortality was determined as described in the legend for Fig. 3. (C) Editing frequency of NDV-PC4 at passages 3 (PC4-3) and passage 7 (PC4-7). mRNA editing was determined from independent clones (Table 1) as described under the legend for Fig. 4.

FIG. 5.

(A) Infectious titers of recombinant NDV-PC4 at passages 3 and 7. Titers were determined as described under the legend for Fig. 2. (B) Pathogenicity of NDV-PC4 at passage 3 (PC4-3) and passage 7 (PC4-7) in SPF chicken embryos. Mortality was determined as described in the legend for Fig. 3. (C) Editing frequency of NDV-PC4 at passages 3 (PC4-3) and passage 7 (PC4-7). mRNA editing was determined from independent clones (Table 1) as described under the legend for Fig. 4.

FIG. 6.

Comparison of putative mRNA-template hybrids of rNDV and NDV-T2 during the editing process. The template negative-strand genomes (top strands) were written 3′ to 5′, and the mRNA chains (bottom strands) were written 5′to 3′. The editing frequency of both viruses for P, V, and W mRNAs are shown in percentage. The only difference between the two viruses at position 2278 (C-U₂₂₇₈) was shown in boldface lowercase. The unique putative base pairings are underlined. As much as 85% of P-gene derived mRNAs in NDV-T2 are edited, whereas only 32% of the mRNAs in the parent virus represent V and W mRNAs.