Genetic evidence for an interaction between a picornaviral cis-acting RNA replication element and 3CD protein.
Internally located, cis-acting RNA replication elements, termed cres, are essential for replication of the genomes of picornaviruses such as human rhinovirus 14 (HRV-14) and poliovirus because they template uridylylation of the protein primer, VPg, by the polymerase 3D(pol). These cres form stem-loop structures sharing a common loop motif, and the HRV-14 cre can substitute functionally for the poliovirus cre in both uridylylation in vitro and RNA replication in vivo. We show, however, that the poliovirus cre is unable to support HRV-14 RNA replication. This lack of complementation maps to the stem of the poliovirus cre and was reversed by single nucleotide substitutions in the stem as well as the base of the loop. Replication-competent, revertant viruses rescued from dicistronic HRV-14 RNAs containing the poliovirus cre, or a chimeric cre containing the poliovirus stem, contained adaptive amino acid substitutions. These mapped to the surface of both the polymerase 3D(pol), at the tip of the "thumb" domain, and the protease 3C(pro), on the side opposing the active site and near the end of an extended strand segment implicated previously in RNA binding. These mutations substantially enhanced replication competence when introduced into HRV-14 RNAs containing the poliovirus cre, and they were additive in their effects. The data support a model in which 3CD or its derivatives 3C(pro) and 3D(pol) interact directly with the stem of the cre during uridylylation of VPg.
Yang
Yan
Y
Department of Microbiology and Immunology, University of Texas Medical Branch, 301 University Boulevard, Galveston, TX 77555-1019, USA.
Rijnbrand
Rene
R
Watowich
Stanley
S
Lemon
Stanley M
SM
D001483:Base Sequence; D016679:Genome, Viral; D006367:HeLa Cells; D006801:Humans; D019426:Integrases / Q000235:genetics*; D008156:Luciferases / Q000378:metabolism; D008957:Models, Genetic; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D010849:Picornaviridae / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D012313:RNA / Q000737:chemistry*; D012367:RNA, Viral; D020218:Response Elements; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000235:genetics*
D012367:RNA, Viral; D014764:Viral Proteins; D012313:RNA; D008156:Luciferases; C045073:Cre recombinase; D019426:Integrases