Solution Structure of the Rous Sarcoma Virus Nucleocapsid Protein: μΨ RNA Packaging Signal Complex

Abstract

The 5′-untranslated region (5′-UTR) of retroviral genomes contains elements required for genome packaging during virus assembly. For many retroviruses, the packaging elements reside in non-contiguous segments that span most or all of the 5′-UTR. The Rous sarcoma virus (RSV) is an exception, in that its genome can be packaged efficiently by a relatively short, 82 nt segment of the 5′-UTR called μΨ. The RSV 5′-UTR also contains three translational start codons (AUG-1, AUG-2 and AUG-3) that have been controvertibly implicated in translation initiation and genome packaging, one of which (AUG-3) resides within the μΨ sequence. We demonstrated recently that μΨ is capable of binding to the cognate RSV nucleocapsid protein (NC) with high affinity (dissociation constant K d ∼ 2 nM), and that residues of AUG-3 are essential for tight binding. We now report the solution structure of the NC:μΨ complex, determined using NMR data obtained for samples containing ( 13C, 15N)-labeled NC and 2H-enriched, nucleotide-specifically protonated RNAs. Upon NC binding, μΨ adopts a stable secondary structure that consists of three stem loops (SL-A, SL-B and SL-C) and an 8 bp stem (O3). Binding is mediated by the two zinc knuckle domains of NC. The N-terminal knuckle interacts with a conserved U(217)GCG tetraloop (a member of the UNCG family; N = A,U,G or C), and the C-terminal zinc knuckle binds to residues that flank SL-A, including residues of AUG-3. Mutations of critical nucleotides in these sequences compromise or abolish viral infectivity. Our studies reveal novel structural features important for NC:RNA binding, and support the hypothesis that AUG-3 is conserved for genome packaging rather than translational control.