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Inhibition of Translation Initiation by Protein 169: A Vaccinia Virus Strategy to Suppress Innate and Adaptive Immunity and Alter Virus Virulence.

Strnadova P, Ren H, Valentine R, Mazzon M, Sweeney TR, Brierley I, Smith GL - PLoS Pathog. (2015)

Bottom Line: Here we show that VACV strain Western Reserve protein 169 is a cytoplasmic polypeptide expressed early during infection that is excluded from virus factories and inhibits the initiation of cap-dependent and cap-independent translation.Ectopic expression of protein 169 causes the accumulation of 80S ribosomes, a reduction of polysomes, and inhibition of protein expression deriving from activation of multiple innate immune signaling pathways.Intranasal infection by vΔ169 caused increased pro-inflammatory cytokines and chemokines, infiltration of pulmonary leukocytes, and lung weight.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of Cambridge, Cambridge, United Kingdom; Department of Virology, Faculty of Medicine, Imperial College London, London, United Kingdom.

ABSTRACT
Vaccinia virus (VACV) is the prototypic orthopoxvirus and the vaccine used to eradicate smallpox. Here we show that VACV strain Western Reserve protein 169 is a cytoplasmic polypeptide expressed early during infection that is excluded from virus factories and inhibits the initiation of cap-dependent and cap-independent translation. Ectopic expression of protein 169 causes the accumulation of 80S ribosomes, a reduction of polysomes, and inhibition of protein expression deriving from activation of multiple innate immune signaling pathways. A virus lacking 169 (vΔ169) replicates and spreads normally in cell culture but is more virulent than parental and revertant control viruses in intranasal and intradermal murine models of infection. Intranasal infection by vΔ169 caused increased pro-inflammatory cytokines and chemokines, infiltration of pulmonary leukocytes, and lung weight. These alterations in innate immunity resulted in a stronger CD8+ T-cell memory response and better protection against virus challenge. This work illustrates how inhibition of host protein synthesis can be a strategy for virus suppression of innate and adaptive immunity.

No MeSH data available.


Related in: MedlinePlus

Protein 169 is expressed early and localizes in cytoplasmic puncta.(A) HeLa cells were either mock-infected or infected at 5 plaque-forming units (PFU)/cell with different VACV strains or cowpox virus. Cell lysates were prepared 16 h p.i., separated by SDS-PAGE and immunoblotted using antibodies against 169, VACV protein D8 or α-tubulin. (B) HeLa cells were infected with v169 at 10 PFU/cell in the presence or absence of AraC (40 μg/ml) for the indicated times. Cell lysates were resolved by SDS-PAGE and immunoblotted using the indicated antibodies. (C) HeLa cells were either mock-infected or infected with indicated viruses at 10 PFU/cell. After 7 h, cells were separated into nuclear and cytoplasmic fractions and analysed by SDS-PAGE and immunoblotting with the indicated antibodies. In all immunoblots, molecular size markers are indicated on the left in kDa. (D) HeLa cells were infected with v169 at 10 PFU/cell for the indicated times and at 2 PFU/cell for 16 h. Cells were fixed and stained with anti-169 purified antibody (green) and DAPI (blue). White lines are scale bars (20 μm).
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ppat.1005151.g001: Protein 169 is expressed early and localizes in cytoplasmic puncta.(A) HeLa cells were either mock-infected or infected at 5 plaque-forming units (PFU)/cell with different VACV strains or cowpox virus. Cell lysates were prepared 16 h p.i., separated by SDS-PAGE and immunoblotted using antibodies against 169, VACV protein D8 or α-tubulin. (B) HeLa cells were infected with v169 at 10 PFU/cell in the presence or absence of AraC (40 μg/ml) for the indicated times. Cell lysates were resolved by SDS-PAGE and immunoblotted using the indicated antibodies. (C) HeLa cells were either mock-infected or infected with indicated viruses at 10 PFU/cell. After 7 h, cells were separated into nuclear and cytoplasmic fractions and analysed by SDS-PAGE and immunoblotting with the indicated antibodies. In all immunoblots, molecular size markers are indicated on the left in kDa. (D) HeLa cells were infected with v169 at 10 PFU/cell for the indicated times and at 2 PFU/cell for 16 h. Cells were fixed and stained with anti-169 purified antibody (green) and DAPI (blue). White lines are scale bars (20 μm).

Mentions: The expression of protein 169 by several VACV strains was investigated by immunoblotting using a rabbit polyclonal antibody raised against VACV WR protein 169 that had been expressed in and purified from E. coli (Methods). This detected a 13-kDa polypeptide in cells infected with VACV strains WR, MVA, Lister, rabbitpox, International Health Department (IHD)-J and Tian Tan, and cowpox virus strain Brighton Red, but not VACV strain Copenhagen, or in mock-infected cells (Fig 1A). VACV infection was confirmed by immunoblotting with a mAb that recognizes the VACV structural protein D8 [37], although this mAb did not detect the D8 protein made by MVA (Fig 1A). Immunoblotting for α-tubulin demonstrated equal loading of samples.


Inhibition of Translation Initiation by Protein 169: A Vaccinia Virus Strategy to Suppress Innate and Adaptive Immunity and Alter Virus Virulence.

Strnadova P, Ren H, Valentine R, Mazzon M, Sweeney TR, Brierley I, Smith GL - PLoS Pathog. (2015)

Protein 169 is expressed early and localizes in cytoplasmic puncta.(A) HeLa cells were either mock-infected or infected at 5 plaque-forming units (PFU)/cell with different VACV strains or cowpox virus. Cell lysates were prepared 16 h p.i., separated by SDS-PAGE and immunoblotted using antibodies against 169, VACV protein D8 or α-tubulin. (B) HeLa cells were infected with v169 at 10 PFU/cell in the presence or absence of AraC (40 μg/ml) for the indicated times. Cell lysates were resolved by SDS-PAGE and immunoblotted using the indicated antibodies. (C) HeLa cells were either mock-infected or infected with indicated viruses at 10 PFU/cell. After 7 h, cells were separated into nuclear and cytoplasmic fractions and analysed by SDS-PAGE and immunoblotting with the indicated antibodies. In all immunoblots, molecular size markers are indicated on the left in kDa. (D) HeLa cells were infected with v169 at 10 PFU/cell for the indicated times and at 2 PFU/cell for 16 h. Cells were fixed and stained with anti-169 purified antibody (green) and DAPI (blue). White lines are scale bars (20 μm).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4559412&req=5

ppat.1005151.g001: Protein 169 is expressed early and localizes in cytoplasmic puncta.(A) HeLa cells were either mock-infected or infected at 5 plaque-forming units (PFU)/cell with different VACV strains or cowpox virus. Cell lysates were prepared 16 h p.i., separated by SDS-PAGE and immunoblotted using antibodies against 169, VACV protein D8 or α-tubulin. (B) HeLa cells were infected with v169 at 10 PFU/cell in the presence or absence of AraC (40 μg/ml) for the indicated times. Cell lysates were resolved by SDS-PAGE and immunoblotted using the indicated antibodies. (C) HeLa cells were either mock-infected or infected with indicated viruses at 10 PFU/cell. After 7 h, cells were separated into nuclear and cytoplasmic fractions and analysed by SDS-PAGE and immunoblotting with the indicated antibodies. In all immunoblots, molecular size markers are indicated on the left in kDa. (D) HeLa cells were infected with v169 at 10 PFU/cell for the indicated times and at 2 PFU/cell for 16 h. Cells were fixed and stained with anti-169 purified antibody (green) and DAPI (blue). White lines are scale bars (20 μm).
Mentions: The expression of protein 169 by several VACV strains was investigated by immunoblotting using a rabbit polyclonal antibody raised against VACV WR protein 169 that had been expressed in and purified from E. coli (Methods). This detected a 13-kDa polypeptide in cells infected with VACV strains WR, MVA, Lister, rabbitpox, International Health Department (IHD)-J and Tian Tan, and cowpox virus strain Brighton Red, but not VACV strain Copenhagen, or in mock-infected cells (Fig 1A). VACV infection was confirmed by immunoblotting with a mAb that recognizes the VACV structural protein D8 [37], although this mAb did not detect the D8 protein made by MVA (Fig 1A). Immunoblotting for α-tubulin demonstrated equal loading of samples.

Bottom Line: Here we show that VACV strain Western Reserve protein 169 is a cytoplasmic polypeptide expressed early during infection that is excluded from virus factories and inhibits the initiation of cap-dependent and cap-independent translation.Ectopic expression of protein 169 causes the accumulation of 80S ribosomes, a reduction of polysomes, and inhibition of protein expression deriving from activation of multiple innate immune signaling pathways.Intranasal infection by vΔ169 caused increased pro-inflammatory cytokines and chemokines, infiltration of pulmonary leukocytes, and lung weight.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of Cambridge, Cambridge, United Kingdom; Department of Virology, Faculty of Medicine, Imperial College London, London, United Kingdom.

ABSTRACT
Vaccinia virus (VACV) is the prototypic orthopoxvirus and the vaccine used to eradicate smallpox. Here we show that VACV strain Western Reserve protein 169 is a cytoplasmic polypeptide expressed early during infection that is excluded from virus factories and inhibits the initiation of cap-dependent and cap-independent translation. Ectopic expression of protein 169 causes the accumulation of 80S ribosomes, a reduction of polysomes, and inhibition of protein expression deriving from activation of multiple innate immune signaling pathways. A virus lacking 169 (vΔ169) replicates and spreads normally in cell culture but is more virulent than parental and revertant control viruses in intranasal and intradermal murine models of infection. Intranasal infection by vΔ169 caused increased pro-inflammatory cytokines and chemokines, infiltration of pulmonary leukocytes, and lung weight. These alterations in innate immunity resulted in a stronger CD8+ T-cell memory response and better protection against virus challenge. This work illustrates how inhibition of host protein synthesis can be a strategy for virus suppression of innate and adaptive immunity.

No MeSH data available.


Related in: MedlinePlus