Inoculation of six brome mosaic virus (BMV) RNA3 transcripts with defined deletions in the coat protein (CP) gene to three Chenopodium spp demonstrated that synthesis of a functional, encapsidation-competent CP is required for the induction of local lesions. The BMV CP open reading frame contains two in-frame AUG codons separated by seven amino acids, resulting in the synthesis of two CPs (CP1 and CP2). To elucidate the biological significance of the N-terminal basic region of BMV CP, RNA3 variants capable of producing either CP1 or CP2 but not both were constructed. Infection phenotypes elicited on three Chenopodium spp by each RNA3 variant revealed that amino-terminal residues 1 to 7 are required to establish chlorotic local lesions and systemic infection in Chenopodium quinoa. Deletion of this region has no effect on infection in barley plants but resulted in the induction of the hypersensitive response on the inoculated leaves of C. quinoa and blocked systemic spread. Analysis of seven additional RNA3 variant transcripts, each having a six-base deletion (two amino acids) in the sequence encoding the N-terminal seven residues, indicated that variants that share a common deletion of positively charged lysine rendered the CP encapsidation-incompetent and failed to establish infection. Taken together, these results suggest that residues 1 to 7 of the BMV CP play an important role in virus-host interactions and contribute differently to the virulence phenotype in different host plants.