Solution structure of the Rous sarcoma virus nucleocapsid protein: muPsi RNA packaging signal complex.
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 muPsi. 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 muPsi sequence. We demonstrated recently that muPsi is capable of binding to the cognate RSV nucleocapsid protein (NC) with high affinity (dissociation constant K(d) approximately 2 nM), and that residues of AUG-3 are essential for tight binding. We now report the solution structure of the NC:muPsi complex, determined using NMR data obtained for samples containing ((13)C,(15)N)-labeled NC and (2)H-enriched, nucleotide-specifically protonated RNAs. Upon NC binding, muPsi 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.
Zhou
Jing
J
Howard Hughes Medical Institute and Department of Chemistry and Biochemistry, University of Maryland Baltimore County, 1000 Hilltop Circle, Baltimore, MD 21250, USA.
Bean
Rebecca L
RL
Vogt
Volker M
VM
Summers
Michael
M
D000818:Animals; D020029:Base Pairing; D001665:Binding Sites; D002460:Cell Line; D002645:Chickens; D016679:Genome, Viral; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019590:Nucleocapsid Proteins / Q000737:chemistry* / Q000235:genetics; D011485:Protein Binding; D017433:Protein Structure, Secondary; D012367:RNA, Viral / Q000737:chemistry* / Q000235:genetics; D052736:Rous sarcoma virus / Q000737:chemistry* / Q000472:pathogenicity; D012996:Solutions / Q000737:chemistry; D013329:Structure-Activity Relationship; D019065:Virus Assembly
D019590:Nucleocapsid Proteins; D012367:RNA, Viral; D012996:Solutions