Research ArticlePLANT SCIENCES

A distinct class of plant and animal viral proteins that disrupt mitosis by directly interrupting the mitotic entry switch Wee1-Cdc25-Cdk1

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Science Advances  13 May 2020:
Vol. 6, no. 20, eaba3418
DOI: 10.1126/sciadv.aba3418
  • Fig. 1 Inhibition of fission yeast cell growth and mitosis by BYDV-GAV 17K.

    (A) The seven ORFs of BYDV-GAV and their corresponding proteins. (B) Effect of inducible BYDV-GAV protein production on fission yeast colony formation. Gene-off, no production of BYDV-GAV protein; gene-on, inducible production of BYDV-GAV protein. VC, vector control. Cells were streaked onto agar plates, incubated at 30°C, and recorded at 5 days after inoculation. (C) Cell elongation induced by 17K expression in fission yeast. Cell image was captured at 24 hours after 17K induction. Average cell length values of the two cultures differed significantly (P < 0.0001, Student’s t test). Scale bars, 10 μm. (D) Distribution of fission yeast cell lengths in low-nitrogen EMM with or without 17K production as analyzed by forward scatter analysis of 10,000 cells per culture. Cells were collected at 40 hours after 17K induction. FSC, forward scatter; SSC, side scatter. (E) Effect of 17K expression on nuclear DNA content of fission yeast cells as determined by flow cytometry at 40 hours after 17K induction. The dotted line indicates polyploid nuclei in the cells expressing 17K. The datasets shown above were each repeated three times with comparable results obtained. Photo credits: Judit Antal and Zsigmond Benko (Children’s Memorial Institute for Education and Research, Northwestern University Feinberg School of Medicine, Chicago, IL 60614, USA).

  • Fig. 2 Suppression of barley mitosis by 17K.

    (A) Organization of DZ, EZ, MZ, and root cap (RC) in barley root tips. Dash lines indicate the cuts for preparing DZ, EZ, and MZ + RC samples. Amplification of barley GAPDH gene served as an internal control. (B) Growth of BYDV-GAV–infected barley seedlings and mock controls examined at 4, 7, and 14 DPI, respectively. (C) Analysis of nuclear DNA contents by flow cytometry using root tip cells from BYDV-GAV–infected barley seedlings or mock controls at 4 or 7 DPI. The means (± SE) were calculated from four separated experiments. *P < 0.05 and **P < 0.01 (Student’s t test). (D) Localization of 17K-GFP in transgenic barley line tB_17K-GFP1. (E) The root growth of barley seedlings was inhibited in 17K transgenic lines (tB_17K-GFP1 and tB_17K-GFP5) comprised to WT and GFP transgenic line (tB_GFP4). (F) Flow cytometry analysis of root tip cells from WT and three transgenic barley lines. The means (± SE) were each calculated from three independent assays, with significantly different values labeled by nonidentical letters [P < 0.05, analysis of variance (ANOVA) and least significant difference test (LSD) for multiple comparisons]. Photo credits: Huaibing Jin and Yanjing Zhang (State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China).

  • Fig. 3 Inhibition of nonhost plant growth and mitosis by 17K.

    (A) Effects of 17K expression on Arabidopsis growth analyzed using transgenic lines (tA_17K-His and tA_17K-GFP) with inducible expression of 17K by ES and a control line expressing free GFP. (B) Reduction of primary root growth by inducible expression of 17K in transgenic Arabidopsis. (C) Measurement of nuclear DNA contents. DNA contents in the root tip cells of tA_GFP, tA_17K-GFP, or WT control cultured with 0.5 μM ES (+ES) or tA_17K-GFP without ES (−ES) were measured by flow cytometry. The means (± SE) were each calculated from three separate experiments, with significantly different values labeled by nonidentical letters (P < 0.05, ANOVA and LSD for multiple comparisons). (D) Compared to the parental marker line pCYCB1;1::GUS (28), CYCB1,1::GUS signals (represented by the blue precipitates) were substantially decreased in root tips of the F1 seedlings derived from crossing pCYCB1.1::GUS with tA_17K-GFP, with 17K expression induced by ES. (E) Validation of PEBV vector–mediated expression of 17K and 17K-GFP in N. benthamiana using immunoblotting (IB). (F) Inhibition of N. benthamiana growth by 17K or 17K-GFP. CK, mock control (infiltrated with water). The datasets presented were each typical of three independent experiments. Photo credits: Huaibing Jin and Zongliang Xia (State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China).

  • Fig. 4 Interruption of mitotic entry switch by 17K in plant cells.

    (A) The interactions detected between 17K and components of the mitotic entry switch. (B to D) Effects of 17K expression on Wee1 protein accumulation. Protein samples, extracted from transgenic Arabidopsis seedlings (B), transgenic barley root tips (C), and BYDV-GAV–infected barley root tips (D), were treated with or without λ-PP, followed by immunoblotting with anti-Wee1 antibody. Arrowhead, phosphorylated Wee1. (E) Inhibition of in vitro phosphatase activities of AtCdc25 and HvCdc25 by 17K. Ten reactions were carried out with the reagents listed on top. To facilitate comparison, the OD410 (optical density at 410 nm) absorbance in reaction 1 was set as 1. Each mean (± SE) was calculated from three independent assays. *P < 0.05, **P < 0.01, and ***P < 0.001 (Student’s t test). BSA, bovine serum albumin; pNPP, para-nitrophenyl phosphate. (F to H) Up-regulation of the CDKA with T14 and Y15 phosphorylation by 17K. Protein samples, prepared as detailed in (B) to (D), were subjected to immunoblotting with the antibodies specific for PSTAIRE, phosphor-T14, or phosphor-Y15. Numerical values represent relative intensities of the corresponding bands calculated by ImageJ. The datasets shown were each repeated three times with comparable results obtained.

  • Fig. 5 Similarities among the secondary structures of BYDV-GAV 17K, HIV-1 Vpr, and ARV p17 and in the inhibition of tobacco growth.

    (A) Secondary structure of 17K, Vpr, or p17 modeled with QUART software (47). (B) CD spectrum of recombinant 17K. Two negative peaks around 208 and 222 nm indicated that 17K was mainly composed of α helices. (C) Inhibition of tobacco growth by 17K, 17K-GFP, Vpr, Vpr-GFP, p17, or p17-GFP expressed using PEBV vector. (D) Nuclear DNA contents in young leaves of the tobacco plants displayed in (C). The means (± SE) were each calculated from three independent flow cytometry assays, with significantly different means labeled by nonidentical letters (P < 0.05, ANOVA and LSD for multiple comparisons). (E to G) Effects of 17K expression on HEK-293Ta cell proliferation (E), cell size distribution (analyzed by flow cytometry with 30,000 cells per culture) (F), and cell cycle progression at 12 and 24 hours after transfection (G). The means (± SE) in (G) were each calculated from four separated experiments. ***P < 0.001 (Student’s t test). The datasets presented were representative of at least three separate experiments. Photo credits: Huaibing Jin (State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China).

  • Fig. 6 Analysis of the three residues conserved between 17K and Vpr and their requirement for efficient disruption of mitosis.

    (A) Amino acid sequence alignment of BYDV-GAV 17K and HIV-1 Vpr. H1 (17-33), H2 (38-50), and H3 (56-77), respectively indicate the three α helices of Vpr. Red arrows mark the three residues that have been reported to function in Vpr-induced G2/M arrest (4042). (B and C) Decreased inhibition of tobacco growth by the 17K mutant 17K-L48A/L96A/R117A and the Vpr mutant Vpr-L22A/L67A/R88A. 17K, Vpr, and their corresponding mutants were expressed in N. benthamiana using the PEBV vector. The graph was taken at 4 weeks after agroinfiltration. (D) Nuclear DNA contents in the young leaves of the tobacco plants shown in (B). The means (± SE) were each calculated from three separated flow cytometry assays, with significantly different means labeled by dissimilar letters (P < 0.05, ANOVA and LSD for multiple comparisons). (E) The 17K mutant 17K-L48A/L96A/R117A and the Vpr mutant Vpr-L22A/L67A/R88A failed to interact with the CDKAs of N. benthamiana, Arabidopsis, and barley in Y2H assays. As a control, WT Vpr showed positive interactions with the same set of CDKAs. The datasets presented above were each typical of three independent assays. Photo credits: Huaibing Jin (State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China).

Supplementary Materials

  • Supplementary Materials

    A distinct class of plant and animal viral proteins that disrupt mitosis by directly interrupting the mitotic entry switch Wee1-Cdc25-Cdk1

    Huaibing Jin, Zhiqiang Du, Yanjing Zhang, Judit Antal, Zongliang Xia, Yan Wang, Yang Gao, Xiaoge Zhao, Xinyun Han, Yanjun Cheng, Qianhua Shen, Kunpu Zhang, Robert E. Elder, Zsigmond Benko, Csaba Fenyvuesvolgyi, Ge Li, Dionne Rebello, Jing Li, Shilai Bao, Richard Y. Zhao, Daowen Wang

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