AN INVESTIGATION INTO THE MOLECULAR BASIS OF DENGUE VIRUS ATTENUATION IN ITS NATURAL HOSTS

Abstract

The molecular basis of dengue virus (DENV) attenuation remains incompletely understood, hindering a rational approach to generate live attenuated dengue vaccines. This thesis took advantage of the DENV2 PDK53 vaccine strain, which recently completed phase 3 clinical trials with good safety and efficacy profiles, to elucidate how this virus is attenuated compared to its wild-type parent, DENV2 16681 strain. The Gibson assembly method coupled with site-directed mutagenesis identified a G53D mutation in the non-structural 1 (NS1) protein as a key attenuating mutation; 16681 NS1 G53D mutant infection activates the host innate immune response that dampens viral infection and dissemination in both Aedes aegypti and mammalian cells to produce the attenuated phenotypes of PDK53. Mechanistically, the NS1 G53D mutation impairs the function of a host dependency protein, the endoplasmic reticulum (ER)-resident ribophorin 1 (RPN1) protein, to properly glycosylate NS1 and hence activates an antiviral response through ER stress. This thesis reveals insights into the genetic and mechanistic basis of DENV attenuation on a clinically proven vaccine strain.