FIG 7.

Analysis of hMPV and VSV mRNA cap methylation by in vitro trans-methylation assay. (A) trans-G-N-7 methylation assay for hMPV and VSV. Five hundred nanograms of mRNA was isolated from virus-infected Vero E6 cells and was trans-methylated by vaccinia virus G-N-7 MTase in the presence of 15 μCi [³H]SAM as described in Materials and Methods. The number of corrected counts per minute (CCPM) of [³H]SAM incorporation from RNA from mock-infected cells was subtracted from the CCPM of each hMPV mutant. Subsequently, the CCPM was normalized by the level of viral mRNA quantified by real-time RT-PCR. The number on the top of each column indicates the ratio of [³H]SAM incorporation of viral mRNAs between mutant and wild-type virus. The averages of four independent experiments are shown. (B) trans-2′-O methylation assay for hMPV and VSV. Five hundred nanograms of mRNA was isolated from virus-infected cells and was subsequently premethylated by vaccinia virus G-N-7 MTase in the presence of 1 mM cold SAM. The RNAs were purified and further trans-methylated by vaccinia virus 2′-O MTase in the presence of 15 μCi [³H]SAM. The number of CCPM of [³H]SAM incorporation for each virus is shown. The number on the top of each column indicates the ratio of [³H]SAM incorporation of viral mRNAs between mutant and wild-type virus. The averages of four independent experiments are shown.