Journal of Virology, September 2003, p. 9244-9258, Vol. 77, No. 17
0022-538X/03/$08.00+0 DOI: 10.1128/JVI.77.17.9244-9258.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Characterization of the RNA-Binding Domains in the Replicase Proteins of Tomato Bushy Stunt Virus
K. S. Rajendran and Peter D. Nagy*
Department of Plant Pathology, University of Kentucky, Lexington, Kentucky 40546
Received 17 January 2003/
Accepted 30 May 2003
Tomato bushy stunt virus (TBSV), a tombusvirus with a nonsegmented, plus-stranded RNA genome, codes for two essential replicase proteins. The sequence of one of the replicase proteins, namely p33, overlaps with the N-terminal domain of p92, which contains the signature motifs of RNA-dependent RNA polymerases (RdRps) in its nonoverlapping C-terminal portion. In this work, we demonstrate that both replicase proteins bind to RNA in vitro based on gel mobility shift and surface plasmon resonance measurements. We also show evidence that the binding of p33 to single-stranded RNA (ssRNA) is stronger than binding to double-stranded RNA (dsRNA), ssDNA, or dsDNA in vitro. Competition experiments with ssRNA revealed that p33 binds to a TBSV-derived sequence with higher affinity than to other nonviral ssRNA sequences. Additional studies revealed that p33 could bind to RNA in a cooperative manner. Using deletion derivatives of the Escherichia coli-expressed recombinant proteins in gel mobility shift and Northwestern assays, we demonstrate that p33 and the overlapping domain of p92, based on its sequence identity with p33, contain an arginine- and proline-rich RNA-binding motif (termed RPR, which has the sequence RPRRRP). This motif is highly conserved among tombusviruses and related carmoviruses, and it is similar to the arginine-rich motif present in the Tat trans-activator protein of human immunodeficiency virus type 1. We also find that the nonoverlapping C-terminal domain of p92 contains additional RNA-binding regions. Interestingly, the location of one of the RNA-binding domains in p92 is similar to the RNA-binding domain of the NS5B RdRp protein of hepatitis C virus.
* Corresponding author. Mailing address: Department of Plant Pathology, University of Kentucky, Plant Science Building, Lexington, KY 40546. Phone: (859) 257-7445, ext. 80726. Fax: (859) 323-1961. E-mail: pdnagy2{at}uky.edu.
Publication 03-12-003 of the Kentucky Agricultural Experiment Station.
Journal of Virology, September 2003, p. 9244-9258, Vol. 77, No. 17
0022-538X/03/$08.00+0 DOI: 10.1128/JVI.77.17.9244-9258.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
This article has been cited by other articles:
-
Li, Z., Barajas, D., Panavas, T., Herbst, D. A., Nagy, P. D.
(2008). Cdc34p Ubiquitin-Conjugating Enzyme Is a Component of the Tombusvirus Replicase Complex and Ubiquitinates p33 Replication Protein. J. Virol.
82: 6911-6926
[Abstract]
[Full Text]
-
Van Wynsberghe, P. M., Chen, H.-R., Ahlquist, P.
(2007). Nodavirus RNA Replication Protein A Induces Membrane Association of Genomic RNA. J. Virol.
81: 4633-4644
[Abstract]
[Full Text]
-
Genoves, A., Navarro, J. A., Pallas, V.
(2006). Functional analysis of the five melon necrotic spot virus genome-encoded proteins.. J. Gen. Virol.
87: 2371-2380
[Abstract]
[Full Text]
-
Jiang, Y., Serviene, E., Gal, J., Panavas, T., Nagy, P. D.
(2006). Identification of essential host factors affecting tombusvirus RNA replication based on the yeast tet promoters hughes collection.. J. Virol.
80: 7394-7404
[Abstract]
[Full Text]
-
Serva, S., Nagy, P. D.
(2006). Proteomics Analysis of the Tombusvirus Replicase: Hsp70 Molecular Chaperone Is Associated with the Replicase and Enhances Viral RNA Replication. J. Virol.
80: 2162-2169
[Abstract]
[Full Text]
-
Serviene, E., Jiang, Y., Cheng, C.-P., Baker, J., Nagy, P. D.
(2006). Screening of the Yeast yTHC Collection Identifies Essential Host Factors Affecting Tombusvirus RNA Recombination. J. Virol.
80: 1231-1241
[Abstract]
[Full Text]
-
Panaviene, Z., Panavas, T., Nagy, P. D.
(2005). Role of an Internal and Two 3'-Terminal RNA Elements in Assembly of Tombusvirus Replicase. J. Virol.
79: 10608-10618
[Abstract]
[Full Text]
-
Panavas, T., Nagy, P. D.
(2005). Mechanism of Stimulation of Plus-Strand Synthesis by an RNA Replication Enhancer in a Tombusvirus. J. Virol.
79: 9777-9785
[Abstract]
[Full Text]
-
Pogany, J., White, K. A., Nagy, P. D.
(2005). Specific Binding of Tombusvirus Replication Protein p33 to an Internal Replication Element in the Viral RNA Is Essential for Replication. J. Virol.
79: 4859-4869
[Abstract]
[Full Text]
-
Boonrod, K., Chotewutmontri, S., Galetzka, D., Krczal, G.
(2005). Analysis of tombusvirus revertants to identify essential amino acid residues within RNA-dependent RNA polymerase motifs. J. Gen. Virol.
86: 823-826
[Abstract]
[Full Text]
-
Samuilova, O., Krogerus, C., Poyry, T., Hyypia, T.
(2004). Specific Interaction between Human Parechovirus Nonstructural 2A Protein and Viral RNA. J. Biol. Chem.
279: 37822-37831
[Abstract]
[Full Text]
-
Pantaleo, V., Rubino, L., Russo, M.
(2004). The p36 and p95 replicase proteins of Carnation Italian ringspot virus cooperate in stabilizing defective interfering RNA. J. Gen. Virol.
85: 2429-2433
[Abstract]
[Full Text]
-
Panaviene, Z., Panavas, T., Serva, S., Nagy, P. D.
(2004). Purification of the Cucumber Necrosis Virus Replicase from Yeast Cells: Role of Coexpressed Viral RNA in Stimulation of Replicase Activity. J. Virol.
78: 8254-8263
[Abstract]
[Full Text]
-
Shapka, N., Nagy, P. D.
(2004). The AU-Rich RNA Recombination Hot Spot Sequence of Brome Mosaic Virus Is Functional in Tombusviruses: Implications for the Mechanism of RNA Recombination. J. Virol.
78: 2288-2300
[Abstract]
[Full Text]
Copyright © 2003 by the American Society for Microbiology. All rights reserved.