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Selection and partial characterization of dengue 2 virus mutants that induce fusion at elevated pH
F Guirakhoo1, A R Hunt, J G Lewis
1Division of Vector-Borne Infectious Diseases, Centers for Disease Control, Fort Collins, Colorado 80522.
Abstract:
Two types of dengue (DEN) 2 virus mutants were selected either by repeated exposure to acidic pH (acid mutant, AM), or by the addition of ammonium chloride to Aedes albopictus C6/36 cells prior to and during viral infection (fusion mutant, FM). Both mutants grew more slowly than the parent strain and induced smaller plaques in Vero cells. The 50% fusion from within index for both mutants occurred at least 0.65 pH units higher than with the wild-type DEN virus. A single amino acid substitution (Asn-153 to Asp) was found in the envelope (E)-glycoprotein of the AM virus. Three amino acid substitutions were detected on the E-glycoprotein of the FM virus: Ile-6 to Met, Asn-134 to Ser, and Asn-153 to Tyr. No mutations were found in the precursor to the membrane protein, prM. The DEN virus E-glycoprotein has two potential glycosylation sites: Asn-67 and Asn-153. The loss of the potential glycosylation site at Asn-153 or the change in the chemical characteristics resultant from the amino acid substitutions in both mutants implicates these regions of the E-glycoprotein in virus-mediated membrane fusion.
Insights
Researchers identified two dengue virus type 2 mutants affecting viral fusion. Mutations in the envelope glycoprotein suggest these regions are crucial for dengue virus membrane fusion.
Area of Science:
- Virology
- Molecular Biology
- Biochemistry
Background:
- Dengue virus (DEN) is a significant global health concern.
- Understanding DEN virus entry mechanisms is vital for antiviral development.
- The envelope (E)-glycoprotein plays a critical role in DEN virus fusion.
Purpose of the Study:
- To investigate the role of specific regions of the DEN virus E-glycoprotein in membrane fusion.
- To characterize DEN virus mutants selected for altered fusion properties.
Main Methods:
- Selection of DEN virus type 2 mutants (acid mutant, AM; fusion mutant, FM) via pH or ammonium chloride treatment.
- Growth kinetics and plaque size analysis in Vero cells.
- Determination of 50% fusion from within index.
- Amino acid sequencing of the E-glycoprotein and precursor to membrane protein (prM).
Main Results:
- Both AM and FM mutants exhibited slower growth and smaller plaques compared to wild-type DEN virus.
- Mutants showed a higher pH threshold for fusion, indicating impaired membrane fusion.
- Specific amino acid substitutions were identified in the E-glycoprotein of both mutants, including at Asn-153.
- No mutations were found in the prM protein.
Conclusions:
- Amino acid substitutions in the DEN virus E-glycoprotein, particularly affecting Asn-153, are implicated in altered virus-mediated membrane fusion.
- The loss of a potential glycosylation site or changes in chemical characteristics at Asn-153 are critical for fusion.
- These findings provide insights into the molecular mechanisms of DEN virus entry.