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Coronavirus non-structural protein 1 is a major pathogenicity factor: implications for the rational design of coronavirus vaccines.

Züst R, Cervantes-Barragán L, Kuri T, Blakqori G, Weber F, Ludewig B, Thiel V - PLoS Pathog. (2007)

Bottom Line: Attenuated viral vaccines can be generated by targeting essential pathogenicity factors.The effect of nsp1 on MHV replication in vitro and in vivo was analyzed using a recombinant MHV encoding a deletion in the nsp1-coding sequence.The recombinant MHV nsp1 mutant grew normally in tissue culture, but was severely attenuated in vivo.

View Article: PubMed Central - PubMed

Affiliation: Research Department, Kantonal Hospital St. Gallen, St. Gallen, Switzerland.

ABSTRACT
Attenuated viral vaccines can be generated by targeting essential pathogenicity factors. We report here the rational design of an attenuated recombinant coronavirus vaccine based on a deletion in the coding sequence of the non-structural protein 1 (nsp1). In cell culture, nsp1 of mouse hepatitis virus (MHV), like its SARS-coronavirus homolog, strongly reduced cellular gene expression. The effect of nsp1 on MHV replication in vitro and in vivo was analyzed using a recombinant MHV encoding a deletion in the nsp1-coding sequence. The recombinant MHV nsp1 mutant grew normally in tissue culture, but was severely attenuated in vivo. Replication and spread of the nsp1 mutant virus was restored almost to wild-type levels in type I interferon (IFN) receptor-deficient mice, indicating that nsp1 interferes efficiently with the type I IFN system. Importantly, replication of nsp1 mutant virus in professional antigen-presenting cells such as conventional dendritic cells and macrophages, and induction of type I IFN in plasmacytoid dendritic cells, was not impaired. Furthermore, even low doses of nsp1 mutant MHV elicited potent cytotoxic T cell responses and protected mice against homologous and heterologous virus challenge. Taken together, the presented attenuation strategy provides a paradigm for the development of highly efficient coronavirus vaccines.

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Effect of nsp1 on IFN-α Production and SignalingC57BL/6 bone marrow–derived (A) or primary (B) splenic cDCs or pDCs were infected with MHV-nsp1Δ99 or MHV-A59 at an MOI of 1. IFN-α secreted into cell culture supernatants was determined by ELISA at the indicated time points. Bone marrow–derived cDCs (C) or inflammatory macrophages (D) from 129Sv mice were treated with 50, 200, 500, or 1,000 U IFN-α/100,000 cells or left untreated. Four hours later, cells were infected with MHV-nsp1Δ99 or MHV-A59 (MOI = 1). Twelve hours p.i., virus titers in culture supernatants were determined by plaque assay. Representative experiments out of two (A, B) or the mean ± SD of two independent experiments (C, D) are shown. Statistical analysis was performed using Student's t-test (***, p < 0.001; **, p < 0.01; *, p < 0.05; n.s., p > 0.05).
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ppat-0030109-g004: Effect of nsp1 on IFN-α Production and SignalingC57BL/6 bone marrow–derived (A) or primary (B) splenic cDCs or pDCs were infected with MHV-nsp1Δ99 or MHV-A59 at an MOI of 1. IFN-α secreted into cell culture supernatants was determined by ELISA at the indicated time points. Bone marrow–derived cDCs (C) or inflammatory macrophages (D) from 129Sv mice were treated with 50, 200, 500, or 1,000 U IFN-α/100,000 cells or left untreated. Four hours later, cells were infected with MHV-nsp1Δ99 or MHV-A59 (MOI = 1). Twelve hours p.i., virus titers in culture supernatants were determined by plaque assay. Representative experiments out of two (A, B) or the mean ± SD of two independent experiments (C, D) are shown. Statistical analysis was performed using Student's t-test (***, p < 0.001; **, p < 0.01; *, p < 0.05; n.s., p > 0.05).

Mentions: We have previously shown that pDCs are the major source of IFN-α in the early stages of MHV infection and that type I IFN responses in CD11c+ cDCs are only weakly triggered by MHV [27]. To test whether nsp1 has an influence on the induction of IFN-α, we infected both cDCs and pDCs with MHV-nsp1Δ99 or wild-type MHV. Both viruses elicited rapid and high IFN-α production in Flt3-L–differentiated bone marrow–derived pDCs (Figure 4A) and FACS-sorted primary pDCs (Figure 4B). Furthermore, both wild-type and mutant MHV elicited only a late and weak IFN-α production in cDCs (Figure 4A and 4B). These results suggest that nsp1 does not affect the induction of type I IFN. To assess a potential impact of nsp1 on type I IFN signaling and antiviral effector mechanisms in target cells that efficiently support MHV replication, cDCs and macrophages were pretreated with different dosages of IFN-α and infected with MHV-nsp1Δ99 or wild-type MHV. In cDCs, IFN-α treatment had a comparable effect on the replication of both MHV-nsp1Δ99 and the wild-type control virus (Figure 4C). However, replication of MHV-nsp1Δ99 was, in a dose-dependent manner, more vulnerable to IFN-α treatment in macrophages (Figure 4D), suggesting that nsp1 might counteract IFN signaling and/or the antiviral activities of IFN-induced effector proteins.


Coronavirus non-structural protein 1 is a major pathogenicity factor: implications for the rational design of coronavirus vaccines.

Züst R, Cervantes-Barragán L, Kuri T, Blakqori G, Weber F, Ludewig B, Thiel V - PLoS Pathog. (2007)

Effect of nsp1 on IFN-α Production and SignalingC57BL/6 bone marrow–derived (A) or primary (B) splenic cDCs or pDCs were infected with MHV-nsp1Δ99 or MHV-A59 at an MOI of 1. IFN-α secreted into cell culture supernatants was determined by ELISA at the indicated time points. Bone marrow–derived cDCs (C) or inflammatory macrophages (D) from 129Sv mice were treated with 50, 200, 500, or 1,000 U IFN-α/100,000 cells or left untreated. Four hours later, cells were infected with MHV-nsp1Δ99 or MHV-A59 (MOI = 1). Twelve hours p.i., virus titers in culture supernatants were determined by plaque assay. Representative experiments out of two (A, B) or the mean ± SD of two independent experiments (C, D) are shown. Statistical analysis was performed using Student's t-test (***, p < 0.001; **, p < 0.01; *, p < 0.05; n.s., p > 0.05).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC1941747&req=5

ppat-0030109-g004: Effect of nsp1 on IFN-α Production and SignalingC57BL/6 bone marrow–derived (A) or primary (B) splenic cDCs or pDCs were infected with MHV-nsp1Δ99 or MHV-A59 at an MOI of 1. IFN-α secreted into cell culture supernatants was determined by ELISA at the indicated time points. Bone marrow–derived cDCs (C) or inflammatory macrophages (D) from 129Sv mice were treated with 50, 200, 500, or 1,000 U IFN-α/100,000 cells or left untreated. Four hours later, cells were infected with MHV-nsp1Δ99 or MHV-A59 (MOI = 1). Twelve hours p.i., virus titers in culture supernatants were determined by plaque assay. Representative experiments out of two (A, B) or the mean ± SD of two independent experiments (C, D) are shown. Statistical analysis was performed using Student's t-test (***, p < 0.001; **, p < 0.01; *, p < 0.05; n.s., p > 0.05).
Mentions: We have previously shown that pDCs are the major source of IFN-α in the early stages of MHV infection and that type I IFN responses in CD11c+ cDCs are only weakly triggered by MHV [27]. To test whether nsp1 has an influence on the induction of IFN-α, we infected both cDCs and pDCs with MHV-nsp1Δ99 or wild-type MHV. Both viruses elicited rapid and high IFN-α production in Flt3-L–differentiated bone marrow–derived pDCs (Figure 4A) and FACS-sorted primary pDCs (Figure 4B). Furthermore, both wild-type and mutant MHV elicited only a late and weak IFN-α production in cDCs (Figure 4A and 4B). These results suggest that nsp1 does not affect the induction of type I IFN. To assess a potential impact of nsp1 on type I IFN signaling and antiviral effector mechanisms in target cells that efficiently support MHV replication, cDCs and macrophages were pretreated with different dosages of IFN-α and infected with MHV-nsp1Δ99 or wild-type MHV. In cDCs, IFN-α treatment had a comparable effect on the replication of both MHV-nsp1Δ99 and the wild-type control virus (Figure 4C). However, replication of MHV-nsp1Δ99 was, in a dose-dependent manner, more vulnerable to IFN-α treatment in macrophages (Figure 4D), suggesting that nsp1 might counteract IFN signaling and/or the antiviral activities of IFN-induced effector proteins.

Bottom Line: Attenuated viral vaccines can be generated by targeting essential pathogenicity factors.The effect of nsp1 on MHV replication in vitro and in vivo was analyzed using a recombinant MHV encoding a deletion in the nsp1-coding sequence.The recombinant MHV nsp1 mutant grew normally in tissue culture, but was severely attenuated in vivo.

View Article: PubMed Central - PubMed

Affiliation: Research Department, Kantonal Hospital St. Gallen, St. Gallen, Switzerland.

ABSTRACT
Attenuated viral vaccines can be generated by targeting essential pathogenicity factors. We report here the rational design of an attenuated recombinant coronavirus vaccine based on a deletion in the coding sequence of the non-structural protein 1 (nsp1). In cell culture, nsp1 of mouse hepatitis virus (MHV), like its SARS-coronavirus homolog, strongly reduced cellular gene expression. The effect of nsp1 on MHV replication in vitro and in vivo was analyzed using a recombinant MHV encoding a deletion in the nsp1-coding sequence. The recombinant MHV nsp1 mutant grew normally in tissue culture, but was severely attenuated in vivo. Replication and spread of the nsp1 mutant virus was restored almost to wild-type levels in type I interferon (IFN) receptor-deficient mice, indicating that nsp1 interferes efficiently with the type I IFN system. Importantly, replication of nsp1 mutant virus in professional antigen-presenting cells such as conventional dendritic cells and macrophages, and induction of type I IFN in plasmacytoid dendritic cells, was not impaired. Furthermore, even low doses of nsp1 mutant MHV elicited potent cytotoxic T cell responses and protected mice against homologous and heterologous virus challenge. Taken together, the presented attenuation strategy provides a paradigm for the development of highly efficient coronavirus vaccines.

Show MeSH
Related in: MedlinePlus