Fig 6.

Cellular mRNAs become hyperadenylated, and the amount of poly(A)-containing mRNAs decreases in the cytoplasm of infected cells. (A) MA104 cells were infected or mock infected with rotavirus RRV, and at 12 hpi, the cells were lysed. Total RNA was extracted with TRIzol, hybridized or not with oligo(dT), and digested with RNase H as described in Materials and Methods. The digested products were resolved on a 0.8% agarose-formaldehyde gel, blotted, and hybridized to a ³²P-labeled grp78 probe. Hyperadenylated species are indicated by the open bracket [hyp(A) grp78]. The ribosomal 18S RNA, which was stained with ethidium bromide, is shown as a loading control. (B) MA104 cells were mock infected or infected with rotavirus RRV, and at 12 hpi, total, nuclear, and cytoplasmic fractions were obtained as described in Materials and Methods. RNA was extracted with TRIzol, and poly(A)-containing mRNAs were purified by affinity chromatography using an Oligotex Direct mRNA minikit (Qiagen). The mRNA content of each fraction was determined by measuring the absorbance at 260 nm in an ND-1000 spectrophotometer. The amount of polyadenylated mRNAs in mock-infected cells was taken as 100%.