Production of Recombinant Snakehead Rhabdovirus: the NV Protein Is Not Required for Viral Replication

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Abstract
The poly(rC) binding protein (PCBP) is a cellular protein required for poliovirus replication. PCBP specifically interacts with two domains of the poliovirus 5′ untranslated region (5′UTR), the 5′ cloverleaf structure, and the stem-loop IV of the internal ribosome entry site (IRES). Using footprinting analysis and site-directed mutagenesis, we have mapped the RNA binding site for this cellular protein within the stem-loop IV domain. A C-rich sequence in a loop at the top of this large domain is required for PCBP binding and is crucial for viral translation. PCBP binds to stem-loop IV RNA with six-times-higher affinity than to the 5′ cloverleaf structure. However, the binding of the viral protein 3CD (precursor of the viral protease 3C and the viral polymerase 3D) to the cloverleaf RNA dramatically increases the affinity of PCBP for this RNA element. The viral protein 3CD binds to the cloverleaf RNA but does not interact directly with stem-loop IV nor with other RNA elements of the viral IRES. Our results indicate that the interactions of PCBP with the poliovirus 5′UTR are modulated by the viral protein 3CD.