Requirements for Assembly of Poliovirus Replication Complexes and Negative-Strand RNA Synthesis

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Abstract
HeLa cells were transfected with several plasmids that encoded all poliovirus (PV) nonstructural proteins. Viral RNAs were transcribed by T7 RNA polymerase expressed from recombinant vaccinia virus. All plasmids produced similar amounts of viral proteins that were processed identically; however, RNAs were designed either to serve as templates for replication or to contain mutations predicted to prevent RNA replication. The mutations included substitution of the entire PV 5′ noncoding region (NCR) with the encephalomyocarditis virus (EMCV) internal ribosomal entry site, thereby deleting the 5′-terminal cloverleaf-like structure, or insertion of three nucleotides in the 3D pol coding sequence. Production of viral proteins was sufficient to induce the characteristic reorganization of intracellular membranes into heterogeneous-sized vesicles, independent of RNA replication. The vesicles were stably associated with viral RNA only when RNA replication could occur. Nonreplicating RNAs localized to distinct, nonoverlapping regions in the cell, excluded from the viral protein-membrane complexes. The absence of accumulation 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 sequences of PV RNA are essential for initiation of minus-strand RNA synthesis at its 3′ end.