FIGURE 1.

Impaired EMCV replication in untreated Krebs-2 S10 extract programmed with the poly(A) tail-deficient EMCV RNA. (A) RNase H/oligo(dT) treatment of EMCV RNA. EMCV RNA that was treated with RNase H/oligo(dT) and blocked at the 3′ end with cordycepin was analyzed along with the intact RNA by denaturing agarose gel electrophoresis and Northern blotting using ³²P-labeled oligo(dT) or a fragment of EMCV cDNA (EMCV) as a probe. (B) Comparison of translation efficiencies of poly(A+) and poly(A−) EMCV RNAs. Intact and deadenylated EMCV RNA, at the indicated concentrations, was translated in untreated extracts in the presence of [³⁵S]methionine as described in Materials and Methods. The samples were analyzed by SDS-PAGE and fluorography. The positions of EMCV-specific proteins are indicated on the right. Products of translation of endogenous cellular mRNA are also shown (zero EMCV RNA concentration). (C) Stability of EMCV RNA. Poly(A+) and poly(A−) EMCV RNA was used at a concentration of 10 μg/mL to program untreated extracts. Total RNA was isolated at the indicated times from aliquots of the reaction mixtures. EMCV RNA integrity was analyzed by formaldehyde–agarose gel electrophoresis and Northern blotting as described in Materials and Methods. Relative amounts of recovered mRNAs are indicated at the bottom (values obtained for time 0 were set as 100%). (D) Poly(A+) and poly(A−) EMCV RNA dose responses of RNA synthesis. Poly(A+) and poly(A−) EMCV RNAs were added to the reaction mixtures at the indicated concentrations. The products of RNA synthesis were labeled with [α-³²P]CTP and analyzed by agarose gel electrophoresis and autoradiography as described in Materials and Methods. (Arrow) Position of single-stranded EMCV RNA. (E) Poly(A+) and poly(A−) EMCV RNA dose responses of virus yield. Reaction mixtures were programmed with the indicated concentrations of poly(A+) or poly(A−) EMCV RNA for 18 h at 32°C, treated with RNase A/T1, and assayed for infectivity. The data are averages (with standard deviation from the mean) of three independent titer determinations.