Dengue virus (DENV) causes over 500,000 infections annually with a spectrum of clinical diseases ranging from subclinical infection to dengue, a mild febrile illness, to life-threatening severe dengue. Vascular leakage without endothelial cell damage is the hallmark symptom of severe dengue illness and is proposed to be directly mediated by soluble inflammatory mediators IL-8 and TNFα. IL-8 production occurs in response to DENV infection, is elevated during severe dengue, is proposed to inhibit interferon, and could potentially recruit target cells to sites of infection. We previously showed that expression of DENV NS5 activates the IL-8 promoter, induces IL-8 transcription, and induces IL-8 protein production in HepG2 and HEK293A cell lines. As multiple DENV proteins are reported to interact with important signaling pathways, we hypothesized that other DENV proteins could contribute to the activation of IL-8. We found that plasmids expressing prM-E together, the GPI-linked variant of NS1 (NS1G), the carboxyl-terminal 112 amino acids of NS4B, as well as NS5 each induced expression from an IL-8 promoter-driven reporter plasmid. Expression of NS5 also induced activation of a RANTES promoter construct and TNFα mRNA expression. Further, we found that the carboxyl-terminal polymerase domain of NS5 was sufficient to induce IL-8 secretion but polymerase function was not required. Like NS5, prM-E and NS1G induced luciferase expression from an AP-1-driven reporter plasmid. We further tested whether activation of the IL-8 promoter depended on any single transcription factor within IL-8 using IL-8 promoter-driven plasmids mutated at the AP-1, C/EBP or NF-κB binding sites. We found that activation of the IL-8 promoter by prM-E, NS1G and NS4B did not depend on activation of any single transcription factor. Our data suggested that AP-1 may be both positively and negatively inducing transcription, fitting with previous theories that DENV regulates IL-8 induction. However, we did not observe any differences in activation of AP-1 subunit c-Jun, or the inhibitory subunits Fra-1 or Fra-2 between DENV and mock-infected cells. These data support a model in which multiple DENV proteins activate the IL-8 promoter, provide a potential basis of IL-8 induction by DENV in multiple cell types, and further supports a mechanism by which DENV contributes to severe dengue illness.
| Identifer | oai:union.ndltd.org:umassmed.edu/oai:escholarship.umassmed.edu:gsbs_diss-1618 |
| Date | 29 May 2012 |
| Creators | Collins, Jacob M. |
| Publisher | eScholarship@UMassChan |
| Source Sets | University of Massachusetts Medical School |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Morningside Graduate School of Biomedical Sciences Dissertations and Theses |
| Rights | Copyright is held by the author, with all rights reserved., select |
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