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Journal of Virology, October 2008, p. 10207-10217, Vol. 82, No. 20
0022-538X/08/$08.00+0     doi:10.1128/JVI.00220-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Mutational Evidence for a Structural Model of the Lassa Virus RNA Polymerase Domain and Identification of Two Residues, Gly1394 and Asp1395, That Are Critical for Transcription but Not Replication of the Genome{triangledown}

Meike Hass,{dagger} Michaela Lelke,{dagger} Carola Busch, Beate Becker-Ziaja, and Stephan Günther*

Department of Virology, Bernhard-Nocht-Institute for Tropical Medicine, 20359 Hamburg, Germany

Received 31 January 2008/ Accepted 23 June 2008

The RNA-dependent RNA polymerase (RdRp) of arenaviruses is an integral part of the L protein, a 200-kDa multifunctional and multidomain protein. In view of the paucity of structural data, we recently proposed a model for the RdRp domain of arenaviruses based on the folding of RdRps of plus-strand viruses (S. Vieth et al., Virology 318:153-168, 2004). In the present study, we have chosen a large-scale mutagenesis approach to gain insight into the structure and function of the Lassa virus RdRp domain. A total of 180 different mutants of the domain were generated by using a novel PCR-based mutagenesis technique and tested in the context of the Lassa virus replicon system. Nearly all residues, which were essential for function, clustered in the center of the three-dimensional model including the catalytic site, while residues that were less important for function mapped to the periphery of the model. The combined bioinformatics and mutagenesis data allowed deducing candidate residues for ligand interaction. Mutation of two adjacent residues in the putative palm-thumb subdomain junction, G1394 and D1395 (strain AV), led to a defect in mRNA synthesis but did not affect antigenomic RNA synthesis. In conclusion, the data provide circumstantial evidence for the existence of an RdRp domain between residues 1040 and 1540 of the Lassa virus L protein and the folding model of the domain. A functional element within the RdRp was identified, which is important for transcription but not replication of the genome.

* Corresponding author. Mailing address: Bernhard-Nocht-Institute of Tropical Medicine, Bernhard-Nocht-Strasse 74, D-20359 Hamburg, Germany. Phone: (49) 40 42818 421. Fax: (49) 40 42818 378. E-mail: guenther{at}bni.uni-hamburg.de

{triangledown} Published ahead of print on 30 July 2008.

{dagger} M.H. and M.L. contributed equally to this study.

Journal of Virology, October 2008, p. 10207-10217, Vol. 82, No. 20
0022-538X/08/$08.00+0     doi:10.1128/JVI.00220-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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