During virus multiplication, the viral genome is recognized and recruited for replication based on specific cis-acting elements. Here we have dissected the important cis-acting sequence elements in Semliki Forest virus RNA, by using a trans-replication system. Since the viral replicase is expressed from a separate plasmid, the template RNA can be freely modified in this system. We show that the cis-acting element at the beginning of the non-structural protein 1 (nsP1) coding region together with the end of the 3′ untranslated region are the minimal requirements for minus strand synthesis. To achieve a high level of replication, the native 5′ untranslated region was also needed. The virus-induced membranous replication compartments or spherules were only detected when a replication-competent template was present with an active replicase and minus strands were produced. No translation could be detected from the minus strands, suggesting that they are segregated from the cytoplasm. Minus strands could not directly be recruited to initiate the replication process. Thus, there is only one defined pathway for replication, starting with plus strand recognition followed by concomitant spherule formation and minus strand synthesis.
- 1183 Växtbiologi, mikrobiologi, virologi