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Mutations which alter the level or structure of nsP4 can affect the efficiency of Sindbis virus replication in a
J A Lemm1, R K Durbin, V Stollar
1Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri 63110-1093.
Abstract:
Two mutants of Sindbis virus have been isolated which grow inefficiently at 34.5 degrees C in mosquito cells yet replicate normally in chicken embryo fibroblast cells at the same temperature. In addition, these mutants exhibit temperature-sensitive growth in both cell types and are RNA- at the nonpermissive temperatures (K.J. Kowal and V. Stollar, Virology 114:140-148, 1981). To clarify the basis of this host restriction, we have mapped the causal mutations for these temperature-dependent, host-restricted mutants. Functional mapping and sequence analysis of the mutant cDNAs revealed several mutations which mapped to the amino terminus of nsP4, the putative polymerase subunit of the viral RNA replicase. These mutations resulted in the following amino acid changes in nsP4: leucine to valine at residue 48, aspartate to glycine at residue 142, and proline to arginine at residue 187. Virus containing any of these mutations was restricted in its ability to replicate in mosquito but not chicken embryo fibroblast cells at 34.5 degrees C. In addition to its temperature-dependent, host-restricted phenotype, virus derived from one cDNA clone also exhibited decreased levels of nsP34 and nsP4 yet contained only a silent change in its genome. This C-to-U mutation occurred at nucleotide 5751, the first nucleotide after the opal termination codon separating nsP3 and nsP4. Our results suggest that this substitution decreases readthrough of the opal codon and diminishes production of nsP34 and nsP4. Such a decrease in synthesis rates might lead to levels of these products which are insufficient for viral RNA replication in mosquito cells at the higher temperature. This work provides the first evidence that nsP4 function can be strongly influenced by the host environment.
Insights
Two Sindbis virus mutants show temperature-sensitive, host-restricted growth in mosquito cells due to mutations in the nsP4 protein, impacting viral RNA replication.
Area of Science:
- Virology
- Molecular Biology
- Genetics
Background:
- Sindbis virus mutants exhibit inefficient growth in mosquito cells at 34.5°C but normal growth in chicken cells.
- These mutants display temperature-sensitive growth in both cell types and are RNA-negative at nonpermissive temperatures.
Purpose of the Study:
- To map the causal mutations responsible for the temperature-dependent, host-restricted phenotype of Sindbis virus mutants.
- To elucidate the molecular basis of host restriction in Sindbis virus replication.
Main Methods:
- Functional mapping and sequence analysis of mutant Sindbis virus cDNAs.
- Identification of mutations within the nsP4 gene, encoding the putative RNA replicase polymerase subunit.
Main Results:
- Mutations in the amino terminus of nsP4 (L48V, D142G, P187R) caused replication restriction in mosquito cells but not chicken cells at 34.5°C.
- A silent C-to-U mutation at nucleotide 5751 decreased readthrough of the nsP3-nsP4 opal termination codon, reducing nsP34 and nsP4 production.
- Reduced synthesis of nsP34 and nsP4 may be insufficient for viral RNA replication in mosquito cells at elevated temperatures.
Conclusions:
- The study identified specific nsP4 mutations as responsible for Sindbis virus host restriction.
- A novel mechanism involving reduced readthrough of a termination codon impacts viral protein synthesis and host adaptation.
- This work demonstrates that nsP4 function is significantly influenced by the host environment, providing insights into viral-host interactions.