Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2010 Nov-Dec;7(6):820-9.
doi: 10.4161/rna.7.6.13781. Epub 2010 Nov 1.

The twenty-nine amino acid C-terminal cytoplasmic domain of poliovirus 3AB is critical for nucleic acid chaperone activity

Divya R Gangaramani  1 , Elizabeth L Eden, Manthan Shah, Jeffrey J Destefano
Affiliations

The twenty-nine amino acid C-terminal cytoplasmic domain of poliovirus 3AB is critical for nucleic acid chaperone activity

Divya R Gangaramani et al. RNA Biol. 2010 Nov-Dec.

Abstract

Poliovirus 3AB protein is the first picornavirus protein demonstrated to have nucleic acid chaperone activity. Further characterization of 3AB demonstrates that the C-terminal 22 amino acids (3B region (also referred to as VPg), amino acid 88-109) of the protein is required for chaperone activity, as mutations in this region abrogate nucleic acid binding and chaperone function. Protein 3B alone has no chaperone activity as determined by established assays that include the ability to stimulate nucleic acid hybridization in a primer-template annealing assay, helix-destabilization in a nucleic acid unwinding assay, or aggregation of nucleic acids. In contrast, the putative 3AB C-terminal cytoplasmic domain (C terminal amino acids 81-109, 3B + the last 7 C-terminal amino acids of 3A, termed 3B+7 in this report) possesses strong activity in these assays, albeit at much higher concentrations than 3AB. The characteristics of several mutations in 3B+7 are described here, as well as a model proposing that 3B+7 is the site of the "intrinsic" chaperone activity of 3AB while the 3A N-terminal region (amino acids 1-58) and/or membrane anchor domain (amino acids 59-80) serve to increase the effective concentration of the 3B+7 region leading to the potent chaperone activity of 3AB.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Possible roles for chaperone activity in poliovirus/picornavirus replication. A schematic representation of the poliovirus genome along with important secondary structures in the non-translated regions (NTRs) is shown (from De Jesus NH, Virol J 2007; 4:70). Possible roles of 3AB or other chaperones are noted in boxed areas. See text for details.
Figure 2
Figure 2
(A–D) Depiction of 3AB sequence and results from FRET unwinding assay. (A) Amino acid sequence of poliovirus 3AB. Regions: 3A, 1–87: 3B (VPg), 88–109 (boxed); C-terminal cytoplasmic domain, 81–109 (referred to as 3B+7 in this proposal); membrane anchor domain, amino acids 59–80 (in bold italics). (B) Substrates for FRET unwinding assay. Complementary stem-loop forming oligos with 5′ fluorescing (FAM) or quenching DABCYL groups are shown. Hybridization leads to quenching of FAM fluorescence. Structures and ΔG values of −7.2 kcal/mol are as predicted from DNA fold. (C) FRET unwinding time course assay with wild type 3AB, 3AB-R104E, 3AB-Y90A, 3AB-R104E/Y90A, VPg (3B) and HIV NC, all at 2 µM. (D) FRET unwinding time course assay with wild type 3AB (2 µM) or 2, 8 and 100 µM 3B+7. Representative results are shown for all experiments and assays were repeated at least once and typically several times.
Figure 3
Figure 3
Primer-template annealing assay with wild type 3AB. A 25 nucleotide 5′ end-labeled RNA primer that was complementary to a 230 nucleotide RNA template was used in the assay. The primer and template were incubated with decreasing amounts of wild type 3AB (l–r: 2, 1, 0.5, 0.25, 0.013, 0.0063 or 0.0031 µM). Samples were processed and run on a 6% native polyacrylamide gel as described under Methods. The positions of the primer and annealed primer-template hybrid are indicated. Three controls (A–C) are as described below the panel. Representative results are shown and the assay was repeated several times.
Figure 4
Figure 4
Primer-template annealing assay with wild type 3B+7. The amount of 3B+7 protein used was: l–r, 200, 100, 50, 25, 12.5, 6.3 or 3.1 µM. See Figure 3 for details.
Figure 5
Figure 5
Gel-shift assay to test protein-nucleic acid binding. Proteins (type and amount as indicated) were incubated with a 40 nucleotide 5′ end-labeled RNA and run on a 6% native polyacrylamide as described in Methods. The positions of the unshifted 40-mer and shifted material are indicated. Two controls are as described below the part. Representative results are shown and the assays were repeated twice with similar results.
Figure 6
Figure 6
Model for in vitro 3AB chaperone activity. See text for description. MA, membrane anchor domain.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Zuniga S, Sola I, Cruz JL, Enjuanes L. Role of RNA chaperones in virus replication. Virus Res. 2009;139:253–266. - PMC - PubMed
    1. Herschlag D. RNA chaperones and the RNA folding problem. J Biol Chem. 1995;270:20871–20874. - PubMed
    1. Levin JG, Guo J, Ioulia R, Musier-Forsyth K. Nucleic acid chaperone activity of HIV-1 nucleocapsid protein: critical role in reverse transcription and molecular mechanism. Prog. Nucleic Acids Res Mol Biol. 2005;80:217–286. - PubMed
    1. Rajkowitsch L, Chen D, Stampfl S, Semrad K, Waldsich C, Mayer O, et al. RNA chaperones, RNA annealers and RNA helicases. RNA Biol. 2007;4:118–130. - PubMed
    1. Henriet S, Sinck L, Bec G, Gorelick RJ, Marquet R, Paillart JC. Vif is a RNA chaperone that could temporally regulate RNA dimerization and the early steps of HIV-1 reverse transcription. Nucleic Acids Res. 2007;35:5141–5153. - PMC - PubMed

Publication types

MeSH terms

LinkOut - more resources