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Published on: July 6, 2013
Molecular analysis of Sindbis virus pathogenesis in neonatal mice by using virus recombinants constructed in vitro
J M Polo1, N L Davis, C M Rice
1Department of Microbiology, North Carolina State University, Raleigh 27695-7615.
Abstract:
Genetic loci affecting Sindbis virus pathogenesis in neonatal mice have been examined by using a full-length cDNA clone of the virus (Toto1101). The full-length cDNA is linked to a bacteriophage SP6 promoter to facilitate the synthesis of infectious RNA transcripts in vitro. Virus derived from Toto1101 showed reduced virulence (attenuation) in neonatal mice. Replacement of the E1 glycoprotein and 6K genes of Toto1101 with cloned E1 and 6K genes derived from a virulent Sindbis virus strain, AR339 (SB), resulted in a new construct, TR2000, that gave rise to virulent virus. Sequence determinations for the entire substituted regions of TR2000, Toto1101, and related virulent and attenuated strains identified three coding differences in E1 between Toto1101 and TR2000. These differences, individually or in combination, may be responsible for the attenuated phenotype. Previous studies in this laboratory identified another attenuating mutation at amino acid position 114 of the E2 glycoprotein (N.L. Davis, F.J. Fuller, W.G. Dougherty, R.A. Olmsted, and R.E. Johnston, Proc. Natl. Acad. Sci. USA 83:6771-6775, 1986). Substitution of Arg-114 in the mutant SB-RL for Ser-114 of SB appears to confer three distinguishing phenotypes: attenuation in neonatal mice, increased sensitivity to specific E2 monoclonal antibodies, and accelerated penetration of BHK cells. Replacement of TR2000 sequences containing the codon for amino acid 114 of E2 with corresponding fragments from cDNA clones of SB or SB-RL produced two strains of Sindbis virus (TR2100 and TR2200) which were isogenic except for the E2 114 codon (Ser and Arg, respectively). The three diagnostic phenotypes cosegregated according to the origin of the codon for amino acid 114 of E2, confirming the dramatic effect of this single amino acid substitution on these three phenotypes.
Insights
This study investigates Sindbis virus pathogenesis by analyzing genetic mutations. A single amino acid change in the E2 glycoprotein significantly impacts viral virulence and cell interaction.
Area of Science:
- Virology
- Molecular Biology
- Genetics
Background:
- Sindbis virus (Sindbis) pathogenesis in neonatal mice is influenced by genetic factors.
- Understanding these factors is crucial for developing effective antiviral strategies.
Purpose of the Study:
- To identify genetic determinants of Sindbis virus virulence.
- To investigate the role of specific viral genes and mutations in pathogenesis.
Main Methods:
- Utilized a full-length Sindbis virus cDNA clone (Toto1101) for in vitro RNA transcript synthesis.
- Constructed recombinant viruses (TR2000, TR2100, TR2200) by gene swapping and site-directed mutagenesis.
- Sequenced viral genes and assessed viral phenotypes in neonatal mice and cell culture.
Main Results:
- Recombinant virus TR2000, with genes from virulent strain AR339, regained virulence.
- Identified three coding differences in the E1 glycoprotein between attenuated and virulent strains.
- A single amino acid substitution (Ser to Arg) at position 114 of the E2 glycoprotein conferred attenuation, altered antibody binding, and increased cell penetration.
Conclusions:
- Specific genetic loci, particularly within the E1 and E2 glycoproteins, significantly influence Sindbis virus pathogenesis.
- A single amino acid substitution in the E2 glycoprotein has a profound effect on multiple viral phenotypes.
- This research provides insights into the molecular basis of viral virulence and host-pathogen interactions.
