Abstract
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. 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.
Highlights
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
The intact TMV contains 2130 copies of the coat protein assembled into a helical rod with a length of 300 nm, an outer diameter of 18 nm, and a pore diameter of 4 nm
Our TMV stock is grown in N. benthamiana eight weeks after germination while the plants are around 11.5 cm tall
Summary
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. The intact TMV contains 2130 copies of the coat protein assembled into a helical rod with a length of 300 nm, an outer diameter of 18 nm, and a pore diameter of 4 nm. Nanoparticles, use an extremophile strain of the Australian plant Nicotiana benthamiana for large-scale production of TMV It is the most widely used host for plant virus replication as it is very susceptible to infection. TMV is well-known to be susceptible to environmental and evolutionary stressors and the emergence of mutants should not be surprising; there is little discussion in the literature of how coat protein mutations might alter the viruses’ physical properties vis-à-vis chemical functionalization and antibody binding. Knowing what to look for, the relative time scale, and how to identify mutation in plant-sourced viruses would be helpful to the broader community
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