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, 7 (11), e49968

Passage of a Sendai Virus Recombinant in Embryonated Chicken Eggs Leads to Markedly Rapid Accumulation of U-to-C Transitions in a Limited Region of the Viral Genome

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Passage of a Sendai Virus Recombinant in Embryonated Chicken Eggs Leads to Markedly Rapid Accumulation of U-to-C Transitions in a Limited Region of the Viral Genome

Asuka Yoshida et al. PLoS One.

Abstract

The P gene of paramyxoviruses is unique in producing not only P but also "accessory" C and/or V proteins. Successful generation of C- or V-deficient recombinant viruses using a reverse genetics technique has been revealing their importance in viral pathogenesis as well as replication. As for Sendai virus (SeV), the C proteins, a nested set of four polypeptides C', C, Y1, and Y2, have been shown to exert multiple functions in escaping from the host innate immunity, inhibiting virus-induced apoptosis, promoting virus assembly and budding, and regulating viral RNA synthesis. In this study, we subjected the 4C(-) recombinant lacking expression of all four C proteins to serial passages through eggs, and found the rapid emergence of a C-recovered revertant virus. Unlike the SeV strains or the recombinants reported previously or tested in this study, this was caused by an exceptionally quick accumulation of U-to-C transitions in a limited region of the 4C(-) genome causing recovery of the C protein expression. These results suggest that a lack of C proteins could lead unexpectedly to strong selective pressures, and that the C proteins might play more critical roles in SeV replication than ever reported.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Characteristics of the recombinant 4C(-) virus used in this study.
(A) Sequences around the initiation codons (underlined) of the P, C’, C, Y1 and Y2 of WT and 4C(-) are shown, and mutated codons to silence the C related proteins in the 4C(-) sequence are highlighted in bold. (B) Schematic representation of C protein of the 4C(-) recombinant. Amino acid changes for the 4C(-) mutant are indicated.
Figure 2
Figure 2. Passage history and profiles of C protein expression for the 4C(-) virus.
(A) The P2 allantoic fluid stock harvested from eggs after two passages of the 4C(-) transfectant recovered from cDNA was further passed through eggs in three individual experiments. The enclosed indicate the fluid stocks with C protein expression. (B, C) LLC-MK2 cells were infected with the indicated viruses and analyzed by Western blotting using anti-P and C pAbs. (D) The amount of each C protein detected in (C) was normalized to that of P protein. The relative ratios of C to P proteins are shown as bar graphs and the ratio of S3-P8 set to 1.
Figure 3
Figure 3. Growth kinetics of the WT and 4C(-) viruses.
LLC-MK2 cells were infected with WT, S3-P4, or S3-P8 at an MOI of 5, and infectivity was determined at various time points.
Figure 4
Figure 4. Nucleotide substitutions during serial passages of the S3 series stocks.
(A) The regions of C ORF containing stop codons introduced in the 4C(-) virus are shown. (B) The electrographs of the region shown in (A) of the S3 series stocks. Appreciable A-to-G (U-to-C) transition substitutions are indicated as arrows, and among these, those causing reversions of introduced stop codons are indicated as stars.
Figure 5
Figure 5. Distribution of nucleotide and amino acid substitutions observed in the S3-P8 virus stock.
(A) Alignment of the 1,801 - 2,100-nt region of S3-P8 with that of the original 4C(-). Initiation codons for P, C, Y1, and Y2 proteins are underlined. Nucleotides of S3-P8 identical to that of 4C(-) are indicated as asterisks. (B) Schematic representation of a 3,700-nt region containing N and P genes of S3-P8. Arrows present positions of nucleotide substitutions. (C) Schematic representations of P and C polypeptides of S3-P8. Arrows present positions of amino acid substitutions compared to that of the original 4C(-). Stars indicate reversions of stop codons introduced into the 4C(-) genome.
Figure 6
Figure 6. Occurrence rates of nucleotide substitutions.
RT-PCR products including 1,801 - 2,100-nt regions were prepared using bulk RNAs prepared from indicated virus stocks and subcloned into pUC18 plasmids. About twenty clones of (A) WT, (B) S3-P6, (C) S3-P7 and (D) S3-P8 were sequenced and compared to those of the WT or original 4C(-). The occurrence rate of each detected nucleotide substitution is shown as bars over (for U-to-C mutations) and under (for the other types of mutations) the line of sequences of the WT or original 4C(-).
Figure 7
Figure 7. The Occupancy rate of the C-recovered mutants in each indicated virus fluid stock was shown as bar graphs.
Figure 8
Figure 8. Distribution of nucleotide and amino acid substitutions observed in the WT and other recombinant virus stocks.
(A) Schematic representation of regions containing N and P genes of indicated virus stocks. Arrows present positions of nucleotide substitutions. Schematic representations of (B) P and (C) C polypeptides. Arrows present positions of amino acid changes compared to that of the original 4C(-), WT, F170S or V(-). Stars indicated reversions of the stop codons introduced in the 4C(-) virus.

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Grant support

This work was supported by JSPS KAKENHI Grant Number 23790505. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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