Maturation of dengue virus nonstructural protein 4B in monocytes induces dengue hemorrhagic fever-associated immunomediators that modulate microvascular endothelial cell adhesion molecules and vascular permeability

Ph.D. University of Hawaii at Manoa 2011.Includes bibliographical references.Dengue virus (DENV) belongs to the Flaviviridae family and exists as four distinct serotypes, DENV-1,-2,-3, and-4. Of the 50-100 million DENV infections worldwide each year, approximately 80% present as mild and self-limiting dengue fever (DF). A limited number of infected patients progress to severe dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS), leading to a half-million hospitalizations annually. Mechanisms explaining why some individuals progress to severe DHF/DSS are unclear. Of the ten DENV proteins, several nonstructural proteins (NS), including NS4B and NS5, are capable of inhibiting interferon signaling. DENV preferentially infects peripheral blood monocytes, which secrete elevated levels of chemokines and cytokines in patients progressing to severe disease. The focus of this dissertation was to study the role of DENV in the induction of chemokines and cytokines and the effects these immunomediators have on endothelial cell adhesion molecules expression and permeability. For the first time, we report that NS4B is a potent inducer of immunomediators associated with severe disease. We demonstrate that sequential processing of the NS4AB polyprotein by the viral protease NS2B3(pro) and via the intermediate 2KNS4B, is significantly more potent than NS4B alone, inducing immunomediators to levels similar to DENV infection. Further, the 2K-signal peptide is not required for the induction of immunomediators yet it plays a synergistic role with NS4B. A classic clinical sign associated with the progression of DHF/DSS is plasma leakage, which involves vascular endothelial cell changes such as increased expression of adhesion molecules and permeability. DHF/DSS patients present with elevated levels of tumor necrosis factor-alpha (TNFα) and interleukin (IL)-8, which can modulate vascular endothelial cell changes. Based on our initial finding, we hypothesized that levels of immunomediators induced by maturation of NS4B would be sufficient to modulate human microvascular endothelial cell (HMVEC) phenotypic changes associated with severe dengue disease. We employ a HMVEC monolayer and transwell permeability model in-vitro and demonstrate that the immunomediator milieu present in the supernatants collected from DENV-infected THP-1 cells increase HMVEC permeability and expression of intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and E-selectin (E-sel), while direct DENV infection of HMVEC does not. Furthermore, we demonstrate that the supernatants from monocytes expressing 2KNS4B are sufficient to increase vascular permeability and expression of adhesion molecules, which appear to be synergistically induced by TNFα and IL-8. Our data demonstrate that maturation of NS4B is primarily responsible for the induction of immunomediators associated with severe dengue disease. Based on clinical data and our findings, therapies to prevent the progression of DHF/DSSassociated phenotypic changes may include antivirals targeting the maturation of NS4B or TNFα inhibitors to neutralize damaging effects caused by their high levels. Given that NS4B topologies are conserved across flaviviruses, NS4B may be an attractive target for the development of Flavivirus-wide therapeutic interventions