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Genetic interactions among the West Nile virus methyltransferase, the RNA-dependent RNA polymerase, and the 5' stem-loop of genomic RNA
- Source :
- Journal of virology. 82(14)
- Publication Year :
- 2008
-
Abstract
- Flavivirus methyltransferase catalyzes both guanine N7 and ribose 2′-OH methylations of the viral RNA cap (GpppA-RNA→m 7 GpppAm-RNA). The methyltransferase is physically linked to an RNA-dependent RNA polymerase (RdRp) in the flaviviral NS5 protein. Here, we report genetic interactions of West Nile virus (WNV) methyltransferase with the RdRp and the 5′-terminal stem-loop of viral genomic RNA. Genome-length RNAs, containing amino acid substitutions of D146 (a residue essential for both cap methylations) in the methyltransferase, were transfected into BHK-21 cells. Among the four mutant RNAs (D146L, D146P, D146R, and D146S), only D146S RNA generated viruses in transfected cells. Sequencing of the recovered viruses revealed that, besides the D146S change in the methyltransferase, two classes of compensatory mutations had reproducibly emerged. Class 1 mutations were located in the 5′-terminal stem-loop of the genomic RNA (a G35U substitution or U38 insertion). Class 2 mutations resided in NS5 (K61Q in methyltransferase and W751R in RdRp). Mutagenesis analysis, using a genome-length RNA and a replicon of WNV, demonstrated that the D146S substitution alone was lethal for viral replication; however, the compensatory mutations rescued replication, with the highest rescuing efficiency occurring when both classes of mutations were present. Biochemical analysis showed that a low level of N7 methylation of the D146S methyltransferase is essential for the recovery of adaptive viruses. The methyltransferase K61Q mutation facilitates viral replication through improved N7 methylation activity. The RdRp W751R mutation improves viral replication through an enhanced polymerase activity. Our results have clearly established genetic interactions among flaviviral methyltransferase, RdRp, and the 5′ stem-loop of the genomic RNA.
- Subjects :
- Models, Molecular
viruses
Immunology
DNA Mutational Analysis
Molecular Sequence Data
Mutation, Missense
RNA-dependent RNA polymerase
Viral Plaque Assay
Biology
medicine.disease_cause
Virus Replication
Microbiology
Cell Line
chemistry.chemical_compound
Viral Proteins
Suppression, Genetic
Virology
RNA polymerase
Cricetinae
Protein Interaction Mapping
medicine
Animals
Replicon
Polymerase
Genetics
Mutation
Base Sequence
RNA
Methyltransferases
Stem-loop
RNA-Dependent RNA Polymerase
Genome Replication and Regulation of Viral Gene Expression
Viral replication
chemistry
Amino Acid Substitution
Insect Science
biology.protein
Nucleic Acid Conformation
RNA, Viral
West Nile virus
Protein Binding
Subjects
Details
- ISSN :
- 10985514
- Volume :
- 82
- Issue :
- 14
- Database :
- OpenAIRE
- Journal :
- Journal of virology
- Accession number :
- edsair.doi.dedup.....0478b37371ebcf740dcdd26ac6ca6697