Supporting Figures

Files in this Data Supplement:

Supporting Figure 5
Supporting Figure 6
Supporting Figure 7

Fig. 5. Effect of AdoMet and SAH concentration on G-N-7 methylation of rVSV and G1674A. Viral mRNA was synthesized in vitro in the presence of altered [AdoMet] (A) or [SAH] (B) and 15 mCi of [a-³²P]GTP without a cell lysate. Purified RNA was digested with 2 units of TAP, and the products were analyzed by TLC on PEI F cellulose sheets. Plates were dried, and the spots were visualized by phosphoimage analysis. The identity of the virus is shown at the bottom of the plate, and the migration of the markers 7^mGp and Gp is indicated. Quantitative analysis was performed on three independent experiments (Lower). For each virus, the released 7^mGp (mean ± SD) was expressed as a percentage of the total released cap structure. These experiments showed that recombinant G1674A requires a higher concentration of AdoMet for G-N-7 methylation compared with rVSV and that methylation is more sensitive to SAH inhibition.

Fig. 6. Effects of [AdoMet] in the presence of a cell lysate on G-N-7 methylation. (A) Viral mRNA was synthesized in vitro in the presence of different concentrations of AdoMet (20, 200, and 1,000 mM), a cell lysate, and 15 mCi of [a-³²P]GTP as described in Materials and Methods. Purified mRNAs were digested with 2 units of TAP, and the products were analyzed by TLC on PEI cellulose sheets. Plates were dried, and the spots were visualized by using a phosphoimager. (B) Quantitative analysis was performed on three independent experiments for each condition. These data show that the inclusion of a cell lysate has no significant effect on G-N-7 methylation (compare Figs. 3 and 6). The TLC plate shown was using a cell lysate supplemented with 20 mM [³H]AdoMet, the exact same conditions that were used to evaluate the effect of these substitutions on 2'-O methylation.

Fig. 7. G1670A and G1672A are defective in G-N-7 methylation but not 2'-O methylation. (A) Viral mRNA was synthesized in vitro in the presence of 15 mCi of [³H]AdoMet and a cell lysate to increase RNA yield. Purified RNA was digested with 10 units of RNase T2 and 2 units of TAP, and the products were analyzed by TLC on PEI cellulose F sheets. The plates were dried, and the spots were visualized by using a phosphoimager. The identity of the virus is shown at the top of the plate, and the migration of the marker 7^mGp is shown on the right. RNase T2 specifically cleaves the phosphodiester bonds on the 3' side of A residues. Complete digestion of the VSV mRNA cap structure (7^mGpppA^mpApCpApGp) with RNase T2 and TAP should yield 7^mGp and pA^mpA if the cap structure is both [G-N-7] and [2'-O]-methylated. For rVSV and G1674A, two products of RNase T2 and TAP cleavage were detected (lanes 1 and 4), one of which comigrated with the 7mGp marker. This is consistent with the presence of the fully methylated cap structure 7^mGpppA^mpA and the data of Fig. 3 D and E. Both of these products were absent when transcription reactions were performed in the presence of SAH, demonstrating that they were methylated. In contrast, for G1670A and G1672A, the major product observed comigrated with the slower product obtained with rVSV and G1674A (lanes 2 and 3). Our data from Fig. 3E predicted that the major product of RNase T2 cleavage would be GpppA^mA and that codigestion of this product with TAP should yield an unlabeled Gp and the labeled pAmpAp. These data thus provide additional support that the cap structures synthesized by G1670A and G1672A were 2'-O-methylated but not G-N-7-methylated and are thus of the form GpppA^mpA. (B) Quantitative analysis was performed on two independent experiments.