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. 2009 Feb;83(4):1800-10.
doi: 10.1128/JVI.02112-08. Epub 2008 Dec 3.

A conserved domain in the leader proteinase of foot-and-mouth disease virus is required for proper subcellular localization and function

Teresa de los Santos  1 , Fayna Diaz-San Segundo, James Zhu, Marla Koster, Camila C A Dias, Marvin J Grubman
Affiliations

A conserved domain in the leader proteinase of foot-and-mouth disease virus is required for proper subcellular localization and function

Teresa de los Santos et al. J Virol. 2009 Feb.

Abstract

The leader proteinase (L(pro)) of foot-and-mouth disease virus (FMDV) is involved in antagonizing the innate immune response by blocking the expression of interferon (IFN) and by reducing the immediate-early induction of IFN-beta mRNA and IFN-stimulated genes. In addition to its role in shutting off cap-dependent host mRNA translation, L(pro) is associated with the degradation of the p65/RelA subunit of nuclear factor kappaB (NF-kappaB). Bioinformatics analysis suggests that L(pro) contains a SAP (for SAF-A/B, Acinus, and PIAS) domain, a protein structure associated in some cases with the nuclear retention of molecules involved in transcriptional control. We have introduced a single or a double mutation in conserved amino acid residues contained within this domain of L(pro). Although three stable mutant viruses were obtained, only the double mutant displayed an attenuated phenotype in cell culture. Indirect immunofluorescence analysis showed that L(pro) subcellular distribution is altered in cells infected with the double mutant virus. Interestingly, nuclear p65/RelA staining disappeared from wild-type (WT) FMDV-infected cells but not from double mutant virus-infected cells. Consistent with these results, NF-kappaB-dependent transcription was not inhibited in cells infected with double mutant virus in contrast to cells infected with WT virus. However, degradation of the translation initiation factor eIF-4G was very similar for both the WT and the double mutant viruses. Since L(pro) catalytic activity was demonstrated to be a requirement for p65/RelA degradation, our results indicate that mutation of the SAP domain reveals a novel separation-of-function activity for FMDV L(pro).

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Figures

FIG. 1.
FIG. 1.
Alignment of FMDV Lpro partial amino acid sequence. Lpro protein sequence was aligned to all available sequences utilizing SMART software. Depicted are sequences in single letters between amino acids 47 and 88, and a schematic diagram displays the approximate location of predicted α-helices (green ovals). Asterisks mark the location of amino acids targeted by mutagenesis. Summary of color coding including consensus sequence: + (positive sign in blue), positive charged amino acids (H, K, and R); b (lowercase letter “b” highlighted in yellow), amino acids with a large or bulky side chain (E, F, H, I, K, L, M, Q, R, W, and Y); c (lowercase letter “c” in pink font), charged amino acids (D, E, H, K, and R); l (lowercase letter “l” highlighted in yellow), aliphatic amino acids (I, L, and V); p (lowercase letter “p” in blue font), polar amino acids (C, D, E, H, K, N, Q, R, S, and T); s (lowercase letter “s” in green font), amino acids with a small side chain (A, C, D, G, N, P, S, T, and V); and gray highlighted, amino acids with ≥60% of the homology in the alignment.
FIG. 2.
FIG. 2.
Kinetics of growth and plaque morphology. (A) Growth curves. BHK-21 or EBK cells were infected with the indicated viruses and, after 1 h, unabsorbed virus was removed by washing with 150 mM NaCl-20 mM MES (pH 6.0), followed by the addition of complete medium. Samples were taken at 1, 3, 6, and 24 hpi, and virus titers were determined by plaque assay on BHK-21 cells. (Reported values display one out of three representative experiments with similar results.) (B) BHK-21 cells were infected with similar amounts of viruses and treated as described in panel A, but medium with a gum tragacanth overlay was added and the plaques were stained at 40 hpi.
FIG. 3.
FIG. 3.
IFA of Lpro during FMDV infection. LF-BK cells were infected at an MOI of 10 with FMDV A12-WT (panels 1, 4, 7, and 10), A12-LLV2 (panels 2, 5, 8, and 11), or double SAP mutant A12#49 (panels 3, 6, 9, and 12) and were fixed at different times postinfection. Viral protein Lpro was detected using a rabbit polyclonal Ab and an Alexa Fluor 488-conjugated secondary Ab. Viral protein VP1 was detected using mouse MAb 6HC4 and an Alexa Fluor 594-conjugated secondary Ab.
FIG. 4.
FIG. 4.
IFA of p65/RelA during FMDV infection. LF-BK cells were infected at an MOI of 10 with WT (panels 1 to 3), A12-LLV2 (panels 4 to 6), and the double SAP mutant A12#49 (panels 7 to 9). As a control, cells were mock infected (panels 10 and 11) or treated with synthetic dsRNA poly[IC] (25 μg/ml) and Lipofectamine (panels 12 and 13). At 6 hpi, cells were fixed and stained. p65/RelA was detected using a rabbit polyclonal Ab (Abcam RB-1638) and an Alexa Fluor 488-conjugated secondary Ab. Viral protein VP1 was detected using mouse MAb 6HC4 and an Alexa Fluor 594-conjugated secondary Ab. Nuclei were stained with DAPI (panels 11 and 13).
FIG. 5.
FIG. 5.
Addition of LMB does not block the nuclear export of FMDV Lpro. LF-BK cells were infected at an MOI of 10 with A12-WT (panels 1, 3, 5, and 7) and the double SAP mutant A12#49 (panels 2, 4, 6, and 8). Where indicated, 200 nM LMB was added to the culture medium. At 6 hpi, cells were fixed and stained. For panels 1 to 4, viral protein Lpro was detected using a rabbit polyclonal Ab and an Alexa Fluor 488-conjugated secondary Ab. Viral protein VP1 was detected using mouse MAb 6HC4 and an Alexa Fluor 594-conjugated secondary Ab. For panels 5 to 8, p65/RelA was detected using rabbit polyclonal Ab Abcam RB-1638 and an Alexa Fluor 488-conjugated secondary Ab.
FIG. 6.
FIG. 6.
Analysis of IFN expression. (A) The expression of IFNβ, TNF-α, RANTES, Mx1, and IRF7 mRNAs was measured by real-time RT-PCR in secondary PK cells infected with A12-WT, A12-LLV2, or A12#49 FMDV at an MOI of 2 for the indicated times. Porcine GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was used as an internal control. The results are expressed as the fold increase in gene expression for virus-infected with respect to mock-infected cells. (Reported values display the findings for one out of three representative experiments with similar results.) (B) pIFNα expression in the supernatants of PK-infected cells determined by ELISA. The values are presented as the mean ± the standard deviation of three independent determinations.
FIG. 7.
FIG. 7.
Processing of cellular proteins during infection with WT and mutant FMDV. (A) LF-BK cells were infected with WT (A12-WT), leaderless (A12-LLV2), and SAP mutant (A12#49) at MOIs of 10 for 6 h. At the indicated times, cytoplasmic extracts were prepared and analyzed by Western blotting using rabbit polyclonal Ab anti-eIF-4G (p220), rabbit polyclonal Ab anti-p65/RelA (RB-1638), mouse MAb 6HC4 (VP1), and mouse MAb anti-tubulin-α (Ab-2 MS-581). CP, p220 cleavage products. (B) Radioimmunoprecipitation of FMDV-infected cell lysates at different times postinfection. [35S]methionine-labeled cell lysates from FMDV-infected LF-BK cells were immunoprecipitated with serum from a convalescent bovine. Samples were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and developed by autoradiography.
FIG. 8.
FIG. 8.
FMDV Lpro catalytic activity is required for p65/RelA degradation. IBRS-2 cells were infected with TMEV-Lb-containing WT or mutant C23A Lpro at an MOI of 10. At 24 hpi, p65/RelA and viral proteins were visualized by IFA. p65/RelA was detected with rabbit polyclonal Ab (RB-1638) and Alexa Fluor 488-conjugated secondary Ab. Lpro was visualized a rabbit polyclonal Ab and Alexa Fluor 488-conjugated secondary Ab. TMEV VP1 was detected with MAb DAmAb2 (28) and Alexa Fluor 594-conjugated secondary Ab.
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