title: A Molecular Approach Designed to Limit the Replication of Mature DENV2 in Host Cells.
abstract: Dengue virus (DENV) is an arthropod-borne virus, which belongs to the Flaviviridae family, and completes its life cycle in two hosts: humans and mosquitoes. For DENV maturation, the surface pre-membrane (prM) protein is cleaved to form a mature membrane protein (M) by furin, which is a cellular enzyme subsequently releasing the mature virus from the host dendritic cell. The objective of the current study was to inhibit mature DENV isotype 2 (DENV2) by RNA-interference in a Vero-81 cell line. Mature DENV2 was propagated in and isolated from U937 cells expressing dendritic cell-specific intracellular adhesion molecule-3-grabbing non-integrin. Maturation of DENV2 was confirmed by Western blot analysis, where virus stock lacking prM was considered mature. Inhibition studies were carried out by transfection of Vero-81 cells with six synthetic siRNAs along with a control siRNA. Reduction in cellular DENV2 was observed also by focus-reduction assay, immunofluorescence assay (IFA), and real-time quantitative polymerase chain reaction (RT-qPCR). Cells transfected with DENV2SsiRNA2, which was targeting the structural region M of mature DENV2, was able to reduce DENV2 titer by up to 85% in focus reduction assays. A significant reduction in mature DENV2 RNA load was observed by RT-qPCR, confirming the previous findings. IFA also revealed reduced levels of cellular DENV2. These results demonstrated that mature DENV2 can be effectively inhibited by synthetic siRNA targeting the structural region of the genome. Mature DENV2 can be successfully inhibited by siRNAs, and specifically high knock-down efficiency is observed by siRNAs against M region of mature DENV2. This study shows that M represents a potential target for RNAi based inhibitory approaches.
authors.affiliation: Atta-ur-Rahman School of Applied Biosciences (ASAB), National University of Sciences and Technology (NUST) , Islamabad, Pakistan .
authors.forename: Ummar
authors.initials: U
authors.lastname: Raheel
authors.affiliation: Atta-ur-Rahman School of Applied Biosciences (ASAB), National University of Sciences and Technology (NUST) , Islamabad, Pakistan .
authors.forename: Muhsin
authors.initials: M
authors.lastname: Jamal
authors.affiliation: Atta-ur-Rahman School of Applied Biosciences (ASAB), National University of Sciences and Technology (NUST) , Islamabad, Pakistan .
authors.forename: Najam Us Sahar Sadaf
authors.initials: NU
authors.lastname: Zaidi
mesh_terms: D000818:Animals; D002460:Cell Line; D002522:Chlorocebus aethiops; D003716:Dengue Virus / Q000235:genetics / Q000502:physiology*; D005455:Fluorescent Antibody Technique; D006801:Humans; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D012367:RNA, Viral / Q000096:biosynthesis / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D014162:Transfection; D019562:Viral Load; D014773:Virology / Q000379:methods; D014779:Virus Replication
chemical_list: D034741:RNA, Small Interfering; D012367:RNA, Viral