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Journal of Virology, March 2007, p. 2849-2860, Vol. 81, No. 6
0022-538X/07/$08.00+0     doi:10.1128/JVI.02078-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Enzymatic Defects of the nsP2 Proteins of Semliki Forest Virus Temperature-Sensitive Mutants{triangledown}

Giuseppe Balistreri, Javier Caldentey,{dagger} Leevi Kääriäinen,* and Tero Ahola

Program in Cellular Biotechnology, Institute of Biotechnology, University of Helsinki, Helsinki, Finland

Received 22 September 2006/ Accepted 6 December 2006

We have analyzed the biochemical consequences of mutations that affect viral RNA synthesis in Semliki Forest virus temperature-sensitive (ts) mutants. Of the six mutations mapping in the multifunctional replicase protein nsP2, three were located in the N-terminal helicase region and three were in the C-terminal protease domain. Wild-type and mutant nsP2s were expressed, purified, and assayed for nucleotide triphosphatase (NTPase), RNA triphosphatase (RTPase), and protease activities in vitro at 24°C and 35°C. The protease domain mutants (ts4, ts6, and ts11) had reduced protease activity at 35°C but displayed normal NTPase and RTPase. The helicase domain mutation ts1 did not have enzymatic consequences, whereas ts13a and ts9 reduced both NTPase and protease activities but in different and mutant-specific ways. The effects of these helicase domain mutants on protease function suggest interdomain interactions within nsP2. NTPase activity was not directly required for protease activity. The similarities of the NTPase and RTPase results, as well as competition experiments, suggest that these two reactions utilize the same active site. The mutations were also studied in recombinant viruses first cultivated at the permissive temperature and then shifted up to the restrictive temperature. Processing of the nonstructural polyprotein was generally retarded in cells infected with viruses carrying the ts4, ts6, ts11, and ts13a mutations, and a specific defect appeared in ts9. All mutations except ts13a were associated with a large reduction in the production of the subgenomic 26S mRNA, indicating that both protease and helicase domains influence the recognition of the subgenomic promoter during virus replication.

* Corresponding author. Mailing address: Institute of Biotechnology, P.O. Box 56, University of Helsinki, FIN-00014 Helsinki, Finland. Phone: 358-9-19159400. Fax: 358-9-19159560. E-mail: leevi.kaariainen{at}helsinki.fi.

{triangledown} Published ahead of print on 3 January 2007.

{dagger} Present address: COST Office, European Science Foundation, 1050 Brussels, Belgium.

Journal of Virology, March 2007, p. 2849-2860, Vol. 81, No. 6
0022-538X/07/$08.00+0     doi:10.1128/JVI.02078-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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