Point mutations were introduced into or near five conserved sequence motifs of the readthrough domain of the beet western yellows virus minor capsid protein P74. The mutant virus was tested for its ability to accumulate efficiently in agroinfected plants and to be transmitted by its aphid vector, Myzus persicae. The stability of the mutants in the agroinfected and aphid-infected plants was followed by sequence analysis of the progeny virus. Only the mutation Y201D was found to strongly inhibit virus accumulation in planta following agroinfection, but high accumulation levels were restored by reversion or pseudoreversion at this site. Four of the five mutants were poorly aphid transmissible, but in three cases successful transmission was restored by pseudoreversion or second-site mutations. The same second-site mutations in the nonconserved motif PVT(32-34) were shown to compensate for two distinct primary mutations (R24A and E59A/D60A), one on each side of the PVT sequence. In the latter case, a second-site mutation in the PVT motif restored the ability of the virus to move from the hemocoel through the accessory salivary gland following microinjection of mutant virus into the aphid hemocoel but did not permit virus movement across the epithelium separating the intestine from the hemocoel. Successful movement of the mutant virus across both barriers was accompanied by conversion of A59 to E or T, indicating that distinct features of the readthrough domain in this region operate at different stages of the transmission process.