Key result
In a poliovirus/human rhinovirus 14 chimera, a specific UAC(60)G tetra loop mutation in subdomain D of the cloverleaf element was required to restore ternary complex formation and virus replication.
The sequence and structure of the tetra loop in subdomain D of the cloverleaf element play an essential role in poliovirus RNA synthesis and replication.
No immediate clinical implications; extends cloverleaf RNA requirements for picornavirus replication in chimeras.
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 C(57)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 (C(57)UAU(60)GG(61)) did not rescue replication of the chimera. However, an additional mutation at position 60 (C(57)UAC(60)GG(61)) 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 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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Rieder et al. (2003) studied this question. Genetic mutation of the cloverleaf subdomain Id (UAC(60)G tetra loop) vs. Wild-type or other mutant cloverleaf structures was evaluated on Virus replication and ternary protein complex formation. In a poliovirus/human rhinovirus 14 chimera, a specific UAC(60)G tetra loop mutation in subdomain D of the cloverleaf element was required to restore ternary complex formation and virus replication.
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