Analysis of Dengue virus NS5 protein - host cell interactions.

Dengue virus (DENV) is a mosquito borne flavivirus that causes the most important arthropod-borne viral disease of humans. Dengue disease ranges from mild fever to the potentially fatal dengue haemorrhagic fever/ shock syndromes (DHF/DSS). It is estimated that at least 50 million dengue infections and 250000 cases of DHF occur annually. DHF is characterised by an increase in vascular permeability, which is believed to be immune mediated. Our current understanding of dengue disease is limited and there are no vaccines or antiviral treatments in use to control the spread of DENV.
Our research is aimed at understanding how DENV replicates and causes disease. The DENV NS5 protein has been found to be essential for replication of the virus and is an important target for antiviral drug development. Recent evidence also suggests that the NS5 protein may play a role in promoting disease. We intend to investigate how the NS5 interacts with the host cell to alter host cell processes, defining the regions of the NS5 protein involved and determining whether these features are common to different strains of DENV.
By determining how individual DENV proteins modulate host cell processes, we will increase our understanding of DENV disease. This knowledge can be used to develop improved DENV vaccines and antiviral agents that are urgently needed. In addition, the research proposed in this application is potentially relevant to our general understanding of how the immune system functions to control microbial disease.

Dengue virus (DENV) is a mosquito borne flavivirus that causes the most important arthropod-borne viral disease of humans. Dengue disease ranges from mild fever to the potentially fatal dengue haemorrhagic fever/ shock syndromes (DHF/DSS). It is estimated that at least 50 million dengue infections and 250000 cases of DHF occur annually. DHF is characterised by an increase in vascular permeability which is believed to be immune mediated and has been associated with elevated levels of inflammatory cytokines in the blood of DENV infected individuals. Our current understanding of dengue pathogenesis is limited and there are no vaccines or antiviral treatments in use to control the spread of DENV.
DENV non-structural protein 5 (NS5) plays a key role in virus replication, containing enzymatic activities required for replication of the DENV RNA genome. Recent studies have shown that the flavivirus NS5 protein may also play a role in viral pathogenesis. The NS5 proteins of a number of flaviviruses have been shown to inhibit interferon signalling. By contrast, the DENV NS5 protein has been shown to induce the production of interleukin-8 (IL-8), a cytokine that has been associated with severe DENV disease.
We hypothesise that the DENV NS5 protein interacts with components of the signalling pathways involved in the regulation of host cell genes, including IL-8, in DENV infected cells. The overall aim of this proposal, is to investigate how the DENV NS5 protein interacts with cellular signalling pathways, define the regions of NS5 involved and determine whether there are differences in the induction of IL-8 by the NS5 protein of different DENV strains. To achieve this aim the effects of expressing i) the NS5 gene from different DENV strains and ii) mutated versions of the NS5 gene on IL-8 gene transcription in cultured cells will be investigated. A monocytic cell line stably expressing NS5 will then be used to identify cellular proteins that interact with NS5 and determine how NS5 induces IL-8 gene transcription.
By understanding how individual DENV gene products modulate host cell processes, we will begin to elucidate the role of viral factors in DENV pathogenesis. This knowledge can be used to develop improved DENV vaccines and antiviral agents. In addition, the research proposed in this application has the potential to identify a novel host-pathogen interaction of general importance.