Interactions of Viral Protein 3CD and Poly(rC) Binding Protein with the 5′ Untranslated Region of the Poliovirus Genome

The highly structured 5′ untranslated region (5′UTR) of picornaviruses plays an important role in the regulation of both viral translation and RNA replication. The regulatory function of this region is mediated by its interaction with cellular and viral proteins (for a review, see references 1, 5, and 12). Using computer analysis and genetic and biochemical tests, the 5′UTR of poliovirus (the prototype member of the picornavirus family) can be divided into six well-defined RNA domains (stem-loops I to VI) (29, 30, 34). Stem-loop I, which folds into a cloverleaf-like structure (30), is essential for viral RNA synthesis (2, 3, 17, 31, 37). This element forms a ternary RNP complex with the cellular poly(rC) binding protein (PCBP; also known as hnRNP E or α-CP) and the uncleaved precursor of the viral protease-polymerase, 3CD (15, 26). Mutations that disrupt complex formation, either within the cloverleaf RNA or within 3CD, impair viral RNA synthesis (3, 4, 31). The mechanism by which the ternary complex participates in RNA replication is poorly understood, but it was suggested that it catalyzes the initiation of positive strand RNA synthesis in trans (2). More recent evidence suggests that the ternary complex has a bifunctional role, participating in both viral translation and RNA replication (14, 33). The binding of PCBP to stem-loop B of the cloverleaf structure enhances viral translation 10-fold, while binding of 3CD decreases translation and promotes negative-strand RNA synthesis (14). Viral translation is directed by an RNA element located within the 5′UTR (stem-loops II to VI) known as the internal ribosomal entry site (IRES). This element allows ribosomes to enter the RNA without scanning from the 5′ end (21, 27, 36). The mechanism by which the translation apparatus recognizes IRES sequences is still unknown, but it has been proposed that several canonical initiation factors, as well as other cellular proteins, participate in this process (24, 28). So far, four noncanonical factors that bind to the poliovirus IRES have been identified: the polypyrimidine tract binding protein (18), the La autoantigen (23), the cellular protein PCBP (7), and the protein encoded by the gene present upstream of N-ras, UNR (20). PCBP was first identified as a component of the α-complex of the human α-globin mRNA, which greatly increases mRNA stability (22). More-recent evidence implicates PCBP in controlling the expression of numerous cellular and viral RNAs (for a review, see reference 25). It has been shown that PCBP specifically represses the expression of the late gene L2 in human papillomavirus (10) and enhances hepatitis A capindependent translation (16). However, the molecular mechanism by which PCBP interacts with the translation apparatus remains undefined. In poliovirus, PCBP specifically interacts with two stem-loops of the 5′UTR: the cloverleaf structure and stem-loop IV (7, 8, 15, 26). A number of observations suggest that PCBP is required for poliovirus translation. In fact, alteration of PCBP binding sites, depletion of PCBP from HeLa cell extracts, or microinjection of anti-PCBP antibodies into Xenopus oocytes inhibits poliovirus translation (8, 15). In the present study, we further analyzed the interactions of 3CD and PCBP with the poliovirus 5′UTR and the interplay between the RNP complexes formed during the viral life cycle. Using footprinting analysis and mobility shift assays we found that PCBP forms RNP complexes with different affinities depending on the presence of the viral protein 3CD. These findings may further our understanding of the molecular mechanism by which PCBP and 3CD participate in viral translation and RNA replication.