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Cellular Response to Infection

The Leader Proteinase of Foot-and-Mouth Disease Virus Inhibits the Induction of Beta Interferon mRNA and Blocks the Host Innate Immune Response

Teresa de los Santos, Sonia de Avila Botton, Rudi Weiblen, Marvin J. Grubman
Teresa de los Santos
Plum Island Animal Disease Center, Agricultural Research Service, U.S. Department of Agriculture, Greenport, New York 11944
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Sonia de Avila Botton
Plum Island Animal Disease Center, Agricultural Research Service, U.S. Department of Agriculture, Greenport, New York 11944
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Rudi Weiblen
Plum Island Animal Disease Center, Agricultural Research Service, U.S. Department of Agriculture, Greenport, New York 11944
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Marvin J. Grubman
Plum Island Animal Disease Center, Agricultural Research Service, U.S. Department of Agriculture, Greenport, New York 11944
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  • For correspondence: marvin.grubman@ars.usda.gov
DOI: 10.1128/JVI.80.4.1906-1914.2006
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  • FIG. 1.
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    FIG. 1.

    End-point titration. BHK-21 and SK6 cells were infected with A12-IC (WT) or A12-LLV2 at an MOI of 10 at 37°C. After 1 h, unabsorbed virus was removed by washing with 150 mM NaCl-20 mM MES (pH 6.0), and MEM was added. Supernatants were collected at 24 hpi, and the virus titers were determined on BHK-21 cells.

  • FIG. 2.
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    FIG. 2.

    IFN secretion after FMDV infection. SK6 (A) or PK (B) cells were infected with A12-IC (WT) or A12-LLV2 at an MOI of 1. Supernatants were collected at different times postinfection, and pIFN-α was measured by antigen capture ELISA. The results are representative of three independent experiments. Error bars refer to the standard deviation for each mean data point (n = 3).

  • FIG. 3.
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    FIG. 3.

    RNAi of porcine PKR. SK6 cells were transfected twice with 200 nM of a pool of siRNAs specific for porcine PKR (siPKR). Transfected cells were infected with A12-IC (WT) or A12-LLV2 at MOIs from 0.0001 to 1, and after 24 h the virus titer was measured on BHK-21 cells (B). A control experiment with siGLO (Risc-free) was performed simultaneously (A). The results are representative of two independent experiments.

  • FIG. 4.
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    FIG. 4.

    IFN-α/β response in porcine cells. IFN-α1/β mRNA expression was measured by real-time RT-PCR in SK6 cells (A and C) or PK cells (B and D) infected at an MOI of 1 with A12-IC (WT) or A12-LLV2. Porcine GAPDH was used as an internal control. Results are expressed as the increase (n-fold) of gene expression for virus-infected cells relative to mock-infected cells at the indicated time points. Error bars indicate the standard deviation for each mean data point (n = 3). Similar results were obtained in at least two independent experiments.

  • FIG. 5.
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    FIG. 5.

    Induction of IFN-β mRNA expression in the presence of cycloheximide. (A) SK6 cells were treated with 10 μg/ml of poly(rI · C) in the absence or presence of 100 μg/ml cycloheximide. (B) SK6 cells were infected with A12-IC (WT) or A12-LLV2 (MOI of 1; 9 h) in the absence or presence of 50 μg/ml cycloheximide. The levels of IFN-β mRNA were measured by real-time RT-PCR as indicated in Fig. 4. Error bars indicate the standard deviation for each mean data point (n = 3). chx, cycloheximide.

  • FIG. 6.
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    FIG. 6.

    Expression of ISG products in response to FMDV infection. SK6 or PK cells were infected with A12-IC (WT) and A12-LLV2 at an MOI of 1. Expression of PKR, OAS, and Mx1 mRNAs was measured by real-time RT-PCR using GAPDH or 18S rRNA as an internal control. Results are expressed as the increase (n-fold) for each specific gene in virus-infected cells relative to mock-infected cells. Error bars indicate the standard deviation for each mean data point (n = 3). Similar results were obtained in at least two independent experiments.

Tables

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  • TABLE 1.

    Oligonucleotide primer and probe sequences for amplification of porcine genes used in real-time RT-PCR

    Gene typePrimer and probe setsSequenceGenBanka
    GAPDHPorcine GAPDH-327Fb5′-CGTCCCTGAGACACGATGGT-3′ AF017079
    Porcine GAPDH-380Rc5′-CCCGATGCGGCCAAAT-3′
    Porcine GAPDH-348Td5′-AAGGTCGGAGTGAACG-3′
    Mx1Porcine Mx1-803Fb5′-GAGGTGGACCCCGAAGGA-3′ M65087
    Porcine Mx1-859Rc5′-CACCAGATCCGGCTTCGT-3′
    Porcine Mx1-824Td5′-AGGACCATCGGGATC-3′
    OASPorcine OAS-889Fb5′-CTGTCGTTGGACGATGTATGCT-3′ AJ225090
    Porcine OAS-954Rc5′-CAGCCGGGTCCAGAATCA-3′
    Porcine OAS-919Td5′-TCAAGAAACCCAGGCCT-3′
    PKRPorcine PKR-968Fb5′-GGAAGAAAACAAACACAGCTTGAA-3′ AB104654
    Porcine PKR-1048Rc5′-CCAAATCCACCTGAGCCAATT-3′
    Porcine PKR-994Td5′-CCAGGTT TGTCGAAGAT-3′
    pIFNα set#1Porcine IFNα-236Fb5′-TGGTGCATGAGATGCTCCA-3′ M28623
    Porcine IFNα-290Rc5′-GCCGAGCCCTCTGTGCT-3′
    Porcine IFNα-256Td5′-CAGACCTTCCAGCTCT-3′
    pIFNα set#2Porcine IFNα-449Fb5′-TCACCCTCTATCTGCAAGAGAAGA-3′ M28623
    Porcine IFNα-514Rc5′-TGACTTCTGCCCTGACGATCT-3′
    Porcine IFNα-478Td5′-AGCCCTGTGCCTG-3′
    pIFNβPorcine IFNβ-11Fb5′-AGTGCATCCTCCAAATCGCT-3′ M86762
    Porcine IFNβ-69Rc5′-GCTCATGGAAAGAGCTGTGGT-3′
    Porcine IFNβ-32Td5′-TCCTGATGTGTTTCTC-3′
    • ↵ a NCBI GenBank accession code.

    • ↵ b Forward primer.

    • ↵ c Reverse primer.

    • ↵ d TaqMan FAM-MGB probe.

  • TABLE 2.

    Antiviral activity in supernatants of porcine cellsa

    Inducing virusbTotal antiviral activity (U)Antiviral activity (U) in cells incubated with:
    Normal serumNeutralizing anti-IFN-αNeutralizing anti-IFN-βNeutralizing anti-IFN-α/β
    A12-LLV21616<216<2
    A12-IC (WT)<2<2NANANA
    • ↵ a Supernatants from infected SK6 cells were incubated with serum for 1 h at RT prior to determination of antiviral activity. Activity is measured in arbitrary units. NA, not applicable.

    • ↵ b Virus was used at an MOI of 1.

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The Leader Proteinase of Foot-and-Mouth Disease Virus Inhibits the Induction of Beta Interferon mRNA and Blocks the Host Innate Immune Response
Teresa de los Santos, Sonia de Avila Botton, Rudi Weiblen, Marvin J. Grubman
Journal of Virology Jan 2006, 80 (4) 1906-1914; DOI: 10.1128/JVI.80.4.1906-1914.2006

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The Leader Proteinase of Foot-and-Mouth Disease Virus Inhibits the Induction of Beta Interferon mRNA and Blocks the Host Innate Immune Response
Teresa de los Santos, Sonia de Avila Botton, Rudi Weiblen, Marvin J. Grubman
Journal of Virology Jan 2006, 80 (4) 1906-1914; DOI: 10.1128/JVI.80.4.1906-1914.2006
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KEYWORDS

Endopeptidases
foot-and-mouth disease virus
Gene Expression Regulation
Immunity, Innate
Interferon-beta

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