Poliovirus-induced inhibition of host RNA synthesis studied in isolated HEp-2 cell nuclei.

Abstract

Nuclei isolated from uninfected HEp-2 cells synthesized RNA for 60 to 90 min. The individual RNA polymerase activities were determined by alpha-amanitin differential inhibition and the RNA products characterized by electron microscope (EM) autoradiography and sucrose gradient centrifugation. In nuclei prepared from poliovirus-infected cells, the capacity to synthesize RNA in vitro decreased with time after infection. RNA polymerase II activity (hnRNA synthesis) was preferentially inhibited more than was the polymerase I activity (rRNA synthesis). Poliovirus-infected cytoplasm (S-30) inhibited in vitro RNA synthesis in uninfected nuclei by selectively affecting the polymerase II activity. Selective inhibition of hnRNA synthesis by the crude extracts could be monitored by EM autoradiography directly. Determinations of individual RNA polymerase activities by differential alpha-amanitin inhibition were done only after treatment of the infected cytoplasm with micrococcal nuclease to abolish virus RNA replication. Selective inhibition of hnRNA synthesis depended on preincubation of the nuclei together with the infected cytoplasm, indicating that inhibitory substances from the infected cytoplasm entered the nuclei. Isolated nuclei therefore provide a useful system for studying the nature of the inhibitor(s) and of host RNA synthesis inhibition by picornaviruses.