Recombinant Dengue Type 1 Virus NS5 Protein Expressed inEscherichia coliExhibits RNA-Dependent RNA Polymerase Activity

Tan, Bernard C. Y.; Fu, Jianlin; Sugrue, Richard J.; Yap, E. H.; Chan, Yee Cheun; Tan, Yee-Joo

doi:10.1006/viro.1996.0067

Cited by 237 publications

(185 citation statements)

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“…Recent approaches include the use of inactivated whole-virion vaccines (23), synthetic peptides (5,121,122), subunit vaccines (31,101,140), vector expression, recombinant live vector systems (23,102), infectious cDNA clone-derived vaccines (16,25,79,80,82,93,113), and naked DNA (24,84). The last two approaches appear to be the most promising.…”

Section: Vaccine Developmentmentioning

confidence: 99%

Dengue and Dengue Hemorrhagic Fever

Gubler¹

1997

Tropical Infectious Diseases

629

756

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Section: Vaccine Developmentmentioning

confidence: 99%

Dengue and Dengue Hemorrhagic Fever

Gubler¹

1997

Tropical Infectious Diseases

629

756

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“…NS5 comprises an N-terminal methyltransferase (MTase) domain that catalyses both guanine N-7 and ribose 29-O methylations during viral RNA cap formation (Zhou et al, 2007) and a C-terminal domain that harbours the RNA-dependent RNA polymerase (RdRp) (Tan et al, 1996;Guyatt et al, 2001;Nomaguchi et al, 2004;Selisko et al, 2006). The crystal structure of the WNV RdRp revealed classic RdRp structure bearing palm, thumb and finger subdomains (Malet et al, 2007).…”

Section: Introductionmentioning

confidence: 99%

Monoclonal antibodies to the West Nile virus NS5 protein map to linear and conformational epitopes in the methyltransferase and polymerase domains

Hall

Tan

Selisko

et al. 2009

Journal of General Virology

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The West Nile virus (WNV) NS5 protein contains a methyltransferase (MTase) domain involved in RNA capping and an RNA-dependent RNA polymerase (RdRp) domain essential for virus replication. Crystal structures of individual WNV MTase and RdRp domains have been solved; however, the structure of full-length NS5 has not been determined. To gain more insight into the structure of NS5 and interactions between the MTase and RdRp domains, we generated a panel of seven monoclonal antibodies (mAbs) to the NS5 protein of WNV (Kunjin strain) and mapped their binding sites using a series of truncated NS5 proteins and synthetic peptides. Binding sites of four mAbs (5D4, 4B6, 5C11 and 6A10) were mapped to residues 354-389 in the fingers subdomain of the RdRp. This is consistent with the ability of these mAbs to inhibit RdRp activity in vitro and suggests that this region represents a potential target for RdRp inhibitors. Using a series of synthetic peptides, we also identified a linear epitope (bound by mAb 5H1) that mapped to a 13 aa stretch surrounding residues 47 and 49 in the MTase domain, a region predicted to interact with the palm subdomain of the RdRp. The failure of one mAb (7G6) to bind both N-and Cterminally truncated NS5 recombinants indicates that the antibody recognizes a conformational epitope that requires the presence of residues in both the MTase and RdRp domains. These data support a structural model of the full-length NS5 molecule that predicts a physical interaction between the MTase and the RdRp domains.

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“…The POL domain consists of the RNA dependent RNA polymerase which is essential for virus RNA synthesis. POL has been shown to initiate de novo synthesis of positive-sense stranded RNA in the absence of a primer (Guyatt et al, 2001;Tan et al, 1996).…”

Section: Introductionmentioning

confidence: 99%

An interaction between the methyltransferase and RNA dependent RNA polymerase domains of the West Nile virus NS5 protein

Tan

Hobson‐Peters

Stoermer

et al. 2013

Journal of General Virology

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The flavivirus nonstructural protein 5 (NS5) is a large protein that is structurally conserved among members of the genus, making it an attractive target for antiviral drug development. The protein contains a methyltransferase (MTase) domain and an RNA dependent RNA polymerase (POL) domain. Previous studies with dengue viruses have identified a genetic interaction between residues 46-49 in the aA3-motif in the MTase and residue 512 in POL. These genetic interactions are consistent with structural modelling of these domains in West Nile virus (WNV) NS5 that predict close proximity of these regions of the two domains, and potentially a functional interaction mediated via the aA3-motif. To demonstrate an interaction between the MTase and POL domains of the WNV NS5 protein, we co-expressed affinity-tagged recombinant MTase and POL proteins in human embryonic kidney cells with simian virus 40 large T antigen (HEK293T cells) and performed pulldown assays using an antibody to the flag tag on POL. Western blot analysis with an anti-MTase mAb revealed that the MTase protein was specifically coimmunoprecipitated with POL, providing the first evidence of a specific interaction between these domains. To further assess the role of the aA3 helix in this interaction, selected residues in this motif were mutated in the recombinant MTase and the effect on POL interaction determined by the pulldown assay. These mutations were also introduced into a WNV infectious clone (FLSDX) and the replication properties of these mutant viruses assessed. While none of the aA3 mutations had a significant effect on the MTase-POL association in pulldown assays, suggesting that these residues were not specific to the interaction, an E46L mutation completely abolished virus viability indicating a critical requirement of this residue in replication. Failure to generate compensatory mutations in POL to rescue replication, even after several passages of the transfection supernatant in Vero cells, precluded further conclusion of the role of this residue in the context of MTase-POL interactions.

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Recombinant Dengue Type 1 Virus NS5 Protein Expressed inEscherichia coliExhibits RNA-Dependent RNA Polymerase Activity

Cited by 237 publications

References 0 publications

Dengue and Dengue Hemorrhagic Fever

Dengue and Dengue Hemorrhagic Fever

Monoclonal antibodies to the West Nile virus NS5 protein map to linear and conformational epitopes in the methyltransferase and polymerase domains

An interaction between the methyltransferase and RNA dependent RNA polymerase domains of the West Nile virus NS5 protein

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