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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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Construction and In Vitro Analysis of MHV-nsp1Δ99(A) Schematic representation of the MHV-nsp1Δ99 genome organization. The replicase gene, comprised of open reading frames (ORFs) 1a and 1b, is depicted together with viral proteinase cleavage sites (arrowheads) that separate nsps 1–16. The 99-nt deletion within the nsp1-coding region of MHV-nsp1Δ99 is illustrated on the nucleotide and amino acid level. The arrowhead (far right) indicates the nsp1/nsp2 cleavage site.(B–E) Growth kinetics of MHV-nsp1Δ99– or MHV-A59–infected (MOI = 1) murine 17Clone1 cells (B), inflammatory macrophages (C), bone marrow–derived cDCs (D), and ex vivo cDCs (E). Experiments (C–E) were performed with cells obtained from C57BL/6 mice. Results represent the mean ±SD of two independent experiments. Statistical analysis was performed using Student's t-test (n.s., p > 0.05).
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ppat-0030109-g002: Construction and In Vitro Analysis of MHV-nsp1Δ99(A) Schematic representation of the MHV-nsp1Δ99 genome organization. The replicase gene, comprised of open reading frames (ORFs) 1a and 1b, is depicted together with viral proteinase cleavage sites (arrowheads) that separate nsps 1–16. The 99-nt deletion within the nsp1-coding region of MHV-nsp1Δ99 is illustrated on the nucleotide and amino acid level. The arrowhead (far right) indicates the nsp1/nsp2 cleavage site.(B–E) Growth kinetics of MHV-nsp1Δ99– or MHV-A59–infected (MOI = 1) murine 17Clone1 cells (B), inflammatory macrophages (C), bone marrow–derived cDCs (D), and ex vivo cDCs (E). Experiments (C–E) were performed with cells obtained from C57BL/6 mice. Results represent the mean ±SD of two independent experiments. Statistical analysis was performed using Student's t-test (n.s., p > 0.05).

Mentions: To assess the role of nsp1 in the context of virus replication, we constructed a recombinant MHV encoding a truncated nsp1 protein using our reverse genetic system [24]. Based on the results shown in Figure 1, we decided to delete MHV nucleotides (nts) 829–927 (99 nts). In the resulting mutant virus, MHV-nsp1Δ99, the replicase gene start codon, the translational reading frame, and the residues required for proteolytic release of nsp1 from the replicase polyprotein were maintained (Figure 2A). As reported for a set of similar MHV mutants by Brockway et al. [25], viral growth and peak titers of MHV-nsp1Δ99 in murine 17Clone1 cells were indistinguishable from that of wild-type virus (Figure 2B). To assess the stability of the recombinant MHV-nsp1Δ99, we analyzed the nsp1-coding region by RT-PCR sequencing after seven passages in tissue culture and no nucleotide changes were detected (unpublished data).


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)

Construction and In Vitro Analysis of MHV-nsp1Δ99(A) Schematic representation of the MHV-nsp1Δ99 genome organization. The replicase gene, comprised of open reading frames (ORFs) 1a and 1b, is depicted together with viral proteinase cleavage sites (arrowheads) that separate nsps 1–16. The 99-nt deletion within the nsp1-coding region of MHV-nsp1Δ99 is illustrated on the nucleotide and amino acid level. The arrowhead (far right) indicates the nsp1/nsp2 cleavage site.(B–E) Growth kinetics of MHV-nsp1Δ99– or MHV-A59–infected (MOI = 1) murine 17Clone1 cells (B), inflammatory macrophages (C), bone marrow–derived cDCs (D), and ex vivo cDCs (E). Experiments (C–E) were performed with cells obtained from C57BL/6 mice. Results represent the mean ±SD of two independent experiments. Statistical analysis was performed using Student's t-test (n.s., p > 0.05).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC1941747&req=5

ppat-0030109-g002: Construction and In Vitro Analysis of MHV-nsp1Δ99(A) Schematic representation of the MHV-nsp1Δ99 genome organization. The replicase gene, comprised of open reading frames (ORFs) 1a and 1b, is depicted together with viral proteinase cleavage sites (arrowheads) that separate nsps 1–16. The 99-nt deletion within the nsp1-coding region of MHV-nsp1Δ99 is illustrated on the nucleotide and amino acid level. The arrowhead (far right) indicates the nsp1/nsp2 cleavage site.(B–E) Growth kinetics of MHV-nsp1Δ99– or MHV-A59–infected (MOI = 1) murine 17Clone1 cells (B), inflammatory macrophages (C), bone marrow–derived cDCs (D), and ex vivo cDCs (E). Experiments (C–E) were performed with cells obtained from C57BL/6 mice. Results represent the mean ±SD of two independent experiments. Statistical analysis was performed using Student's t-test (n.s., p > 0.05).
Mentions: To assess the role of nsp1 in the context of virus replication, we constructed a recombinant MHV encoding a truncated nsp1 protein using our reverse genetic system [24]. Based on the results shown in Figure 1, we decided to delete MHV nucleotides (nts) 829–927 (99 nts). In the resulting mutant virus, MHV-nsp1Δ99, the replicase gene start codon, the translational reading frame, and the residues required for proteolytic release of nsp1 from the replicase polyprotein were maintained (Figure 2A). As reported for a set of similar MHV mutants by Brockway et al. [25], viral growth and peak titers of MHV-nsp1Δ99 in murine 17Clone1 cells were indistinguishable from that of wild-type virus (Figure 2B). To assess the stability of the recombinant MHV-nsp1Δ99, we analyzed the nsp1-coding region by RT-PCR sequencing after seven passages in tissue culture and no nucleotide changes were detected (unpublished data).

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