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Title: Catalytic core of alphavirus nonstructural protein nsP4 possesses terminal adenylyltransferase activity
Authors: Tomar, Shailly
Hardy R.W.
Smith J.L.
Kuhn R.J.
Published in: Journal of Virology
Abstract: The RNA-dependent RNA polymerase nsP4 is an integral part of the alphavirus replication complex. To define the role of nsP4 in viral RNA replication and for a structure-function analysis, we expressed Sindbis virus nsP4 in Escherichia coli. The core catalytic domain of nsP4 (?97nsP4, a deletion of the N-terminal 97 amino acids), which consists of the predicted polymerase domain containing the GDD amino acid motif required for viral RNA synthesis, was stable against proteolytic degradation during expression. Therefore, the recombinant core domain and selected mutants were expressed and purified to homogeneity. We determined that ?97nsP4 possesses terminal adenylyltransferase (TATase) activity, as it specifically catalyzed the addition of adenine to the 3? end of an acceptor RNA in the presence of divalent cations. Furthermore, ?97nsP4 is unable to transfer other nucleotides (UTP, CTP, GTP, and dATP) to the acceptor RNA in the absence or presence of other nucleotides. ?97nsP4 possessing a GDD-to-GAA mutation completely inactivates the enzymatic activity. However, a GDD-to-SNN mutation did not inactivate the enzyme but reduced its activity to ?45% of that of the wild type in the presence of Mg2+. Investigation of the TATase of the GDD-to-SNN mutant revealed that it had TATase equivalent to that of the wild type in the presence of Mn2+. Identification of ?97nsP4 TATase activity suggests a novel function of the alphavirus RNA-dependent RNA polymerase in the maintenance and repair of the poly(A) tail, an element required for replication of the viral genome. Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Citation: Journal of Virology(2006), 80(20): 9962-9969
Issue Date: 2006
ISSN: 0022538X
Author Scopus IDs: 57203506001
Author Affiliations: Tomar, S., Department of Biological Sciences, Purdue University, West Lafayette, IN, United States
Hardy, R.W., Department of Biology, Indiana University, Bloomington, IN, United States
Smith, J.L., Life Sciences Institute, University of Michigan, Ann
Corresponding Author: Kuhn, R.J.; Department of Biological Sciences, Purdue University, 915 W. State Street, West Lafayette, IN 47907-2054, United States; email:
Appears in Collections:Journal Publications [BT]

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