PDBsum entry: 2ihx

Viral protein/RNA

PDB id: 2ihx

Contents

Protein chain

50 a.a. *

DNA/RNA

Metals

_ZN ×2

* Residue conservation analysis

PDB id:

2ihx

Name:

Viral protein/RNA

Title:

Solution structure of the rous sarcoma virus nucleocapsid protein:upsi RNA packaging signal complex

Structure:

Nucleocapsid (nc) protein. Chain: a. Fragment: nucleocapsid domain (residues 503-563). Engineered: yes. Upsi RNA. Chain: b. Fragment: minimal RNA packaging signal in the 5'- untranslated region (utr) of rous sarcoma virus (rsv). Engineered: yes

Source:

Rous sarcoma virus. Organism_taxid: 11886. Strain: praguE C (pr-c). Gene: gag. Expressed in: escherichia coli. Expression_system_taxid: 562. Synthetic: yes. Other_details: RNA was prepared by in vitro t7 RNA transcription. The sequence occurs naturally in rous

NMR struc:

20 models

Authors:

J.Zhou,M.Summers

Key ref:

J.Zhou et al. (2007). Solution structure of the Rous sarcoma virus nucleocapsid protein: muPsi RNA packaging signal complex. J Mol Biol, 365, 453-467. PubMed id: 17070546 DOI: 10.1016/j.jmb.2006.10.013

Date:

27-Sep-06 Release date: 16-Jan-07

Protein chain

P03322  (GAG_RSVP) -  Gag-Pro polyprotein

Seq: 701 a.a.

Struc: 50 a.a.

 Gene Ontology (GO) functional annotation 

• Biochemical function: nucleic acid binding (2 terms)

DOI no: 10.1016/j.jmb.2006.10.013

J Mol Biol 365:453-467 (2007)

PubMed id: 17070546

Solution structure of the Rous sarcoma virus nucleocapsid protein: muPsi RNA packaging signal complex.

J.Zhou, R.L.Bean, V.M.Vogt, M.Summers.

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 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.

Selected figure(s)

Figure 8.
Figure 8. Surface (a) and stick (b) representations of the C-terminal NC zinc knuckle (NC-F2) bound to the linker residues (orange) proximal to stem–loop SL-A (purple). (c) Expanded view of the NC-F2 binding site showing the packing of A168 and A197 against the side-chains of Leu49 (green) and His55 (blue), respectively. Hydrogen bonds are shown as broken green lines. Figure 8. Surface (a) and stick (b) representations of the C-terminal NC zinc knuckle (NC-F2) bound to the linker residues (orange) proximal to stem–loop SL-A (purple). (c) Expanded view of the NC-F2 binding site showing the packing of A168 and A197 against the side-chains of Leu49 (green) and His55 (blue), respectively. Hydrogen bonds are shown as broken green lines.

Figure 9.
Figure 9. Virus infectivity monitored by RT assays. Compared with wild-type virus (circles), mutant virus in which the SL-C tetraloop sequence was changed from UGCG[217-220] to GAGA exhibited slower growth kinetics (squares). Mutant viruses in which the sequences AUC[168-170] and/or AUG[197-199] on either side of SL-A were changed to CCU were not infectious (diamonds, crosses, or plus symbols). Figure 9. Virus infectivity monitored by RT assays. Compared with wild-type virus (circles), mutant virus in which the SL-C tetraloop sequence was changed from UGCG[217-220] to GAGA exhibited slower growth kinetics (squares). Mutant viruses in which the sequences AUC[168-170] and/or AUG[197-199] on either side of SL-A were changed to CCU were not infectious (diamonds, crosses, or plus symbols).

The above figures are reprinted from an Open Access publication published by Elsevier: J Mol Biol (2007, 365, 453-467) copyright 2007.

Figures were selected by the author.