Semliki Forest Virus Replication Complex Capable of Synthesizing 42S and 26S Nascent RNA Chains

Abstract

A complex synthesizing Semliki Forest virus (SFV)-specific RNA was purified from infected [human cervical carcinoma] HeLa cells. During purification, the RNA-synthesizing complex was monitored by the presence of RNA chains synthesized during a 1 min pulse in vivo and the ability to synthesize 42S and 26S RNA in vitro. The protein composition of the replication complex was analyzed. The 30-40% of the pulse-labeled RNA and 10-25% of the polymerase activity present in the postnuclear supernatant were recovered in smooth membranes. At this stage of purification single stranded 42S and 26S RNA were synthesized and released from the replication complex in vitro. After treatment of the smooth membrane fraction with Triton X-100 the replication complex was solubilized. When analyzed by sucrose gradient centrifugation, the solubilized replication complex distributed heterogeneously. It had reduced RNA polymerase activity, but was still able to synthesize both 42S and 26S nascent RNA chains which were not released from RI [replicative intermediate] and RF [replicative forms]. The non-structural protein ns70 was the major virus-specified component associated with the replication complex.