Research Article Open Access

The Murine Charged Multivesicular Body Protein 2A, CHMP2A Interacts with the 5' and 3' Terminal Regions of Dengue Virus Complementary Minus-strand RNA

Suchita Chaudhry1, Sathyamangalam Swaminathan1 and Navin Khanna1
  • 1 International Centre for Genetic Engineering and Biotechnology, India

Abstract

Dengue (DEN) viruses, of which there are four distinct serotypes (DEN-1, -2, -3 and –4), belong to the Flaviviridae family. Their ‘plus’ sense RNA genomes contain Non-Translated Regions (NTRs) at their 5’ and 3’ ends. Replication of viral genomic RNA, which takes place in close association with host cell membranes, involves the initial synthesis of the complementary minus sense RNA intermediate. The NTRs, which have the potential to form stable stem-loop structures, are implicated to play a key role in the viral life cycle through binding to viral and/or host proteins. We have screened a mouse macrophage cDNA expression library with a mixture of radioactive plus and minus sense DEN-2 virus NTR transcripts and identified a ~25 kDa protein. A BLAST analysis of the cDNA sequence encoding this protein showed it to be identical to human CHarged Multivesicular body Protein 2A, CHMP2A (also known as CHromatin Modifying Protein 2A), implicated in sorting proteins into endosome-derived vesicles. Recombinant murine CHMP2A was expressed in E. coli as a 6x His tagged protein, purified to homogeneity and shown to interact preferentially with the minus sense 5’ and 3’ NTRs of all four DEN virus serotypes. Using DEN-2 virus 5’ (-) and 3’ (-) NTRs we could demonstrate the specificity of this binding activity in vitro by ultraviolet cross-linking and electrophoretic mobility shift assays, carried out in the absence and presence of specific and non-specific competitors. Finally, this unique RNA/protein interaction was verified in vivo using a yeast-based interaction assay.

American Journal of Infectious Diseases
Volume 2 No. 1, 2006, 18-27

DOI: https://doi.org/10.3844/ajidsp.2006.18.27

Submitted On: 25 January 2006 Published On: 31 March 2006

How to Cite: Chaudhry, S., Swaminathan, S. & Khanna, N. (2006). The Murine Charged Multivesicular Body Protein 2A, CHMP2A Interacts with the 5' and 3' Terminal Regions of Dengue Virus Complementary Minus-strand RNA. American Journal of Infectious Diseases, 2(1), 18-27. https://doi.org/10.3844/ajidsp.2006.18.27

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Keywords

  • Dengue virus
  • non-translated regions
  • chromatin modifying protein 2A (CHMP2A)
  • charged multivesicular body protein
  • RNA replication