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Journal of Virology, October 2001, p. 9633-9643, Vol. 75, No. 20
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.20.9633-9643.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Mutagenesis of the Dengue Virus Type 2 NS3 Protein
within and outside Helicase Motifs: Effects on Enzyme Activity and
Virus Replication
Anita E.
Matusan,
Melinda J.
Pryor,
Andrew D.
Davidson, and
Peter J.
Wright*
Department of Microbiology, Monash
University, Clayton, Victoria 3168, Australia
Received 27 March 2001/Accepted 12 July 2001
The protein NS3 of Dengue virus type 2 (DEN-2) is
the second largest nonstructural protein specified by the virus and is
known to possess multiple enzymatic activities, including a serine
proteinase located in the N-terminal region and an
NTPase-helicase in the remaining 70% of the protein.
The latter region has seven conserved helicase motifs found in all
members of the family Flaviviridae. DEN-2 NS3 lacking
the proteinase region was synthesized as a fusion protein with
glutathione S-transferase in Escherichia
coli. The effects of 10 mutations on ATPase and RNA
helicase activity were examined. Residues at four sites within enzyme
motifs I, II, and VI were substituted, and six sites outside motifs
were altered by clustered charged-to-alanine mutagenesis. The mutations
were also tested for their effects on virus replication by
incorporation into genomic-length cDNA. Two mutations, both
in motif I (G198A and K199A) abolished both ATPase and helicase
activity. Two further mutations, one in motif VI (R457A,R458A) and the
other a clustered charged-to-alanine substitution at
R376KNGK380, abolished helicase activity only.
No virus was detected for any mutation which prevented helicase
activity, demonstrating the requirement of this enzyme for virus
replication. The remaining six mutations resulted in various
levels of enzyme activities, and four permitted virus replication. For
the two nonreplicating viruses encoding clustered changes at
R184KR186 and
D436GEE439, we propose that the substituted residues are surface located and that the viruses are defective through
altered interaction of NS3 with other components of the viral
replication complex. Two of the replicating viruses displayed a
temperature-sensitive phenotype. One contained a clustered mutation at
D334EE336 and grew too poorly for further
characterization. However, virus with an M283F substitution in motif II
was examined in a temperature shift experiment (33 to 37°C) and
showed reduced RNA synthesis at the higher temperature.
*
Corresponding author. Mailing address: Department of
Microbiology, P.O. Box 53, Monash University, Victoria 3800, Australia. Phone: 61 3 9905 4828. Fax: 61 3 9905 4811. E-mail:
Peter.Wright{at}med.monash.edu.au.
Journal of Virology, October 2001, p. 9633-9643, Vol. 75, No. 20
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.20.9633-9643.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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