Towards understanding of the replication and pathogenesis of dengue infection

Abstract

Dengue is the most important human viral disease transmitted by arthropod vectors and over half of the world's population live in areas at risk of infection. Currently there is neither specific treatment nor vaccine to tackle this emerging disease. The work described in this thesis has been conducted at the Novartis Institute for Tropical Diseases (NITD) which aims to find small molecule inhibitors for dengue. In keeping with the goals of the institute, the aim of this thesis was to identify viral and host factors that are important for dengue replication and pathogenesis. Chapter 1 of this thesis describes the features of dengue disease and reviews the molecular studies of the causative organism- the dengue virus. It also lists out the multifaceted efforts to control dengue and the need to gain comprehensive knowledge of the viral and host factors that influence replication of the dengue virus. Of the seven non structural proteins described for dengue, the roles of only NS5 and NS3 have been fully explored. Chapter 2 details the characterisation of NS4B, a small non structural protein of dengue, whose role in dengue replication was previously unexplored. A physical interaction was identified between NS4B and the helicase domain of NS3 using the yeast two-hybrid assay. This interaction was further confirmed by biochemical pull down and immuno-precipitation assays, both with purified proteins and with dengue virus infected cell lysates. Furthermore, NS4B dissociated NS3 from single stranded RNA and consequently enhanced the helicase activity of NS3 in an in vitro unwinding assay. A single amino acid mutation in NS4B (Hanley et al., 2003) that abolished its interaction with NS3 altered the viral replication. In addition, NS4B co-localized with NS3 in the peri-nuclear region of infected human cells suggesting the in vivo significance of this interaction. These results suggest that NS4B modulates dengue replication via its interaction with NS3. Severe dengue has long been speculated to be a result of a complex combination of viral, epidemiological and host factors in the context of a secondary infection. Chapter 3 unveils for the first time, a role for viral genomic variations in dengue pathogenesis via modulation of the response to type I IFN. A strain-dependent difference was detected in gene expression levels of the type I interferon response pathway between two closely related DEN2 strains NGC and TSV01. Activation of type I anti viral responses such as PKR, OAS, ADAR and Mx, were prevalent in infection with TSV01 but not NGC. Biochemical dissection further revealed that NGC but not TSV01 suppressed STAT-1 activation in response to type I IFN but these two strains did not differ in their response to type II IFN. An extension of this study to low passage clinical isolates of various serotypes indicated that this ability to suppress IFN response is independent of serotype as well as viral load. Furthermore, the inability of one such clinical isolate SG 167 (isolated during a recent dengue outbreak which was relatively mild with very few severe dengue cases) to suppress IFN response indicated that type I IFN response could be a prime factor that determines the clinical outcome to dengue infection. Virus-induced apoptosis mediated by the unfolded protein response (UPR) is hypothesized to be a crucial pathogenic event in dengue infection. Chapter 4 of this thesis is one of the first reports on the initial events in dengue infection mediated UPR. Phosphorylation of EIF2α was induced in dengue infection but simultaneously, the expression of GADD34 (which dephosphorylates EIF2α) was also enhanced. An inhibitor of GADD34 reduced dengue replication in infected cells indicating that this viral “compensatory” event is required for viral survival. Both the XBP1 and ATF6 pathways of the UPR were also activated by dengue infection. In addition, modulation of the EIF2α and the XBP1 pathways altered dengue replication indicating that UPR pathway components affect the outcome of infection. Chapter 5 summarizes the conclusions from these studies and discusses some of the future work that can arise from these results. Finally, it is hoped that knowledge gained in this thesis will expedite the quest for an anti-dengue drug.