Requirements for assembly of poliovirus replication complexes and negative-strand RNA synthesis

HeLa cells were transfected with several plasmids that encoded all poliovir us (PV) nonstructural proteins. Viral RNAs were transcribed by T7 RNA polym erase expressed from recombinant vaccinia virus. All plasmids produced simi lar amounts of viral proteins that were processed identically; however, RNA s were designed either to serve as templates for replication or to contain mutations predicted to prevent RNA replication. The mutations included subs titution of the entire PV 5' noncoding region (NCR) with the encephalomyoca rditis virus (EMCV) internal ribosomal entry site, thereby deleting the 5'- terminal cloverleaf-like structure, or insertion of three nucleotides in th e 3D(pol) coding sequence. Production of viral proteins was sufficient to i nduce the characteristic reorganization of intracellular membranes into het erogeneous-sized vesicles, independent of RNA replication. The vesicles wer e stably associated,vith viral RNA only when RNA replication could occur. N onreplicating RNAs localized to distinct, nonoverlapping regions in the cel l, excluded from the viral protein-membrane complexes. The absence of accum ulation of positive-strand RNA from both mutated RNAs in transfected cells was documented. In addition, no minus-strand RNA was produced from the EMCV chimeric template RNA in vitro. These data show that the 5'-terminal seque nces of PV RNA are essential for initiation of minus-strand RNA synthesis a t its 3' end.