A Crystal Structure of the Dengue Virus Non-structural Protein 5 (NS5) Polymerase Delineates Interdomain Amino Acid Residues That Enhance Its Thermostability and de Novo Initiation Activities

Lim, Siew Pheng; Koh, Jolene Hong Kiew.; Seh, Cheah Chen; Liew, Chong Wai; Davidson, Andrew D.; Chua, leng Shiew; Chandrasekaran, Ramya; Cornvik, Tobias; Shi, Pei Yong; Lescar, Julien

doi:10.1074/jbc.m113.508606

Cited by 82 publications

(98 citation statements)

References 27 publications

(40 reference statements)

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…The extended DENV-3 and DENV-4 RdRp domains, which contain additional residues at the N terminus of the protein, were expressed with an N-terminal AVI-tag for site-specific biotinylation during expression in E. coli. The additional residues at the N terminus of the RdRp domain improve the stability and enzymatic activity of the domain (15). The additional residues also improved the stability of the protein on the chip surface and reduced nonspecific binding (data not shown).…”

Section: Jf-31-mg46 Binds To Denv-3 and Denv-4 Rdrp By Spr-mentioning

confidence: 86%

“…DENV-4 NS5 was expressed and purified as described previously (10). DENV-3 and -4 RdRp constructs for surface plasmon resonance (SPR) analyses comprised N-terminal AviTag sequences (14) linked to residues 263-900 (DENV-3) or residues 264 -900 (DENV-4) via PCR (primers are listed in supplemental Table S1) and cloned into the vector pET28a bearing a PreScission protease cleavage site (15) inserted downstream of the vector N-terminal His tag sequence. Expression plasmids for AviTag DENV3 or DENV4 RdRp proteins were co-transformed into BL21 cells with an expression plasmid for biotin protein ligase, BirA of E. coli, and proteins were purified as described previously (13).…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

A Conserved Pocket in the Dengue Virus Polymerase Identified through Fragment-based Screening

Noble

Lim

Arora

et al. 2016

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

We performed a fragment screen on the dengue virus serotype 3 RNA-dependent RNA polymerase using x-ray crystallography. A screen of 1,400 fragments in pools of eight identified a single hit that bound in a novel pocket in the protein. This pocket is located in the polymerase palm subdomain and conserved across the four serotypes of dengue virus. The compound binds to the polymerase in solution as evidenced by surface plasmon resonance and isothermal titration calorimetry analyses. Related compounds where a phenyl is replaced by a thiophene show higher affinity binding, indicating the potential for rational design. Importantly, inhibition of enzyme activity correlated with the binding affinity, showing that the pocket is functionally important for polymerase activity. This fragment is an excellent starting point for optimization through rational structurebased design.Dengue virus (DENV) 3 is the most widespread mosquitoborne viral infection. Disease symptoms of DENV-infected patients range from a mild fever to severe plasma leakage and hemorrhagic shock (1). There are four serotypes of DENV (DENV-1 to -4) that concurrently circulate around the world in tropical and subtropical regions. The vast majority of clinical cases are not reported, and there are estimated to be approximately 390 million human cases of dengue worldwide per year (2). There is currently no licensed vaccine or antiviral to treat DENV infection, underlining the urgency for the development of safe therapeutics (3). DENV is a member of the Flavivirus genus that also includes other viruses that are pathogenic to humans such as West Nile virus (WNV), yellow fever virus, Japanese encephalitis virus (JEV), and tick-borne encephalitis virus.One of the most attractive antiviral targets is the DENV RNA-dependent RNA polymerase (RdRp) because (i) viral polymerases are clinically proven therapeutic targets and (ii) the RdRp is the most conserved viral protein among the four serotypes of DENV so that the likelihood of a single compound with pan-serotype activity is higher than compounds targeting other viral proteins. The DENV RdRp activity resides in the C-terminal two-thirds of the viral nonstructural protein 5 (NS5) (reviewed in Ref. 4), whereas the N-terminal one-third of DENV NS5 encodes a methyltransferase (5).Crystal structures of the RdRp domain and full-length NS5 have been determined (6, 7). The overall architecture of the polymerase resembles a right hand with fingers, palm, and thumb subdomains. DENV RdRp catalyzes de novo initiation, as well as elongation. Like other RdRps that perform de novo RNA synthesis, DENV RdRp has a fully encircled active site (8). These polymerases undergo a conformational change from a closed to an open conformation during the transition from de novo initiation to elongation. During this process, the initiation loop (also known as the priming loop) is thought to move out of the active site, in a manner similar to the hepatitis C virus polymerase (9).A number of in vitro enzyme assays have been developed for DENV Rd...

show abstract

Section: Jf-31-mg46 Binds To Denv-3 and Denv-4 Rdrp By Spr-mentioning

confidence: 86%

Section: Methodsmentioning

confidence: 99%

A Conserved Pocket in the Dengue Virus Polymerase Identified through Fragment-based Screening

Noble

Lim

Arora

et al. 2016

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

show abstract

“…This de novo initiation assay (dnI) enabled us to perform a screening campaign with a more thermo-stable DENV4 RdRp domain and to develop a flow-chart for hit followup activities (Smith et al, 2014;Lim et al, 2013). In this report, we describe the detailed characterization of dengue polymerase dnI activity and the conditions that stabilized the protein.…”

Section: Introductionmentioning

confidence: 95%

Stabilization of dengue virus polymerase in de novo initiation assay provides advantages for compound screening

et al. 2015

Self Cite

View full text Add to dashboard Cite

“…Small-angle X-ray scattering (SAXS) studies suggested that NS5 could adopt different conformations permitted by a flexible linker (13). Interestingly, an Nterminally extended RdRp spanning residues from 265 to 900 improved thermostability and RdRp activity compared with a shorter RdRp spanning residues 273 to 900 (14). More recently, the structures of the full-length NS5 protein from DENV3 (15) and Japanese encephalitis virus (JEV) full-length NS5 (PDB code 4K6M) (16) have been resolved.…”

Section: N Onstructural Protein 5 (Ns5) Is a Multifunctional Protein mentioning

confidence: 99%

Flexibility of NS5 Methyltransferase-Polymerase Linker Region Is Essential for Dengue Virus Replication

Zhao

Soh

Chan

et al. 2015

J Virol

Self Cite

View full text Add to dashboard Cite

We examined the function of the conserved Val/Ile residue within the dengue virus NS5 interdomain linker (residues 263 to 272) by site-directed mutagenesis. Gly substitution or Gly/Pro insertion after the conserved residue increased the linker flexibility and created slightly attenuated viruses. In contrast, Pro substitution abolished virus replication by imposing rigidity in the linker and restricting NS5's conformational plasticity. Our biochemical and reverse genetics experiments demonstrate that NS5 utilizes conformational regulation to achieve optimum viral replication. N onstructural protein 5 (NS5) is a multifunctional protein essential for dengue virus (DENV) replication. NS5 consists of an N-terminal S-adenosyl-L-methionine (SAM)-dependent methyltransferase (MTase) domain (1) and a C-terminal RNAdependent RNA polymerase (RdRp) domain (2-5). The MTase domain catalyzes the formation of type I RNA cap, which is critical for efficient translation and subverting the host immune surveillance (1, 6-8). The MTase domain may also be responsible for guanylyltransferase activity (9, 10). The RdRp domain (NS5 RdRp) synthesizes both negative-strand RNA from the input genomic RNA (gRNA) and the progeny plus-strand gRNA (5, 11).Previous structural studies on DENV NS5 individual domains revealed electron density covering residues 6 to 262 (for the MTase domain) (PDB code 3P97) (12), and residues 273 to 882 (for RdRp) (PDB code 2J7U) (5). Limited proteolysis of full-length NS5 (NS5 FL) in combination with mass determination suggested that the residues 263 to 272 (5) form the interdomain linker, and sequence comparison across various flaviviruses showed that this region is poorly conserved (Fig. 1). Small-angle X-ray scattering (SAXS) studies suggested that NS5 could adopt different conformations permitted by a flexible linker (13). Interestingly, an Nterminally extended RdRp spanning residues from 265 to 900 improved thermostability and RdRp activity compared with a shorter RdRp spanning residues 273 to 900 (14). More recently, the structures of the full-length NS5 protein from DENV3 (15) and Japanese encephalitis virus (JEV) full-length NS5 (PDB code 4K6M) (16) have been resolved. Extensive comparisons between the two structures have been reported (15). Notably, unlike JEV NS5, the DENV NS5 structure revealed a hydrophilic interdomain interface and a linker region composed of a 3 10 -helix (amino acids [aa] 263 to 266) followed by a short loop (aa 267 to 272) ( Fig. 1A and B). In DENV3 full-length NS5, the 3 10 -helix linker adopts a compact conformation compared to JEV full-length NS5 or DENV2 MTase (1 to 296) (PDB code 1L9K) (Fig. 1C). Functionally, the MTase domain stimulates both de novo initiation and elongation activities of the RdRp domain, which implies a critical role for an interdomain linker in controlling NS5 RNA polymerization activities (17). Despite the various conformations adopted by NS5 in solution, position 264 (in DENV3 NS5) is found to be exclusively Val in flaviviruses, except for a homologous su...

show abstract

A Crystal Structure of the Dengue Virus Non-structural Protein 5 (NS5) Polymerase Delineates Interdomain Amino Acid Residues That Enhance Its Thermostability and de Novo Initiation Activities

Cited by 82 publications

References 27 publications

A Conserved Pocket in the Dengue Virus Polymerase Identified through Fragment-based Screening

A Conserved Pocket in the Dengue Virus Polymerase Identified through Fragment-based Screening

Stabilization of dengue virus polymerase in de novo initiation assay provides advantages for compound screening

Flexibility of NS5 Methyltransferase-Polymerase Linker Region Is Essential for Dengue Virus Replication

Contact Info

Product

Resources

About