Please use this identifier to cite or link to this item:

Studying the role of highly conserved residues in the stem region of DENV1 envelope protein on assembly

File Description Size Format  
Pires Amanda r.pdf Version for non-UH users. Copying/Printing is not permitted 2.42 MB Adobe PDF View/Open
Pires Amanda uh.pdf Version for UH users 2.55 MB Adobe PDF View/Open

Item Summary Pires, Amanda Michiko 2016-02-19T22:05:58Z 2016-02-19T22:05:58Z 2012-12
dc.description M.S. University of Hawaii at Manoa 2012.
dc.description Includes bibliographical references.
dc.description.abstract Dengue virus (DENV), a flavivirus with four serotypes (DENV1 to DENV4), is the leading cause of arboviral diseases worldwide. Despite decades of studies on dengue, no antiviral against DENV is currently available. The envelope (E) protein is involved in virus entry and assembly. The Cterminus of E protein contains two alpha-helices (EH1 and EH2) separated by conserved sequences (CS) in the stem region and two transmembrane domains in the anchor region. Previous cryo-electron microscopic (EM) study revealed that EH1 and EH2 are partially buried in the outer leaflet of viral membrane. The stem region contains 16 residues absolutely conserved among diverse flaviviruses. We hypothesize that the absolutely conserved stem residues are involved in critical steps of virus life cycle including assembly. In this study, we investigated the roles of the absolutely conserved stem residues on virus assembly and the mechanisms involved. Our long-term goal is to explore the stem region of DENV E protein as novel targets for the development of antivirals. In the first specific aim, we investigated the role of the absolutely conserved stem residues on virus assembly of DENV1 by examining the production of virus-like particles (VLPs). VLPs are similar to infectious virions in the structural, biochemical and antigenic properties, and can be generated by co-expression of precursor membrane (prM) and E proteins. Using a series of DENV1 prM/E expressing constructs containing a single proline or alanine substitution for each of the 16 highly conserved stem residues by site-directed mutagenesis we were able to examine the role that these play in assembly. After transfection to 293T cells, cell lysates and pellets derived from ultracentrifugation of cultural supernatants were subjected to Western blot analysis and a quantitative capture-ELISA using known concentrations of a recombinant E protein as standard. Compared with those of the wild type (WT), the amounts of E protein in pellets relative to cell lysates were reduced in 15 proline mutants at the EH1, CS and EH2 domains and greatly reduced in 9 mutants at the CS and EH2 domains, suggesting the importance of overall helical structure on assembly and the critical role of residues at the CS and EH2 domains, In the second specific aim, we investigated the mechanisms of impairment in assembly of DENV1 mutants. Immunoprecipitation assay revealed that alanine substitutions do not affect the prM-E interaction. Enzyme digestion experiment showed that these alanine substitutions do not affect the glycosylation of E protein and both WT and mutant prM/E proteins were primarily located in the ER. Subcellular fractionation experiment revealed that the amounts of prM/E proteins of the alanine mutants in the soluble fraction relative to membrane fraction were either less or greater than that of WT, suggesting that some alanine mutations affect the budding of VLPs from the ER membrane to the lumen of ER, whereas other alanine mutations affect the release of VLPs from ER lumen or other intracellular vesicles to outside of the cells. We also set up a transmission EM and immuno-gold EM to explore the morphological defects of these mutants. In summary, our findings indicate several absolutely conserved stem residues are involved in the assembly step of DENV life cycle; in particular, 9 residues in the CS and EH2 domains of the stem region are critical. While considerable efforts have been made to develop antivirals against DENV and other flaviviruses, there are no licensed antivirals against DENV currently available. The absolutely conserved nature of these residues may be potential targets for antiviral strategy to block virus assembly.
dc.language.iso eng
dc.publisher [Honolulu] : [University of Hawaii at Manoa], [December 2012]
dc.relation Theses for the degree of Master of Science (University of Hawaii at Manoa). Biomedical Sciences (Tropical Medicine).
dc.subject DENV1
dc.subject dengue virus
dc.subject absolutely conserved stem residues
dc.subject antiviral
dc.title Studying the role of highly conserved residues in the stem region of DENV1 envelope protein on assembly
dc.type Thesis
dc.type.dcmi Text
Appears in Collections: M.S. - Biomedical Sciences (Tropical Medicine)

Please email if you need this content in ADA-compliant format.

Items in ScholarSpace are protected by copyright, with all rights reserved, unless otherwise indicated.