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. 2015 Apr 13;11(4):e1004817. doi: 10.1371/journal.ppat.1004817

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© 2015 Kazakov et al

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

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Fig 3 — (A) Serine protease active site mutants SGR-Gluc(3m1) and SGR-Gluc(3m2) do not replicate and are not complemented in trans. Values represent mean ± SD from transfections done in triplicate and normalized to untransfected controls. This experiment was performed two times with similar results. (B) Helicase RNA binding and NTPase active site mutants SGR-Gluc(3m3) and SGR-Gluc(3m4) do not replicate and are not complemented in trans. Values represent mean ± SD from transfections done in triplicate and normalized to untransfected controls. For comparison, the SGR-Gluc samples, which were performed in parallel, are reproduced from panel A. This experiment was performed two times with similar results. (C) RNA helicase base stacking mutant SGR-Gluc(3m5) does not replicate but is complemented in trans with an efficiency similar to SGR-Gluc(5m1). For comparison, the SGR-Gluc samples, which were performed in parallel, are reproduced from panel A. Values represent mean ± SD from transfections done in triplicate and normalized to untransfected controls; *, p < 0.05; **, p <0.01 by Student’s t-test, comparing matched time points from SGR-Gluc(3m5) in Huh-7.5[VEEV/NS3–5B] vs. Huh-7.5[VEEV/GFP] cells. This experiment was performed two times with similar results. (D) Western blot to confirm that the serine protease active site mutant SGR-Gluc(3m1) does not undergo polyprotein cleavage and that the RNA helicase mutants SGR-Gluc(3m2), SGR-Gluc(3m3), SGR-Gluc(3m4), and SGR-Gluc(3m5) do not exhibit defects in NS3 accumulation. NS3 was detected by using anti-NS3 monoclonal antibody 4E11; values represent relative NS3 levels compared to SGR-Gluc. Because these mutants do not replicate, expression was driven by the vaccinia-T7 RNA polymerase system. (E) Design of the Jc1-Gluc(3–5Arc) mutant. (F) Jc1-Gluc(3–5Arc) replicates efficiently. Values represent mean ± SD from transfections done in triplicate and normalized to untransfected controls; n.s., p >0.05, *, p ≤ 0.05 by Student’s t-test. This experiment was performed two times with similar results (G) Model showing that SGR-Gluc(3m1) and SGR-Gluc(3m2) do not undergo proteolytic processing, produce misfolded, inactive polyproteins, and are not complemented in trans; grey signifies the nonfunctional serine protease domains; blue signifies the recoded NS3-5B polyprotein. (H) Model showing that SGR-Gluc(3m3) and SGR-Gluc(3m4) are not trans-complemented and may have a defect in RNA recruitment (compare to Fig 1B); grey signifies the nonfunctional helicase domains; blue signifies the recoded NS3-5B polyprotein.