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Analysis of a pair of END+ and END- viruses derived from the same bovine viral diarrhea virus stock reveals the amino acid determinants in Npro responsible for inhibition of type I interferon production.

Kozasa T, Abe Y, Mitsuhashi K, Tamura T, Aoki H, Ishimaru M, Nakamura S, Okamatsu M, Kida H, Sakoda Y - J. Vet. Med. Sci. (2014)

Bottom Line: The Exaltation of Newcastle disease virus (END) phenomenon is induced by the inhibition of type I interferon in pestivirus-infected cells in vitro, via proteasomal degradation of cellular interferon regulatory factor (IRF)-3 with the property of the viral autoprotease protein N(pro).Reportedly, the amino acid residues in the zinc-binding TRASH motif of N(pro) determine the difference in characteristics between END-phenomenon-positive (END(+)) and END-phenomenon-negative (END(-)) classical swine fever viruses (CSFVs).The END assay, measurements of induced type I interferon and IRF-3 detection in cells infected with these viruses revealed that the aspartic acid at position 136 in the zinc-binding TRASH motif of N(pro) was required to inhibit the production of type I interferon via the degradation of cellular IRF-3, consistently with CSFV.

View Article: PubMed Central - PubMed

Affiliation: Food Safety and Consumer Affairs Bureau, Ministry of Agriculture, Forestry and Fisheries, Chiyoda-ku, Tokyo 100-8950, Japan.

ABSTRACT
The Exaltation of Newcastle disease virus (END) phenomenon is induced by the inhibition of type I interferon in pestivirus-infected cells in vitro, via proteasomal degradation of cellular interferon regulatory factor (IRF)-3 with the property of the viral autoprotease protein N(pro). Reportedly, the amino acid residues in the zinc-binding TRASH motif of N(pro) determine the difference in characteristics between END-phenomenon-positive (END(+)) and END-phenomenon-negative (END(-)) classical swine fever viruses (CSFVs). However, the basic mechanism underlying this function in bovine viral diarrhea virus (BVDV) has not been elucidated from the genomic differences between END(+) and END(-) viruses using reverse genetics till date. In the present study, comparison of complete genome sequences of a pair of END(+) and END(-) viruses isolated from the same virus stock revealed that there were only four amino acid substitutions (D136G, I2623V, D3148G and D3502Y) between two viruses. Based on these differences, viruses with and without mutations at these positions were generated using reverse genetics. The END assay, measurements of induced type I interferon and IRF-3 detection in cells infected with these viruses revealed that the aspartic acid at position 136 in the zinc-binding TRASH motif of N(pro) was required to inhibit the production of type I interferon via the degradation of cellular IRF-3, consistently with CSFV.

No MeSH data available.


Related in: MedlinePlus

Detection of IRF-3 in BFM cells infected with parent or mutant viruses. IRF-3 was notdetected in western blots of the lysates of cells infected with GBK_E+,vGBK_E+ or vGBK_E+/I2623V; D3148G; D3502Y, whereas clear bandsof IRF-3 appeared in western blots of the lysates of cells infected withGBK_E−, vGBK_E+/D136G or vGBK_E+/D136G; I2623V;D3148G; D3502Y.
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fig_003: Detection of IRF-3 in BFM cells infected with parent or mutant viruses. IRF-3 was notdetected in western blots of the lysates of cells infected with GBK_E+,vGBK_E+ or vGBK_E+/I2623V; D3148G; D3502Y, whereas clear bandsof IRF-3 appeared in western blots of the lysates of cells infected withGBK_E−, vGBK_E+/D136G or vGBK_E+/D136G; I2623V;D3148G; D3502Y.

Mentions: Detection of IRF-3 in cells infected with the parental or mutant viruses:To investigate the expression levels of IRF-3 in BVDV-infected cells, IRF-3 in the lysatesof BFM cells infected with GBK_E+, GBK_E−, vGBK_E+ or oneof the three mutant viruses was detected by western blotting. IRF-3 was not detected in thelysates of cells infected with GBK_E+, vGBK_E+ orvGBK_E+/I2623V; D3148G; D3502Y, whereas clear bands of IRF-3 appeared in thelysates of cells infected with the END− GBK_E− virus or one of theNpro mutant viruses (vGBK_E+/D136G and vGBK_E+/D136G;I2623V; D3148G; D3502Y) (Fig. 3Fig. 3.


Analysis of a pair of END+ and END- viruses derived from the same bovine viral diarrhea virus stock reveals the amino acid determinants in Npro responsible for inhibition of type I interferon production.

Kozasa T, Abe Y, Mitsuhashi K, Tamura T, Aoki H, Ishimaru M, Nakamura S, Okamatsu M, Kida H, Sakoda Y - J. Vet. Med. Sci. (2014)

Detection of IRF-3 in BFM cells infected with parent or mutant viruses. IRF-3 was notdetected in western blots of the lysates of cells infected with GBK_E+,vGBK_E+ or vGBK_E+/I2623V; D3148G; D3502Y, whereas clear bandsof IRF-3 appeared in western blots of the lysates of cells infected withGBK_E−, vGBK_E+/D136G or vGBK_E+/D136G; I2623V;D3148G; D3502Y.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4478729&req=5

fig_003: Detection of IRF-3 in BFM cells infected with parent or mutant viruses. IRF-3 was notdetected in western blots of the lysates of cells infected with GBK_E+,vGBK_E+ or vGBK_E+/I2623V; D3148G; D3502Y, whereas clear bandsof IRF-3 appeared in western blots of the lysates of cells infected withGBK_E−, vGBK_E+/D136G or vGBK_E+/D136G; I2623V;D3148G; D3502Y.
Mentions: Detection of IRF-3 in cells infected with the parental or mutant viruses:To investigate the expression levels of IRF-3 in BVDV-infected cells, IRF-3 in the lysatesof BFM cells infected with GBK_E+, GBK_E−, vGBK_E+ or oneof the three mutant viruses was detected by western blotting. IRF-3 was not detected in thelysates of cells infected with GBK_E+, vGBK_E+ orvGBK_E+/I2623V; D3148G; D3502Y, whereas clear bands of IRF-3 appeared in thelysates of cells infected with the END− GBK_E− virus or one of theNpro mutant viruses (vGBK_E+/D136G and vGBK_E+/D136G;I2623V; D3148G; D3502Y) (Fig. 3Fig. 3.

Bottom Line: The Exaltation of Newcastle disease virus (END) phenomenon is induced by the inhibition of type I interferon in pestivirus-infected cells in vitro, via proteasomal degradation of cellular interferon regulatory factor (IRF)-3 with the property of the viral autoprotease protein N(pro).Reportedly, the amino acid residues in the zinc-binding TRASH motif of N(pro) determine the difference in characteristics between END-phenomenon-positive (END(+)) and END-phenomenon-negative (END(-)) classical swine fever viruses (CSFVs).The END assay, measurements of induced type I interferon and IRF-3 detection in cells infected with these viruses revealed that the aspartic acid at position 136 in the zinc-binding TRASH motif of N(pro) was required to inhibit the production of type I interferon via the degradation of cellular IRF-3, consistently with CSFV.

View Article: PubMed Central - PubMed

Affiliation: Food Safety and Consumer Affairs Bureau, Ministry of Agriculture, Forestry and Fisheries, Chiyoda-ku, Tokyo 100-8950, Japan.

ABSTRACT
The Exaltation of Newcastle disease virus (END) phenomenon is induced by the inhibition of type I interferon in pestivirus-infected cells in vitro, via proteasomal degradation of cellular interferon regulatory factor (IRF)-3 with the property of the viral autoprotease protein N(pro). Reportedly, the amino acid residues in the zinc-binding TRASH motif of N(pro) determine the difference in characteristics between END-phenomenon-positive (END(+)) and END-phenomenon-negative (END(-)) classical swine fever viruses (CSFVs). However, the basic mechanism underlying this function in bovine viral diarrhea virus (BVDV) has not been elucidated from the genomic differences between END(+) and END(-) viruses using reverse genetics till date. In the present study, comparison of complete genome sequences of a pair of END(+) and END(-) viruses isolated from the same virus stock revealed that there were only four amino acid substitutions (D136G, I2623V, D3148G and D3502Y) between two viruses. Based on these differences, viruses with and without mutations at these positions were generated using reverse genetics. The END assay, measurements of induced type I interferon and IRF-3 detection in cells infected with these viruses revealed that the aspartic acid at position 136 in the zinc-binding TRASH motif of N(pro) was required to inhibit the production of type I interferon via the degradation of cellular IRF-3, consistently with CSFV.

No MeSH data available.


Related in: MedlinePlus