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Journal of Virology, November 2009, p. 10869-10876, Vol. 83, No. 21
0022-538X/09/$08.00+0     doi:10.1128/JVI.01271-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Mutational Analysis Reveals a Noncontractile but Interactive Role of Actin and Profilin in Viral RNA-Dependent RNA Synthesis{triangledown}

Mary Harpen, Tiasha Barik, Alla Musiyenko, and Sailen Barik*

Department of Biochemistry and Molecular Biology, University of South Alabama, College of Medicine, 307 University Blvd., Mobile, Alabama 36688-0002

Received 19 June 2009/ Accepted 18 August 2009

As obligatory parasites, viruses co-opt a variety of cellular functions for robust replication. The expression of the nonsegmented negative-strand RNA genome of respiratory syncytial virus (RSV), a significant pediatric pathogen, absolutely requires actin and is stimulated by the actin-regulatory protein profilin. As actin is a major contractile protein, it was important to determine whether the known functional domains of actin and profilin were important for their ability to activate RSV transcription. Analyses of recombinant mutants in a reconstituted RSV transcription system suggested that the divalent-cation-binding domain of actin is critically needed for binding to the RSV genome template and for the activation of viral RNA synthesis. In contrast, the nucleotide-binding domain and the N-terminal acidic domain were needed neither for template binding nor for transcription. Specific surface residues of actin, required for actin-actin contact during filamentation, were also nonessential for viral transcription. Unlike actin, profilin did not directly bind to the viral template but was recruited by actin. Mutation of the interactive residues of actin or profilin, resulting in the loss of actin-profilin binding, also abolished profilin's ability to stimulate viral transcription. Together, these results suggest that actin acts as a classical transcription factor for the virus by divalent-cation-dependent binding to the viral template and that profilin acts as a transcriptional cofactor, in part by associating with actin. This essential viral role of actin is independent of its contractile cellular role.

* Corresponding author. Mailing address: Department of Biochemistry and Molecular Biology, University of South Alabama, College of Medicine, 307 University Blvd., Mobile, AL 36688-0002. Phone: (251) 460-6860. Fax: (251) 460-6850. E-mail: sbarik{at}jaguar1.usouthal.edu

{triangledown} Published ahead of print on 26 August 2009.

Journal of Virology, November 2009, p. 10869-10876, Vol. 83, No. 21
0022-538X/09/$08.00+0     doi:10.1128/JVI.01271-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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