Figure 3: Mutational analysis of the N-terminal 13aa motif of STMV CP.

(A) Characteristic features of the agroconstruct of CP 13aa is shown in Fig. 1C. The N-terminal 13aa sequence of the CP is indicated below the line diagram. In the clone 13aa, the location of the positively charged amino acids are indicated by a + sign. In each variant clone the location of the engineered alanine mutation is indicated by an underline. The nomenclature used to designate each variant is exemplified by 13aa/XA, where X is characterized by an alanine (A) substitution for arginine (R, at positions 3 and 10) or lysine (K at positions 5, 7 and 11). (B) Progeny analysis of N-terminal 13aa motif variants by Northern blot and RT-PCR. Duplicate blots containing total RNA of N. benthamiana leaves infiltrated with the indicated samples (lanes 1 through 8) were hybridized with a ³²P-labelled STMV probe to detect either plus (panel i) or minus (panel ii) sense RNA. Ribosomal RNA (rRNA) represents the loading control. Conditions used for Northern blot hybridization and RT-PCR is as described in the Fig. 2A legend. Positions of STMV and STMVΔ150 are indicated to the right. (C) Progeny RNA quantification. Northern blots shown in panel B were scanned using a Phosphoimager. Since each mutation was engineered into the genetic background of 13aa, the absolute values of accumulated (+) and (−)-strand progeny RNA for each mutant were compared internally to the 13aa.