ARTICLE ABSTRACT
								DOI: 10.1099/vir.0.19013-0
								Online 14 May 2003

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

Elizabeth Rieder,† Wenkai Xiang,‡ Aniko Paul and Eckard Wimmer

Department of Molecular Genetics and Microbiology, School of Medicine, Stony Brook University, Stony Brook, NY 11794-5222, USA

†Present address: US Department of Agriculture, Agricultural Research Service, Plum Island Animal Disease Center, Greenport, NY 11944, USA.
†Present address: The Wharton School of the University of Pennsylvania, Philadelphia, PA, USA.

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 3CD^pro–3AB or 3CD^pro–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 C₅₇UAU₆₀-G, the underlined nucleotides forming the loop that is shorter by 1 nt when compared to the corresponding PV structure (UUGC₆₀GG). Insertion of a G nucleotide to form a tetra loop (C₅₇UAU₆₀GG₆₁) did not rescue replication of the chimera. However, an additional mutation at position 60 (C₅₇UAC₆₀GG₆₁) yielded a replicating genome. Only the mutant PV/HRV14 CL with the UAC₆₀G tetra loop formed ternary complexes efficiently with either PV proteins 3CD^pro–3AB or 3CD^pro–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 3D^pol–3CD^pro-dependent uridylylation of VPg are not linked.

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This article is now available in the August 2003 print issue of JGV (vol. 84, 2203–2216). The complete issue of the journal may be seen in electronic form on JGV Online.