Abstract
BackgroundThe enzyme activities catalysed by flavivirus non-structural protein 3 (NS3) are essential for virus replication. They are distributed between the N-terminal protease domain in the first one-third and the C-terminal ATPase/helicase and nucleoside 5′ triphosphatase domain which forms the remainder of the 618-aa long protein.Methodology/Principal FindingsIn this study, dengue full-length NS3 protein with residues 49 to 66 of NS2B covalently attached via a flexible linker, was used as bait in biopanning with a naïve human Fab phage-display library. Using a range of truncated constructs spanning the NS2B cofactor region and the full-length NS3, 10 unique Fab were identified and characterized. Of these, monoclonal Fab 3F8 was shown to bind α3″ (residues 526 through 531) within subdomain III of the helicase domain. The antibody inhibits the ATPase and helicase activites of NS3 in biochemical assays and reduces DENV replication in HEK293 cells that were previously transfected with Fab 3F8 compared with mock transfected cells.Conclusions/SignificanceAntibodies such as 3F8 are valuable tools for studying the molecular mechanisms of flaviviral replication and for the monospecific detection of replicating dengue virus in vivo.
Highlights
Dengue virus belongs to the Flaviviridae family and is the etiological agent of dengue fever, dengue hemorrhagic fever and dengue shock syndrome
Dengue non-structural protein 3 (NS3) is an essential, multifunctional, viral enzyme with two distinct domains; a protease domain required for processing of the viral polyprotein, and a helicase domain required for replication of the viral genome
In this study ten unique human antibody fragments (Fab) that bind dengue NS3 were isolated from a diverse library of Fab clones using phage display technology
Summary
Dengue virus belongs to the Flaviviridae family and is the etiological agent of dengue fever, dengue hemorrhagic fever and dengue shock syndrome It is the most prevalent arthropod transmitted infectious disease in humans and has four antigenically distinct viral serotypes (DENV 1–4) [1]. Recent crystal structures of full-length NS3 from DENV and the related flavivirus Murray Valley encephalitis virus, reveal that the protease and helicase domains are linked by an interdomain linker (residues 169–179 in DENV) as illustrated in Figure 1 [8,9]. The enzyme activities catalysed by flavivirus non-structural protein 3 (NS3) are essential for virus replication. They are distributed between the N-terminal protease domain in the first one-third and the C-terminal ATPase/helicase and nucleoside 59 triphosphatase domain which forms the remainder of the 618-aa long protein
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