Towards understanding of the replication and pathogenesis of dengue infection

Dengue is the most important human viral disease transmitted by arthropod vectors \nand over half of the world's population live in areas at risk of infection. Currently there \nis neither specific treatment nor vaccine to tackle this emerging disease. The work \ndescribed in this thesis has been conducted at the Novartis Institute for Tropical \nDiseases (NITD) which aims to find small molecule inhibitors for dengue. In keeping \nwith the goals of the institute, the aim of this thesis was to identify viral and host \nfactors that are important for dengue replication and pathogenesis. \nChapter 1 of this thesis describes the features of dengue disease and reviews the \nmolecular studies of the causative organism- the dengue virus. It also lists out the \nmultifaceted efforts to control dengue and the need to gain comprehensive knowledge \nof the viral and host factors that influence replication of the dengue virus. \nOf the seven non structural proteins described for dengue, the roles of only NS5 and \nNS3 have been fully explored. Chapter 2 details the characterisation of NS4B, a small \nnon structural protein of dengue, whose role in dengue replication was previously \nunexplored. A physical interaction was identified between NS4B and the helicase \ndomain of NS3 using the yeast two-hybrid assay. This interaction was further \nconfirmed by biochemical pull down and immuno-precipitation assays, both with \npurified proteins and with dengue virus infected cell lysates. Furthermore, NS4B \ndissociated NS3 from single stranded RNA and consequently enhanced the helicase \nactivity of NS3 in an in vitro unwinding assay. A single amino acid mutation in NS4B \n(Hanley et al., 2003) that abolished its interaction with NS3 altered the viral \nreplication. In addition, NS4B co-localized with NS3 in the peri-nuclear region of \ninfected human cells suggesting the in vivo significance of this interaction. These results \nsuggest that NS4B modulates dengue replication via its interaction with NS3. \nSevere dengue has long been speculated to be a result of a complex combination of \nviral, epidemiological and host factors in the context of a secondary infection. Chapter \n3 unveils for the first time, a role for viral genomic variations in dengue pathogenesis \n \nvia modulation of the response to type I IFN. A strain-dependent difference was \ndetected in gene expression levels of the type I interferon response pathway between \ntwo closely related DEN2 strains NGC and TSV01. Activation of type I anti viral \nresponses such as PKR, OAS, ADAR and Mx, were prevalent in infection with TSV01 \nbut not NGC. Biochemical dissection further revealed that NGC but not TSV01 \nsuppressed STAT-1 activation in response to type I IFN but these two strains did not \ndiffer in their response to type II IFN. An extension of this study to low passage clinical \nisolates of various serotypes indicated that this ability to suppress IFN response is \nindependent of serotype as well as viral load. Furthermore, the inability of one such \nclinical isolate SG 167 (isolated during a recent dengue outbreak which was relatively \nmild with very few severe dengue cases) to suppress IFN response indicated that type I \nIFN response could be a prime factor that determines the clinical outcome to dengue \ninfection. \nVirus-induced apoptosis mediated by the unfolded protein response (UPR) is \nhypothesized to be a crucial pathogenic event in dengue infection. Chapter 4 of this \nthesis is one of the first reports on the initial events in dengue infection mediated UPR. \nPhosphorylation of EIF2α was induced in dengue infection but simultaneously, the \nexpression of GADD34 (which dephosphorylates EIF2α) was also enhanced. An \ninhibitor of GADD34 reduced dengue replication in infected cells indicating that this \nviral “compensatory” event is required for viral survival. Both the XBP1 and ATF6 \npathways of the UPR were also activated by dengue infection. In addition, modulation \nof the EIF2α and the XBP1 pathways altered dengue replication indicating that UPR \npathway components affect the outcome of infection. \nChapter 5 summarizes the conclusions from these studies and discusses some of the \nfuture work that can arise from these results. Finally, it is hoped that knowledge gained \nin this thesis will expedite the quest for an anti-dengue drug.