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Journal of Virology, April 2000, p. 3888-3891, Vol. 74, No. 8
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Mutation Analysis of the GDD Sequence Motif of a Calicivirus RNA-Dependent RNA Polymerase

Ana López Vázquez, José M. Martín Alonso, and Francisco Parra*

Departamento de Bioquímica y Biología Molecular, Instituto Universitario de Biotecnología de Asturias (CSIC), Universidad de Oviedo, 33006 Oviedo, Spain

Received 15 September 1999/Accepted 20 January 2000

The RNA-dependent RNA polymerase from rabbit hemorrhagic disease virus, a calicivirus, is known to have a conserved GDD amino acid motif and several additional regions of sequence homology with all types of polymerases. To test whether both aspartic acid residues are in fact involved in the catalytic activity and metal ion coordination of the enzyme, several defined mutations have been made in order to replace them by glutamate, asparagine, or glycine. All six mutant enzymes were produced in Escherichia coli, and their in vitro poly(U) polymerase activity was characterized. The results demonstrated that the first aspartate residue was absolutely required for enzyme function and that some flexibility existed with respect to the second, which could be replaced by glutamate.

* Corresponding author. Mailing address: Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Universidad de Oviedo, 33006 Oviedo, Spain. Phone: 34 985 103563. Fax: 34 985 103157. E-mail: parra{at}biosun.medicina.uniovi.es.

Journal of Virology, April 2000, p. 3888-3891, Vol. 74, No. 8
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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