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<p>Geminiviral C2 protein is a multifunctional protein that serves as a versatile regulatory hub, orchestrating the trans-activation of viral promoters while simultaneously subverting host immunity by inhibiting gene silencing pathways (TGS/PTGS) and disrupting the ubiquitin–proteasome system via the COP9 signalosome. Despite these critical roles, the structural architecture of C2 remains unelucidated, and the presence of predicted intrinsically disordered regions (IDRs), which may facilitate such functional plasticity, has not been explored in detail. In this study, we employ computational modeling to predict the three-dimensional structure of Tomato yellow leaf curl virus (TYLCV) C2 and characterize its disordered regions. Our findings provide a structural rationale for how this small protein coordinates diverse protein–protein interactions, offering new insights into the molecular mechanisms by which geminiviruses hijack host cellular machinery. Computational analyses predicted prominent intrinsically disordered regions between residues 40–120 and highlighted the zinc finger domain as a key structural element. Based on these computational predictions, domain deletion mutants (del NLS, del ZnFn, del AD) and zinc finger point mutants (C37A, C39A) were generated and transiently expressed in <i>Nicotiana benthamiana</i> as CFP fusion proteins. Subcellular localization was assessed by confocal microscopy, while HR induction and H<sub>2</sub>O<sub>2</sub> accumulation were evaluated by visual inspection and DAB staining. Wild-type C2 localized predominantly to the nucleus and was associated with HR-like responses. In contrast, deletion of the NLS, Zinc finger, or activation domain, or cysteine mutations in the zinc finger were associated with loss of detectable HR-like responses. Molecular dynamics simulations further showed that deletion of the zinc finger destabilized the protein, whereas removal of the NLS or activation domain produced more compact conformations. Overall, these findings indicate that the zinc finger domain might be important for structural integrity and biological activity, while the NLS appears to be important for nuclear localization, highlighting a potential interplay between ordered domains and predicted intrinsically disordered regions in TYLCV C2.</p>

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In silico structural and disorder prediction of the tomato yellow leaf curl virus C2 protein and experimental assessment of subcellular localization and HR-like response

  • 期刊论文

  • 发表日期：

    2026年6月15日

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  • Sam A. Chandran,
  • Kishore Nagasubramanian,
  • Hagit Hak,
  • Yael Levy,
  • Eduard Belausov,
  • Yedidya Gafni

摘要

Geminiviral C2 protein is a multifunctional protein that serves as a versatile regulatory hub, orchestrating the trans-activation of viral promoters while simultaneously subverting host immunity by inhibiting gene silencing pathways (TGS/PTGS) and disrupting the ubiquitin–proteasome system via the COP9 signalosome. Despite these critical roles, the structural architecture of C2 remains unelucidated, and the presence of predicted intrinsically disordered regions (IDRs), which may facilitate such functional plasticity, has not been explored in detail. In this study, we employ computational modeling to predict the three-dimensional structure of Tomato yellow leaf curl virus (TYLCV) C2 and characterize its disordered regions. Our findings provide a structural rationale for how this small protein coordinates diverse protein–protein interactions, offering new insights into the molecular mechanisms by which geminiviruses hijack host cellular machinery. Computational analyses predicted prominent intrinsically disordered regions between residues 40–120 and highlighted the zinc finger domain as a key structural element. Based on these computational predictions, domain deletion mutants (del NLS, del ZnFn, del AD) and zinc finger point mutants (C37A, C39A) were generated and transiently expressed in Nicotiana benthamiana as CFP fusion proteins. Subcellular localization was assessed by confocal microscopy, while HR induction and H2O2 accumulation were evaluated by visual inspection and DAB staining. Wild-type C2 localized predominantly to the nucleus and was associated with HR-like responses. In contrast, deletion of the NLS, Zinc finger, or activation domain, or cysteine mutations in the zinc finger were associated with loss of detectable HR-like responses. Molecular dynamics simulations further showed that deletion of the zinc finger destabilized the protein, whereas removal of the NLS or activation domain produced more compact conformations. Overall, these findings indicate that the zinc finger domain might be important for structural integrity and biological activity, while the NLS appears to be important for nuclear localization, highlighting a potential interplay between ordered domains and predicted intrinsically disordered regions in TYLCV C2.