Identification and physical characterization of a spontaneous mutation

Virus-like particles are an emerging class of nano-biotechnology with the Tobacco Mosaic Virus (TMV) having found a wide range of applications in imaging, drug delivery, and vaccine development. TMV is typically produced in planta, and, as an RNA virus, is highly susceptible to natural mutation that may impact its properties. Over the course of 2 years, from 2018 until 2020, our laboratory followed a spontaneous point mutation in the TMV coat protein—first observed as a 30 Da difference in electrospray ionization mass spectrometry (ESI–MS). The mutation would have been difficult to notice by electrophoretic mobility in agarose or SDS-PAGE and does not alter viral morphology as assessed by transmission electron microscopy. The mutation responsible for the 30 Da difference between the wild-type (wTMV) and mutant (mTMV) coat proteins was identified by a bottom-up proteomic approach as a change from glycine to serine at position 155 based on collision-induced dissociation data. Since residue 155 is located on the outer surface of the TMV rod, it is feasible that the mutation alters TMV surface chemistry.


Fig: (A) Deconvoluted ESI–MS spectra of denatured TMV showing the emergence of a coat protein mutant from Jan 2018 till Jan 2020. The theoretical mass of wild-type TMV coat protein (wTMV) is 17,534 Da (including acetylation of the N-terminus). The mutant TMV coat protein (mTMV) is measuring approximately 30 Da larger. The relative abundance of the mTMV increased over time. (B) 10% SDS-PAGE and 1% agarose gel electrophoresis showing that the wTMV and mTMV migrate at the same rate. ‘w’ is mostly wTMV, ‘m’ is mostly mTMV, and ‘w + m’ is a mixture. (C) TEM micrographs of wTMV and mTMV showing that the rod-shaped viral morphology is not impacted by the mutation.

However, enzyme-linked immunosorbent assays found no difference in binding between mTMV and wTMV. Functionalization of a nearby residue, tyrosine 139, with diazonium salt, also appears unaffected. Overall, this study highlights the necessity of standard workflows to quality-control viral stocks. We suggest that ESI–MS is a straightforward and low-cost way to identify emerging mutants in coat proteins.

Lumata, J.L., Ball, D., Shahrivarkevishahi, A. et al. Identification and physical characterization of a spontaneous mutation of the tobacco mosaic virus in the laboratory environment. Sci Rep 11, 15109 (2021).

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