Enzymatic Defects of the nsP2 Proteins of Semliki Forest Virus Temperature-Sensitive Mutants
| Autor: | Leevi Kääriäinen, Giuseppe Balistreri, Javier Caldentey, Tero Ahola |
|---|---|
| Rok vydání: | 2007 |
| Předmět: |
viruses
medicine.medical_treatment Amino Acid Motifs Immunology RNA-dependent RNA polymerase Semliki Forest virus medicine.disease_cause Microbiology 03 medical and health sciences Virology medicine RNA triphosphatase Point Mutation Amino Acid Sequence RNA Messenger Amino Acids 030304 developmental biology Subgenomic mRNA Recombination Genetic 0303 health sciences Mutation Binding Sites Protease biology Point mutation 030302 biochemistry & molecular biology Temperature Nucleoside-Triphosphatase biology.organism_classification Semliki forest virus Genome Replication and Regulation of Viral Gene Expression Acid Anhydride Hydrolases Protein Structure Tertiary Cysteine Endopeptidases Amino Acid Substitution Viral replication Biochemistry Insect Science Peptide Hydrolases |
| Zdroj: | Journal of Virology |
| ISSN: | 1098-5514 0022-538X |
| DOI: | 10.1128/jvi.02078-06 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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