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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 inhibits protein expression after activation of several innate immune signaling pathways.(A) HEK 293T cells were transfected in triplicate with an NF-κB reporter plasmid, TK-renilla luciferase and plasmids for expression of the indicated proteins. After 1 d the cells were stimulated with 75 ng/ml of TNF-α for 7 h or treated with the same medium lacking TNF-α. The luminescence of cell lysates was measured using a luminometer. These data are from one representative experiment (n = 3) and results are presented as the fold increase in luciferase expression. Firefly luciferase was normalized to renilla luciferase (internal control) and further normalized to the unstimulated samples ± SD. Statistical analysis was performed using a two-tailed Student’s t-test with Welch’s correction where necessary, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. (B) Performed as in (A) but using an ISG56.1 reporter plasmid (responsive to IRF-3) and cells were transfected with poly I:C or Lipofectamine only. (C) Performed as in (A) but using an ISRE reporter plasmid. The cells were stimulated with 100 U/ml of IFN-α for 7 h. (D) HEK 293T cells were transfected with plasmids for expression of the indicated proteins in triplicate, and the following day the cells were mock-infected or infected with SeV for 24 h. CXCL10 in the supernatant was measured by ELISA. Data shown are from one representative experiment (n = 2) and results are expressed as concentration of CXCL10, estimated from a nonlinear standard curve, ± SD. Statistical comparison to GFP control used a two-tailed Student’s t-test with Welch’s correction where necessary, *** p < 0.001, **** p < 0.0001. (E, F, G) A549 cells were transfected with plasmids for expression of the indicated proteins in triplicate and, after 24 h cells, were mock-stimulated or stimulated with 50 ng/ml of TNF-α for 7 h. Then mRNAs were extracted, cDNAs were prepared and RT-q-PCR was performed using ViiA 7 Real-Time PCR System (Life Technologies) using primers specific for IL-6 (E), NFκBia (F) and ICAM-1 (G). Data shown are from one representative experiment (n = 2) and results are expressed as cycle threshold (CT) values compared to HPRT levels ± SD. Statistical analysis was performed using a two-tailed Student’s t-test with Welch’s correction where necessary, * p < 0.05, ** p <0.01.
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ppat.1005151.g004: Protein 169 inhibits protein expression after activation of several innate immune signaling pathways.(A) HEK 293T cells were transfected in triplicate with an NF-κB reporter plasmid, TK-renilla luciferase and plasmids for expression of the indicated proteins. After 1 d the cells were stimulated with 75 ng/ml of TNF-α for 7 h or treated with the same medium lacking TNF-α. The luminescence of cell lysates was measured using a luminometer. These data are from one representative experiment (n = 3) and results are presented as the fold increase in luciferase expression. Firefly luciferase was normalized to renilla luciferase (internal control) and further normalized to the unstimulated samples ± SD. Statistical analysis was performed using a two-tailed Student’s t-test with Welch’s correction where necessary, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. (B) Performed as in (A) but using an ISG56.1 reporter plasmid (responsive to IRF-3) and cells were transfected with poly I:C or Lipofectamine only. (C) Performed as in (A) but using an ISRE reporter plasmid. The cells were stimulated with 100 U/ml of IFN-α for 7 h. (D) HEK 293T cells were transfected with plasmids for expression of the indicated proteins in triplicate, and the following day the cells were mock-infected or infected with SeV for 24 h. CXCL10 in the supernatant was measured by ELISA. Data shown are from one representative experiment (n = 2) and results are expressed as concentration of CXCL10, estimated from a nonlinear standard curve, ± SD. Statistical comparison to GFP control used a two-tailed Student’s t-test with Welch’s correction where necessary, *** p < 0.001, **** p < 0.0001. (E, F, G) A549 cells were transfected with plasmids for expression of the indicated proteins in triplicate and, after 24 h cells, were mock-stimulated or stimulated with 50 ng/ml of TNF-α for 7 h. Then mRNAs were extracted, cDNAs were prepared and RT-q-PCR was performed using ViiA 7 Real-Time PCR System (Life Technologies) using primers specific for IL-6 (E), NFκBia (F) and ICAM-1 (G). Data shown are from one representative experiment (n = 2) and results are expressed as cycle threshold (CT) values compared to HPRT levels ± SD. Statistical analysis was performed using a two-tailed Student’s t-test with Welch’s correction where necessary, * p < 0.05, ** p <0.01.

Mentions: The 169R gene is located in a terminal variable region of the VACV genome, is expressed early during infection and is non-essential for virus replication in cell culture. These properties are characteristic of VACV genes encoding immunevasins, such as the type I IFN binding protein [41, 42], the 3-β-hydroxysteroid dehydrogenase [43, 44] and the intracellular inhibitors of NF-κB activation [9, 10, 12–18]. Therefore, we hypothesized that protein 169 might be an immunevasin and this was tested by reporter gene assays. A plasmid in which firefly luciferase expression is driven by either an NF-κB, IRF-3 (ISG56.1), or interferon-stimulated response element (ISRE) responsive promoter was transfected separately into HEK 293T cells together with TK renilla luciferase (internal control), and plasmids expressing 169, FLAG-tagged 169 (FLAG-169) or other control proteins. The controls chosen were (i) VACV strain WR protein B14 that inhibits the NF-κB signaling by binding to IKKβ [15], (ii) VACV protein C6 that inhibits IRF-3 signaling by binding to TBK-1 adaptors [45], and (iii) paramyxovirus protein PiV5-V that inhibits type I IFN-induced signaling by degrading STAT1 [46]. Luciferase activity was measured by luminescence after stimulation with TNF-α (NF-κB Luc), IFN-α (ISRE Luc) or after transfection with poly (I:C) (IRF-3 Luc). Protein 169 and FLAG-169 inhibited NF-κB, IRF-3 and ISRE pathways as well as, or better than, known inhibitors of these pathways (Fig 4A–4C). The inhibition of all these pathways was surprising, and contrasted with the controls that generally inhibit specific pathways only. Interestingly, protein 169 also caused reduced expression of TK renilla, suggesting a general reduction in protein expression.


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 inhibits protein expression after activation of several innate immune signaling pathways.(A) HEK 293T cells were transfected in triplicate with an NF-κB reporter plasmid, TK-renilla luciferase and plasmids for expression of the indicated proteins. After 1 d the cells were stimulated with 75 ng/ml of TNF-α for 7 h or treated with the same medium lacking TNF-α. The luminescence of cell lysates was measured using a luminometer. These data are from one representative experiment (n = 3) and results are presented as the fold increase in luciferase expression. Firefly luciferase was normalized to renilla luciferase (internal control) and further normalized to the unstimulated samples ± SD. Statistical analysis was performed using a two-tailed Student’s t-test with Welch’s correction where necessary, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. (B) Performed as in (A) but using an ISG56.1 reporter plasmid (responsive to IRF-3) and cells were transfected with poly I:C or Lipofectamine only. (C) Performed as in (A) but using an ISRE reporter plasmid. The cells were stimulated with 100 U/ml of IFN-α for 7 h. (D) HEK 293T cells were transfected with plasmids for expression of the indicated proteins in triplicate, and the following day the cells were mock-infected or infected with SeV for 24 h. CXCL10 in the supernatant was measured by ELISA. Data shown are from one representative experiment (n = 2) and results are expressed as concentration of CXCL10, estimated from a nonlinear standard curve, ± SD. Statistical comparison to GFP control used a two-tailed Student’s t-test with Welch’s correction where necessary, *** p < 0.001, **** p < 0.0001. (E, F, G) A549 cells were transfected with plasmids for expression of the indicated proteins in triplicate and, after 24 h cells, were mock-stimulated or stimulated with 50 ng/ml of TNF-α for 7 h. Then mRNAs were extracted, cDNAs were prepared and RT-q-PCR was performed using ViiA 7 Real-Time PCR System (Life Technologies) using primers specific for IL-6 (E), NFκBia (F) and ICAM-1 (G). Data shown are from one representative experiment (n = 2) and results are expressed as cycle threshold (CT) values compared to HPRT levels ± SD. Statistical analysis was performed using a two-tailed Student’s t-test with Welch’s correction where necessary, * p < 0.05, ** p <0.01.
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ppat.1005151.g004: Protein 169 inhibits protein expression after activation of several innate immune signaling pathways.(A) HEK 293T cells were transfected in triplicate with an NF-κB reporter plasmid, TK-renilla luciferase and plasmids for expression of the indicated proteins. After 1 d the cells were stimulated with 75 ng/ml of TNF-α for 7 h or treated with the same medium lacking TNF-α. The luminescence of cell lysates was measured using a luminometer. These data are from one representative experiment (n = 3) and results are presented as the fold increase in luciferase expression. Firefly luciferase was normalized to renilla luciferase (internal control) and further normalized to the unstimulated samples ± SD. Statistical analysis was performed using a two-tailed Student’s t-test with Welch’s correction where necessary, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. (B) Performed as in (A) but using an ISG56.1 reporter plasmid (responsive to IRF-3) and cells were transfected with poly I:C or Lipofectamine only. (C) Performed as in (A) but using an ISRE reporter plasmid. The cells were stimulated with 100 U/ml of IFN-α for 7 h. (D) HEK 293T cells were transfected with plasmids for expression of the indicated proteins in triplicate, and the following day the cells were mock-infected or infected with SeV for 24 h. CXCL10 in the supernatant was measured by ELISA. Data shown are from one representative experiment (n = 2) and results are expressed as concentration of CXCL10, estimated from a nonlinear standard curve, ± SD. Statistical comparison to GFP control used a two-tailed Student’s t-test with Welch’s correction where necessary, *** p < 0.001, **** p < 0.0001. (E, F, G) A549 cells were transfected with plasmids for expression of the indicated proteins in triplicate and, after 24 h cells, were mock-stimulated or stimulated with 50 ng/ml of TNF-α for 7 h. Then mRNAs were extracted, cDNAs were prepared and RT-q-PCR was performed using ViiA 7 Real-Time PCR System (Life Technologies) using primers specific for IL-6 (E), NFκBia (F) and ICAM-1 (G). Data shown are from one representative experiment (n = 2) and results are expressed as cycle threshold (CT) values compared to HPRT levels ± SD. Statistical analysis was performed using a two-tailed Student’s t-test with Welch’s correction where necessary, * p < 0.05, ** p <0.01.
Mentions: The 169R gene is located in a terminal variable region of the VACV genome, is expressed early during infection and is non-essential for virus replication in cell culture. These properties are characteristic of VACV genes encoding immunevasins, such as the type I IFN binding protein [41, 42], the 3-β-hydroxysteroid dehydrogenase [43, 44] and the intracellular inhibitors of NF-κB activation [9, 10, 12–18]. Therefore, we hypothesized that protein 169 might be an immunevasin and this was tested by reporter gene assays. A plasmid in which firefly luciferase expression is driven by either an NF-κB, IRF-3 (ISG56.1), or interferon-stimulated response element (ISRE) responsive promoter was transfected separately into HEK 293T cells together with TK renilla luciferase (internal control), and plasmids expressing 169, FLAG-tagged 169 (FLAG-169) or other control proteins. The controls chosen were (i) VACV strain WR protein B14 that inhibits the NF-κB signaling by binding to IKKβ [15], (ii) VACV protein C6 that inhibits IRF-3 signaling by binding to TBK-1 adaptors [45], and (iii) paramyxovirus protein PiV5-V that inhibits type I IFN-induced signaling by degrading STAT1 [46]. Luciferase activity was measured by luminescence after stimulation with TNF-α (NF-κB Luc), IFN-α (ISRE Luc) or after transfection with poly (I:C) (IRF-3 Luc). Protein 169 and FLAG-169 inhibited NF-κB, IRF-3 and ISRE pathways as well as, or better than, known inhibitors of these pathways (Fig 4A–4C). The inhibition of all these pathways was surprising, and contrasted with the controls that generally inhibit specific pathways only. Interestingly, protein 169 also caused reduced expression of TK renilla, suggesting a general reduction in protein expression.

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