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The N-terminal region containing the zinc finger domain of tobacco streak virus coat protein is essential for the formation of virus-like particles

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Abstract

Tobacco streak virus (TSV), a member of the genus Ilarvirus (family Bromoviridae), has a tripartite genome and forms quasi-isometric virions. All three viral capsids, encapsidating RNA 1, RNA 2 or RNA 3 and subgenomic RNA 4, are constituted of a single species of coat protein (CP). Formation of virus-like particles (VLPs) could be observed when the TSV CP gene was cloned and the recombinant CP (rCP) was expressed in E. coli. TSV VLPs were found to be stabilized by Zn2+ ions and could be disassembled in the presence of 500 mM CaCl2. Mutational analysis corroborated previous studies that showed that an N-terminal arginine-rich motif was crucial for RNA binding; however, the results presented here demonstrate that the presence of RNA is not a prerequisite for assembly of TSV VLPs. Instead, the N-terminal region containing the zinc finger domain preceding the arginine-rich motif is essential for assembly of these VLPs.

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Acknowledgments

We thank Dr. S. S. Indi, Dept. of Microbiology and Cell Biology, IISc, for his assistance in electron microscopy. We thank Department of Biotechnology and Department of Science and Technology (J.C. Bose fellowship to HSS), New Delhi, India, for the financial support. CM thanks CSIR, New Delhi, for Junior and Senior Research fellowships. We thank Prof. M.R.N. Murthy for critical reading of the manuscript.

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Authors and Affiliations

  1. Department of Biochemistry, Indian Institute of Science, Bangalore, 560 012, India

    Chhavi Mathur, Kalyani Mohan & Handanahal S. Savithri

  2. Division of Plant Pathology, Indian Institute of Horticulture, Bangalore, 560 089, India

    T. R. Usha Rani & M. Krishna Reddy

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  1. Chhavi Mathur
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Correspondence to Handanahal S. Savithri.

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Supplementary material 1 (TIFF 1223 kb) Supplementary Table S1: List of primers used for cloning and RT-PCR

705_2013_1822_MOESM2_ESM.tif

Supplementary material 2 (TIFF 612 kb) Supplementary Fig. S1 Analysis of TSV CP (a) Mass spectrometric analysis of purified recombinant TSV CP (molecular sizes of monomer and dimer peaks are indicated). (b) Glutaraldehyde crosslinking of TSV CP. TSV coat protein (10 μg) was incubated with 0.01 % glutaraldehyde (Sigma) for 1 h in the dark. The samples were then separated by 10 % SDS-PAGE and stained with CBB R250. Lane 1, TSV CP; lane 2, glutaraldehyde-crosslinked TSV CP

705_2013_1822_MOESM3_ESM.tif

Supplementary material 3 (TIFF 3595 kb) Supplementary Fig. S2 Deletion mutants of TSV CP (a) Amino acid sequence of TSV CP indicating the positions where deletions were created. Underlined, zinc-finger domain; bold, RNA-binding N-ARM. (b) SDS-PAGE analysis of TSV CP and its deletion mutants. Lane 1, molecular mass (kDa) markers; lane 2, purified Ndel48 CP; lane 3, purified Ndel26 CP; lane 4, purified Ndel86 CP. (c) Lane 1, uninduced fraction – TSV CP; lane 2, induced fraction – TSV CP; lane 3, uninduced fraction –Cdel38 CP; lane 4, induced fraction – Cdel38 CP; lane 5, uninduced fraction – Cdel55 CP; lane 6, induced fraction – Cdel55 CP; lane 7, uninduced fraction – Cdel74 CP; lane 8, induced fraction – Cdel74 CP; lane 9, molecular mass (kDa) markers

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Mathur, C., Mohan, K., Usha Rani, T.R. et al. The N-terminal region containing the zinc finger domain of tobacco streak virus coat protein is essential for the formation of virus-like particles. Arch Virol 159, 413–423 (2014). https://doi.org/10.1007/s00705-013-1822-4

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