Tobacco necrosis virus-A ^C single coat protein amino acid substitutions determine host-specific systemic infections of Nicotiana benthamiana and soybean

Plant viruses often infect several distinct host species. Sometimes, viruses can systemically infect a specific host, whereas in other cases, only local infections occur in other species. How viral and host factors interact to determine systemic infections among different hosts is largely unknown, particularly for icosahedral positive-stranded RNA viruses. Tobacco necrosis virus-A Chinese isolate (TNV-A^C) belongs to the genus Alphanecrovirus in the family Tombusviridae. In this study, we investigated variations in systemic infections of TNV-A^C in Nicotiana benthamiana and Glycine max (soybean) by alanine-scanning mutagenesis of the viral coat protein (CP), which is essential for systemic movement of TNV-A^C. We found that three amino acids, R¹⁶⁹, K¹⁷⁷ and Q²³³, are key residues that mediate varying degrees of systemic infections of N. benthamiana and soybean. Further analysis revealed that variations in systemic trafficking of TNV-A^C CP mutants in N. benthamiana and soybean are associated with virion assembly and stability. The CP amino acids K¹⁷⁷ and Q²³³ are highly conserved among all TNV-A isolates and are replaced by Q and K in the TNV-D isolates. We demonstrated that systemic infectivity of either TNV-A^C K177A and Q233A or K177Q and Q233K mutants are correlated with the binding affinity of the mutated CPs to the host-specific Hsc70-2 protein. These results expand our understanding of host-dependent long-distance movement of icosahedral viruses in plants.