Single Amino Acid Change at Two Conserved Residues in Tomato Brown Rugose Fruit Virus Coat Protein Compromises Virion Assembly and Viral Systemic Infection

<i>Tomato brown rugose fruit virus</i> (ToBRFV; <i>Tobamovirus fructirugosum</i>) is a recently discovered new species in the genus <i>Tobamovirus</i> that causes an ongoing pandemic and threatens tomato production worldwide. In this study, we determined the complete genome sequences of three ToBRFV isolates collected from greenhouse tomato plants in Canada in 2019, analyzed their possible phylogenetic relationships, and developed corresponding full-length infectious cDNA clones. Using the ToBRFV infectious clone, we generated gene-specific mutants via site-directed mutagenesis, followed by an infection assay. We confirmed that both the movement protein and the coat protein (CP) are indispensable for ToBRFV long-distance movement. Moreover, we found that alanine substitution of amino acid D89 or R114, both of which are highly conserved among tobamoviral CPs, compromised ToBRFV systemic infection. Confocal and electron microscopy analyses further revealed that either D89A or R114A substitution disrupted CP self-interactions and virion assembly. Additionally, we demonstrated that the ToBRFV 126-kDa replicase has RNA silencing suppression activity. Taken together, our data contribute to a better understanding of ToBRFV-encoded proteins at the molecular level, and the ToBRFV full-length infectious cDNA clones developed from this study are a useful tool to facilitate ToBRFV research. [Formula: see text] © 2026 His Majesty the King in Right of Canada, as represented by the Minister of Agriculture and Agri-Food Canada. This is an open access article distributed under the CC BY-NC-ND 4.0 International license.