| dc.description.abstract | Dengue fever is a global health problem that affects approximately 3.9 billion people worldwide. Dengue virus (DENV) comprises four serotypes (DENV-1,-2,-3, and -4) which belong to the genus flavivirus, family flaviviridae. DENV consists of three structural proteins and seven non-structural proteins. Non-structural proteins form the replication complex responsible for viral RNA synthesis. Besides of role in RNA replication, little is known regarding the role of DENV-2 non-structural proteins, e.g. non-structural protein 4A (NS4A) and 4B (NS4B), during virus life cycle. Two related projects regarding NS4A and NS4B are included in the thesis. The title of first study is “Mutagenesis of the Dengue Virus NS4A Protein Reveals a Novel Cytosolic N-terminal Domain Responsible for Virus-Induced Cytopathic Effects and Intramolecular Interactions within the N-terminus of NS4A”. The title of second study is “A Dengue Virus Type 2 (DENV-2) NS4B-interacting Host Factor, SERP1, Reduces DENV-2 Production by Suppressing Viral RNA Replication”.
The NS4A protein of DENV-2 has a cytosolic N-terminus and four transmembrane domains. NS4A participates in RNA replication and the host antiviral response. However, the roles of amino acid residues within the N-terminus of NS4A during DENV life cycle are not clear. We explored the function of DENV-2 NS4A by introducing a series of alanine substitutions into the N-terminus of NS4A in the context of a DENV-2 infectious clone or subgenomic replicon. Nine of seventeen NS4A mutants displayed a lethal phenotype due to the impairment of RNA replication. M2 and M14 displayed a more than 10,000-fold reduction in viral yields and moderate defects in viral replication by a replicon assay. Sequencing analyses of pseudorevertant viruses derived from M2 and M14 viruses revealed one consensus reversion mutation, A21V, within NS4A. The A21V mutation apparently rescued viral RNA replication in the M2 and M14 mutants although not to wild-type (WT) levels but resulted in 100- and 1,000-fold lower titers than that of the WT, respectively. M2 Rev1 (M2 + A21V) and M14 Rev1 (M14+ A21V) mutants displayed phenotypes of smaller plaque size and WT-like assembly/secretion by a transpackaging assay. A defect in the virus-induced cytopathic effect (CPE) was observed in HEK-293 cells infected with either M2 Rev1 or M14 Rev1 mutant virus by MitoCapture staining, cell proliferation, and ATP levels. In conclusion, the results revealed the essential roles of the N-terminal NS4A in both RNA replication and virus-induced CPE. Intramolecular interactions in the N-terminus of NS4A were implicated.
Host cells infected with DENV often trigger endoplasmic reticulum (ER) stress, a key process that allows viral reproduction without killing host cells until the late stage of the virus life cycle. However, little is known regarding which DENV viral proteins interact with ER machinery to support viral replication. We identified and charecterized a novel host factor, stress-associated ER protein 1 (SERP1), that interacts with the DENV-2 NS4B protein by several assays, e.g. yeast two-hybrid, subcellular localization, NanoBiT complementation, and co-immunoprecipitation. A drastic increase (34.5-fold) in SERP1 gene expression was observed in DENV-2-infected or replicon-transfected Huh7.5 cells. SERP1 overexpression inhibited viral yields (37-fold) in DENV-2-infected Huh7.5 cells. In contrast, shRNAi-knockdown and knockout of SERP1 increased viral yields (3.4- and 16-fold, respectively) in DENV-2-infected HEK-293 and Huh7.5 cells, respectively. DENV-2 viral RNA replication was severely reduced in stable SERP1-expressing Huh7.5 cells transfected with DENV-2 replicon plasmids. Overexpression of DENV-2 NS4B alleviated the inhibitory effect of SERP1 on DENV-2 RNA replication. Taking these results together, we hypothesized that SERP1 may serve as an antiviral player during ER stress to restrict DENV-2 infection. Our studies revealed novel anti-DENV drug targets that may facilitate anti-DENV drug discovery. | en_US |