IDENTIFICATION OF THE MINIMAL REPLICASE AND THE MINIMAL PROMOTER OF (-)-STRAND SYNTHESIS, FUNCTIONAL IN ROTAVIRUS RNA REPLICATION IN-VITRO

An in vitro replication system supporting the initiation and synthesis of complete rotavirus (-)-strands on (+)-strand template RNA (Chen et al., J Virol 68: 7030, 1994) was used to examine several parameters r elated to rotavirus RNA replication. Coexpression of VP1/2/3 in all po ssible combinations from baculovirus vectors revealed: [i] Virus-like particles (VLPs) were formed only if VP2 was present, and [ii] VP1/2 a nd VP1/2/3 VLPs had replicase activity in the in vitro system whereas VP2/3 and VP2 VLPs did not. Thus, the minimal replicase is composed of VPI and VP2 and replicase activity is associated with VP1. In vitro r eplication reactions, using T7 transcripts of porcine rotavirus OSU ge nome segment 9 as reporter template, were performed to map cis-acting elements that regulate replication. Internal deletions and terminal tr uncations of the reporter RNA localized a replication signal, conferri ng full template activity, to the 5'-terminal 27 nucleotides (nt 1-27) and the 3'-terminal 26 nucleotides (nt 1037-1062). Further analysis s howed that a minimal promoter of (-)-strand synthesis was contained in the 3'-terminal 7 nucleotides (nt 1056-1062); the sequence conserved at the 3'-terminus of all rotavirus genes. Hybrid constructs with this promoter had minimal, but detectable, template activity. This result indicated that upstream sequences between nucleotides 1037-1055 positi vely regulate the activity of the minimal promoter.