Altered responses of Dengue virus infected cells to TNF-α and induction of GRP78 and HSP70 - in vitro studies.

Abstract

Dengue virus (DENV) infection of humans is characterised by immunopathology with elevated levels of many inflammatory mediators. Tumour necrosis factor alpha (TNF-α) plays a significant role in the pathogenesis of DENV infection with elevated levels of TNF-α in the sera of DENV infected patients that parallel the severity of disease and release of TNF-α coincident with the peak of DENV production from infected monocytederived-macrophages (MDM) in vitro. However, the effect of TNF-α on DENV replication is not fully clarified. In this study we aimed to determine (1) the effect of TNF-α on DENV replication and (2) the changes in host cell protein expression, in response to DENV-infection. Since macrophages are a primary cell target in vivo for DENV-infection, this study mainly used primary monocyte-derived-macrophages (MDM) and macrophagelike cell lines (K562, U937) to represent this cell type. Initially methods were developed for specific analysis of DENV replication, including a tagged RT-PCR method for quantitation of DENV positive (+ ve) and negative (- ve) strand RNA. Next the potential antiviral role of TNF-α in regulating DENV replication in MDM was investigated. While pre-treatment of MDM with TNF-α had a minor inhibitory effect, addition of TNF-α to MDM with established DENV-infection had no effect on DENV replication as measured by DENV RNA levels or virion production. Blocking endogenous TNF-α using TNF-α antibodies or TNF-α siRNA also had no effect on infectious DENV production or RNA synthesis. Together, these results demonstrate that DENV replication in MDM is not affected by TNF-α. Additionally, normal cellular TNF-α signalling, measured by quantitation of TNF-α-induced stimulation of transcription from a nuclear factor-kappa B (NF-kB) responsive reporter plasmid or NF-kB protein nuclear translocation, was blocked in DENV-infected MDM. Thus, DENV replication in MDM is not affected by TNF-α, and infected cells do not respond normally to TNF-α stimulation. It is therefore unlikely that the increased production of TNF-α seen in DENV-infection and correlating with DENV pathology contributes directly to DENV clearance by inducing anti-viral defence mechanisms and reducing DENV replication in MDM. These results also highlight an example of viral subversion of potential anti-viral cellular responses. Secondly, the host cell response to DENV-infection was analysed, presenting the first proteomic analysis on the cellular response to DENV-infection. The differential proteomes of K562 cells with or without DENV infection were resolved and quantitated with two dimensional differential gel electrophoresis (2D PAGE). One 72 kDa protein, was identified by mass spectrometry to be GRP78 (a member of HSP70 protein family) and was up-regulated 2 to 3 fold in infected cells. Up-regulation of GRP78 in DENV-infected cells was confirmed by immuno-staining and confocal microscopy. GRP78 and HSP70 have previously been identified as a component of the DENV receptor complex and blocking of these proteins has been found to inhibit DENV entry into the cell. By confocal microscopy we found that cytoplasmic GRP78 and HSP70 were also up-regulated in DENV-infected cells. The role of cytoplasmic GRP78 and HSP70 in DENV-infected cells has not been established; however there are precedents in other viral infections that cytoplasmic GRP78 and HSP70 could enhance viral protein production. Thus, this thesis shows that (1) the high levels of circulating TNF-α seen in DENV-infection does not influence DENV replication (2) the cellular responses to TNF-α are altered in DENV-infected cells and (3) we have identified two protein chaperones and stress response proteins (GRP78 and HSP70) that are up-regulated during DENV-infection. With the advancement in proteomic techniques since initiation of this project future proteomic analysis could further identify other novel host factors that may either regulate DENV-infection or be involved in a host cell response to DENV-infection and help our understanding of DENV pathogenesis at the protein level.