Abstract
The replication-associated (Rep) proteins of pathogenic begomoviruses, including cotton leaf curl Multan virus (CLCuMuV) and pedilanthus leaf curl virus (PeLCV), interact with the DNA replication machinery of their eukaryotic hosts. The analysis of Rep protein sequences showed that there is 13–28% sequence variation among CLCuMuV and PeLCV isolates, with phylogenetic clusters that can separated at least in part based on the country of origin of the respective viruses. To identify specific host factors involved in the virus replication cycle, we conducted yeast two-hybrid assays to detect possible interactions between the CLCuMuV and PeLCV Rep proteins and 30 protein components of the Saccharomyces cerevisiae DNA replication machinery. This showed that the proliferating cell nuclear antigen (PCNA) protein of S. cerevisiae interacts with Rep proteins from both CLCuMuV and PeLCV. We used the yeast PCNA sequence in BLAST comparisons to identify two PCNA orthologs each in Gossypium hirsutum (cotton), Arabidopsis thaliana (Arabidopsis), and Nicotiana benthamiana (tobacco). Sequence comparisons showed 38–40% identity between the yeast and plant PCNA proteins, and > 91% identity among the plant PCNA proteins, which clustered together in one phylogenetic group. The expression of the six plant PCNA proteins in the yeast two-hybrid system confirmed interactions with the CLCuMuV and PeLCV Rep proteins. Our results demonstrate that the interaction of begomovirus Rep proteins with eukaryotic PCNA proteins is strongly conserved, despite significant evolutionary variation in the protein sequences of both of the interacting partners.
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Acknowledgements
The authors are thankful to all lab members for their support in conducting research experiments. The research was funded by the International Foundation for Science (IFS grant no, C/5434-1) and the Higher Education Commission (HEC) of Pakistan (project no 1682). Sara Shakir was supported by the HEC indigenous scholarship program and the International Research Support Initiative Program (IRSIP).
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Fig. S1 Multiple sequence alignment of Rep. A typical depiction of multiple sequence alignment of replication-associated protein (Rep) from representative strains of cotton leaf curl Multan virus (CLCuMuV) and pedilanthus leaf curl virus (PeLCV). Asterisks and spaces, respectively, indicate conserved and nonconserved regions. The sequence alignment was performed with Mega 6 (Tamura et al. 2013) using the MUSCLE algorithm and the resulting image was edited in PowerPoint. Fig. S2 Multiple sequence alignment of PCNA. A typical depiction of a multiple sequence alignment of proliferating cell nuclear antigen (PCNA) of S. cerevisiae and begomovirus infecting plant species used in the current study. Asterisks and spaces, respectively indicate conserved and nonconserved regions. The sequence alignment was performed with Mega 6 (Tamura et al. 2013) using the MUSCLE algorithm and the resulting image was edited in PowerPoint (DOCX 7348 kb)
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Shakir, S., Jander, G., Nahid, N. et al. Interaction of eukaryotic proliferating cell nuclear antigen (PCNA) with the replication-associated protein (Rep) of cotton leaf curl Multan virus and pedilanthus leaf curl virus. 3 Biotech 11, 14 (2021). https://doi.org/10.1007/s13205-020-02499-5
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DOI: https://doi.org/10.1007/s13205-020-02499-5