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A Single Amino Acid Substitution Within the Paramyxovirus Sendai Virus Nucleoprotein Is a Critical Determinant for Production of Interferon-Beta-Inducing Copyback-Type Defective Interfering Genomes

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A Single Amino Acid Substitution Within the Paramyxovirus Sendai Virus Nucleoprotein Is a Critical Determinant for Production of Interferon-Beta-Inducing Copyback-Type Defective Interfering Genomes

Asuka Yoshida et al. J Virol.

Abstract

One of the first defenses against infecting pathogens is the innate immune system activated by cellular recognition of pathogen-associated molecular patterns (PAMPs). Although virus-derived RNA species, especially copyback (cb)-type defective interfering (DI) genomes, have been shown to serve as real PAMPs, which strongly induce interferon-beta (IFN-β) during mononegavirus infection, the mechanisms underlying DI generation remain unclear. Here, for the first time, we identified a single amino acid substitution causing production of cbDI genomes by successful isolation of two distinct types of viral clones with cbDI-producing and cbDI-nonproducing phenotypes from the stock Sendai virus (SeV) strain Cantell, which has been widely used in a number of studies on antiviral innate immunity as a representative IFN-β-inducing virus. IFN-β induction was totally dependent on the presence of a significant amount of cbDI genome-containing viral particles (DI particles) in the viral stock, but not on deficiency of the IFN-antagonistic viral accessory proteins C and V. Comparison of the isolates indicated that a single amino acid substitution found within the N protein of the cbDI-producing clone was enough to cause the emergence of DI genomes. The mutated N protein of the cbDI-producing clone resulted in a lower density of nucleocapsids than that of the DI-nonproducing clone, probably causing both production of the DI genomes and their formation of a stem-loop structure, which serves as an ideal ligand for RIG-I. These results suggested that the integrity of mononegaviral nucleocapsids might be a critical factor in avoiding the undesirable recognition of infection by host cells.IMPORTANCE The type I interferon (IFN) system is a pivotal defense against infecting RNA viruses that is activated by sensing viral RNA species. RIG-I is a major sensor for infection with most mononegaviruses, and copyback (cb)-type defective interfering (DI) genomes have been shown to serve as strong RIG-I ligands in real infections. However, the mechanism underlying production of cbDI genomes remains unclear, although DI genomes emerge as the result of an error during viral replication with high doses of viruses. Sendai virus has been extensively studied and is unique in that its interaction with innate immunity reveals opposing characteristics, such as high-level IFN-β induction and strong inhibition of type I IFN pathways. Our findings provide novel insights into the mechanism of production of mononegaviral cbDI genomes, as well as virus-host interactions during innate immunity.

Keywords: Sendai virus; defective interfering genome; innate immunity; interferons; nucleocapsid; paramyxovirus.

Figures

FIG 1
FIG 1
Comparison of SeV strains in terms of the content of cbDI particles in their working stocks, induction of IFN-β and the antiviral state, and counteraction against the type I IFN pathway. (A) The amounts of cbDI genomes and fullGNM in the working stocks of the indicated viruses were analyzed by one-step qRT-PCR. The cbDI/fullGNM ratios are shown. The ratio of the wZ sample was set to 1. The amounts of IFN-β and beta-actin mRNAs in the infected HeLa cells were also analyzed by one-step qRT-PCR in cells infected with the indicated viruses. The ratios of IFN-β to beta-actin mRNAs are shown. The ratio in the uninfected sample was set to 1. (B) HeLa cells were infected with the indicated viruses. At 6 h p.i., the media were replaced with fresh media containing IFN-α (1,000 IU/ml) or no IFN-α. After an additional 6-h incubation, the cells were superinfected with rVSV-GFP. After further incubation for 6 h, GFP expression derived from rVSV-GFP replication was observed under a fluorescence microscope. (C) Western blotting of the cell lysate samples in panel B using anti-GFP, -SeV P, and -SeV C polyclonal antibodies. (D) Schematic representation of the C proteins of the indicated SeV strains. Dashes indicate that the amino acids are identical to those of Z strain. (E) 293T cells were cotransfected with the C proteins derived from the indicated SeV strains, together with a reporter plasmid, pISRE-EGFP. At 18 h p.t., the cells were treated with IFN-α (1,000 IU/ml) for 8 h, and then the expression level of EGFP was analyzed by Western blotting using an anti-GFP antibody. The amount of EGFP in the cell lysates was quantitated and graphed. The value of mock-transfected and non-IFN-α-treated samples was set to 1. (F) 293T cells were cotransfected with MDA5-WT or -CA, together with a reporter plasmid, p55C1B-EGFP. At 24 h p.t., GFP fluorescence was measured using a fluorometer. (G) 293T cells were cotransfected with the V or P proteins derived from the indicated SeV strains, together with a reporter plasmid, p55C1B-EGFP, and pCAG-FL-MDA5-CA. At 24 h p.t., the expression level of EGFP was analyzed by Western blotting using an anti-GFP antibody. The amounts of EGFP in the cell lysates were quantitated and graphed. The value of the sample receiving FL-MDA5-CA but no V protein was set to 1. (H) HeLa cells were infected with SeV strain Z at an MOI of 5. At 6 h p.i., the cells were superinfected with strain Cantell, Z-4C(−), or Z-V(−). After an additional 24 h of incubation, the amounts of IFN-β and beta-actin mRNAs were analyzed by one-step qRT-PCR. The ratios of IFN-β to beta-actin mRNAs are shown. The ratio in the cells without superinfection was set to 1. All of the bar graphs represent the averages of three independent experiments, and the error bars represent the standard deviations. n.s., nonsignificant (P > 0.05); **, P < 0.01 by one-way analysis of variance (ANOVA) with Bonferroni post hoc test.
FIG 2
FIG 2
Identification of cbDI genomes in the wC stock. (A) RT-PCR was performed using a virion RNA sample from the wC stock as a template and Tr80CNT as a primer. The RT-PCR product was analyzed by agarose gel electrophoresis. (B) Schematic representation of the major species of the H4-type cbDI genome.
FIG 3
FIG 3
Characterization of viral clones (Cln) isolated from the wZ and wC stocks. (A) The stocks of wZ and wC were applied to three consecutive limiting dilutions as described in Materials and Methods. Representative viral samples are shown with the viral titers. (B) The relative cbDI/fullGNM ratios of the clones, as well as the parental viral stocks, are shown as in Fig. 1A. The ratio of the wZ sample was set to 1.
FIG 4
FIG 4
Effects of serial passage of the viral clones on the cbDI genome content and IFN-β inducibility. (A) Ratios of IFN-β to beta-actin mRNA in cells infected with the indicated viruses. The ratio in the uninfected sample was set to 1. (B and C) The indicated clones were passaged through Vero-TMPRSS2 cells at an MOI of 10 (B) and a volume of 200 μl (C) five times. The cbDI/fullGNM ratio of each sample is shown as in Fig. 1A. The ratio in the wZ stock was set to 1. (D) Ratios of IFN-β to beta-actin mRNAs shown as in Fig. 1A. The ratio in the uninfected sample was set to 1.
FIG 5
FIG 5
Density gradient fractionation of the nucleocapsids purified from the SeV clones. (A) Nucleocapsids of the indicated viruses were purified from virion samples as described in Materials and Methods and analyzed by SDS-10% PAGE. The densities of the N and P protein bands were measured by densitometry, and the P/N ratios relative to that of cZ are shown. (B) The purified nucleocapsid samples were subjected to 20% to 45% CsCl density gradient ultracentrifugation. Then, 21 fractions were collected from the top of the sample and analyzed by Western blotting using anti-SeV antibody to detect the nucleocapsid-associated N protein. (C) The amount of N protein in each fraction was quantitated, and the ratio of each fraction to the sum of all the fractions was plotted against the density of each fraction. (D) N mutants summarized schematically. The strength of the intermolecular interactions of each of the N mutants was examined using a bimolecular fluorescence complementation technique as described in Materials and Methods. The fluorescence intensity observed in the N-WT sample was set to 1. The graphs represent the averages of three independent experiments, and the error bars represent standard deviations. n.s., nonsignificant (P > 0.05); *, P < 0.05 by one-way ANOVA with Bonferroni post hoc test.
FIG 6
FIG 6
Effects of N mutations on production of cbDI genomes during viral replication. (A) Viral genome structures of the N-mutated recombinants summarized schematically. One-step growth titers of the N recombinants in LLC-MK2 cells at 48 h p.i. were determined as described in Materials and Methods. (B) cbDI/fullGNM ratios in cells infected with the indicated viruses shown as in Fig. 1A. The ratio in the wZ stock was set to 1. (C) Effect of transcomplementation of the N mutants on production of the cbDI genomes during cC virus replication. At 6 h p.i. of the cC clone on LLC-MK2 cells, the indicated pCAGGS-N plasmids were transfected. At 48 h after cC infection, the cbDI/fullGNM ratios in the cells were analyzed and shown as in Fig. 1A; the error bars represent standard deviations.
FIG 7
FIG 7
(A and B) Survival curves (A) and percent weight change (B) over a 2-week period for mice infected with cC and cCdi at the indicated doses. (C) Amounts of IFN-β in BAL fluids harvested from mice infected with the indicated viruses at 1 day p.i. determined as described in Materials and Methods. n.s., nonsignificant (P > 0.05); ***, P < 0.001 by two-way ANOVA with Bonferroni post hoc test; the error bars represent standard deviations.

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