Identification of a cis-acting replication element within the poliovirus coding region

The replication of poliovirus, a positive-stranded RNA virus, requires tran slation of the infecting genome followed by virus-encoded VPg and 3D polyme rase-primed synthesis of a negative-stranded template. RNA sequences involv ed in the tatter process are poorly defined. Since many sequences involved in picornavirus replication form RNA structures, we searched the genome, ot her than the untranslated regions, for predicted local secondary structural elements and identified a 61-nucleotide (nt) stem-leap in the region encod ing the 2C protein, Covariance analysis suggested the structure was well co nserved in the Enterovirus genus of the Picornaviridae. Site-directed mutag enesis, disrupting the structure without affecting the 2C product, destroye d genome viability and suggested that the structure was required in the pos itive sense for function. Recovery of revertant viruses suggested that inte grity of the structure was critical for function, and analysis of replicati on demonstrated that nonviable mutants did not synthesize negative strands. Our conclusion, that this RNA secondary structure constitutes a novel poli ovirus cis-acting replication element (CRE), is supported by the demonstrat ion that subgenomic replicons bearing lethal mutations in the native struct ure can be restored to replication competence by the addition of a second c opy of the 61-nt wild-type sequence at another location within the genome. This poliovirus CRE functionally resembles an element identified in rhinovi rus type 14 (K. L. McKnight and S. M. Lemon, RNA 4:1569-1584, 199) and the cardioviruses (P. E. Lobert, N. Escriou, J. Ruelle, and T. Michiels, proc. Natl. Acad. Sci. USA 96:11560-11565, 1999) but differs in sequence, structu re, and location. The functional role and evolutionary significance of CREs in the replication of positive-sense RNA viruses is discussed.