@article{mbs:/content/journal/jgv/10.1099/vir.0.19013-0, author = "Rieder, Elizabeth and Xiang, Wenkai and Paul, Aniko and Wimmer, Eckard", title = "Analysis of the cloverleaf element in a human rhinovirus type 14/poliovirus chimera: correlation of subdomain D structure, ternary protein complex formation and virus replication", journal= "Journal of General Virology", year = "2003", volume = "84", number = "8", pages = "2203-2216", doi = "https://doi.org/10.1099/vir.0.19013-0", url = "https://www.microbiologyresearch.org/content/journal/jgv/10.1099/vir.0.19013-0", publisher = "Microbiology Society", issn = "1465-2099", type = "Journal Article", abstract = "RNA genomes of enteroviruses and rhinoviruses contain a 5′-terminal structure, the cloverleaf (CL), which serves as signal in RNA synthesis. Substitution of the poliovirus [PV1(M)] CL with that of human rhinovirus type 2 (HRV2) was shown previously to produce a viable chimeric PV, whereas substitution with the HRV14 CL produced a null phenotype. Fittingly, the HRV14 CL failed to form a complex with PV-specific proteins 3CDpro–3AB or 3CDpro–PCBP2, considered essential for RNA synthesis. It was reported previously ( Rohll et al., J Virol 68, 4384–4391, 1994 ) that the major determinant for the null phenotype of a PV/HRV14 chimera resides in subdomain Id of the HRV14 CL. Using a chimeric PV/HRV14 CL in the context of the PV genome, stem–loop Id of HRV14 CL was genetically dissected. It contains the sequence C57 UAU 60-G, the underlined nucleotides forming the loop that is shorter by 1 nt when compared to the corresponding PV structure (UUGC 60 GG). Insertion of a G nucleotide to form a tetra loop (C57 UAU 60 GG61) did not rescue replication of the chimera. However, an additional mutation at position 60 (C57 UAC 60 GG61) yielded a replicating genome. Only the mutant PV/HRV14 CL with the UAC 60 G tetra loop formed ternary complexes efficiently with either PV proteins 3CDpro–3AB or 3CDpro–PCBP2. Thus, in the context of PV RNA synthesis, the presence of a tetra loop in subdomain D of the CL per se is not sufficient for function. The sequence and, consequently, the structure of the tetra loop plays an essential role. Biochemical assays demonstrated that the function of the CL element and the function of the cis-acting replication element in the 3Dpol–3CDpro-dependent uridylylation of VPg are not linked.", }