Towards the design of drugs against the dengue virus RNA dependent RNA polymerase : mechanistic studies of enzyme activities and substrate binding.

Dengue NS5 consists of 2 domains- methyltransferase and RNA-dependent RNA polymerase (RdRp), interspersed by a 9 amino acid linker. Currently, the full-length (FL) NS5 has not been structurally determined due to the flexibility of this linker, impeding drug discovery efforts. Here, FL NS5 with alanine mutation and RdRp domain with variable N-terminal extension (NE-RdRp) were expressed. The ability of these proteins to carry out de novo initiation (dnI) and elongation were then examined. Results showed decreased enzymatic activities for FL Glu271Ala, Pro273Ala and Lys272Ala proteins, while FL Thr270Ala, Ser265Ala and Glu269Ala proteins displayed increased dnI activity but no change in elongation activity. NE-RdRp domain proteins generally demonstrated increased activity compared to D274 NE-RdRp domain protein and more remarkably, greater enhancement for longer proteins. These combined findings suggest Glu271, Lys272 and Pro273 residues play a critical role in maintaining orientation between the two domains, while Glu269, Thr270 and Ser265 residues are important for local protein folding. Also, it indicates that the linker plays an important role in modulating polymerase activity. With these, a “lever” model was proposed for the linker region. Together, this pioneer study provides valuable insight on the biological function of the linker and information for rational drug design.Bachelor of Science in Biological Science

A novel crystal structure of the Dengue virus NS5 polymerase delineates inter-domain amino acids residues that enhance its thermostability and de novo initiation activities.

The dengue virus (DENV) NS5 protein comprises a N-terminal methyltransferase (MTase) and a C-terminal RNA-dependent RNA polymerase (RdRp) domain. Both enzymatic activities form attractive targets for antiviral development. Available crystal structures of NS5 fragments indicate that residues 263-271 (using the DENV serotype 3 numbering) located between the two globular domains of NS5 could be flexible. We observed that the addition of linker residues to the N-terminal end of the DENV RdRp core domain stabilize DENV1-4 proteins and improve their de novo polymerase initiation activities, by enhancing the turnover of the RNA and NTP substrates. Mutation studies of linker residues also indicate their importance for viral replication. We report the structure at 2.6 Å resolution of a RdRp fragment from DENV3, spanning residues 265-900 which has enhanced catalytic properties compared to the RdRp fragment (residues 272-900) reported previously. This new orthorhombic crystal form (space-group P21212,) comprises two polymerase molecules arranged as a dimer around a non-crystallographic dyad. The enzyme adopts a closed “pre-initiation” conformation similar to the one that was captured previously in space-group C2221 with one molecule per asymmetric unit. The structure reveals that residues 269-271 interact with the RdRp domain and suggests that residues 263-268 of the NS5 protein from DENV3 are the major contributors to the flexibility between its MTase and RdRp domains. Together, these results should inform the screening and development of antiviral inhibitors directed against the DENV RdRp

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

The dengue virus (DENV) non-structural protein 5 (NS5) comprises an N-terminal methyltransferase and a C-terminal RNA-dependent RNA polymerase (RdRp) domain. Both enzymatic activities form attractive targets for antiviral development. Available crystal structures of NS5 fragments indicate that residues 263–271 (using the DENV serotype 3 numbering) located between the two globular domains of NS5 could be flexible. We observed that the addition of linker residues to the N-terminal end of the DENV RdRp core domain stabilizes DENV1–4 proteins and improves their de novo polymerase initiation activities by enhancing the turnover of the RNA and NTP substrates. Mutation studies of linker residues also indicate their importance for viral replication. We report the structure at 2.6-Å resolution of an RdRp fragment from DENV3 spanning residues 265–900 that has enhanced catalytic properties compared with the RdRp fragment (residues 272–900) reported previously. This new orthorhombic crystal form (space group P2(1)2(1)2) comprises two polymerases molecules arranged as a dimer around a non-crystallographic dyad. The enzyme adopts a closed “preinitiation” conformation similar to the one that was captured previously in space group C222(1) with one molecule per asymmetric unit. The structure reveals that residues 269–271 interact with the RdRp domain and suggests that residues 263–268 of the NS5 protein from DENV3 are the major contributors to the flexibility between its methyltransferase and RdRp domains. Together, these results should inform the screening and development of antiviral inhibitors directed against the DENV RdRp