Structural & functional characterization of the dengue virus non-structural protein 5 (NS5)

Dengue virus (DENV) is the most important arthropod-borne pathogens capable of causing human mortality and morbidity. Currently, there are no antiviral drugs available for treatment of dengue infections. Although a tetravalent DENV vaccine has recently been licensed for use, it has limited efficacy. For DENV, NS5 is the best characterized and most conserved multi-functional protein comprising an N-terminal methyltransferase (MTase) and a C-terminal RNA-dependent RNA polymerase (RdRp). Both play essential roles in viral replication in the host cell. The crystal structure of the DENV full-length NS5 revealed a well-ordered linker region and an inter-domain interface mostly formed by polar residues. Using a combination of biochemical and reverse genetic approaches, the biological relevance of the flexible linker between MTase and RdRp in the DENV-3 NS5 FL and their intra-molecular interactions was investigated. Several conserved interface residues were shown to be important for viral replication, through influencing either MTase or RdRp activities. Other NS5 alanine mutants displayed comparable enzymatic activities as wild-type, but were either less competent or lethal for virus production, suggesting that they play vital but non-enzymatic roles in viral replication and infectivity. Alanine mutations of the linker region showed that the third and fourth residues of the short 310-helix regulate polymerase de novo initiation activity for viral replication in cells. In addition, linker swapping experiment demonstrated that the unique amino acid composition of the linker controls NS5 conformation flexibility for cross-talk between the two domains and for interaction with viral and host proteins in a serotype/virus-specific manner. By solving crystal structures of ternary complexes between DENV-3 NS5 protein, an authentic cap-0-viral RNA substrate, S-adenosyl-L-homocysteine (SAH) and/or RdRp allosteric inhibitors, we functionally probed these inhibitor and substrate binding sites in the RdRp and MTase with biochemical, biophysical and reverse genetic tools. Based on the catalytically-competent NS5-SAH-cap-0-viral RNA methylation complex, mutagenesis studies targeting the highly conserved capped-RNA binding groove in the MTase domain was performed. The importance of the polar interaction between NS5 residue E111 and G2 base of RNA for viral replication as well as the positional requirement G2 for virus growth were identified. Moreover, residues lining the RNA binding groove exhibited differential reduction in 2’-O methylation activity, indicating that these residues are critical for capped-RNA binding and 2’-O methyl transfer reaction. Using compound and fragment-based screening coupled with structure-guided design, we identified two classes of allosteric inhibitors that bound either to the F1 motif, or to the thumb subdomain and priming loop (termed “N-pocket”) of the DENV RdRp. Antiviral activities of F1 motif and N-pocket inhibitors were primarily due to an impact on polymerase de novo initiation activity rather than elongation during RNA synthesis. Additionally, kinetic characterization showed that the N-pocket inhibitors exhibited mixed inhibition profiles when compete against the RNA or GTP substrate. Resistant mutants raised from these inhibitors were also mapped to the N-pocket of RdRp, confirming that they bind specifically to this pocket to block viral replication. The proposed mode of action for N-pocket compounds is to prevent NS5 RdRp de novo initiation and block conformation changes during transition from initiation to elongation. In order to examine how the viral RNA is recognized and replicated as well as to facilitate drug discovery and design targeting the RdRp, we attempted to obtain crystal structure of NS5 RdRp bound to RNA. A novel fluorescence polarization (FP)-based assay was developed to profile various distinct RNA constructs for their suitability in co-crystallization. Several RNA substrates demonstrated good binding affinity to NS5 protein and were capable of forming functional elongation complexes. Crystallization trials using commercial screening kits were set up, but no crystal structure with bound RNA was obtained. Future work will aim at optimizing the conditions during assembly and reaction in order to attain more soluble and stable elongation complexes for crystallization. Overall, these findings provide valuable information on the functions and dynamics of NS5 as well as its molecular interactions with substrates and inhibitors, and have significant implications for the development of antiviral drugs targeting flaviviruses.​Doctor of Philosophy (SBS

NS5 from dengue virus serotype 2 can adopt a conformation analogous to that of its Zika virus and Japanese encephalitis virus homologues

Flavivirus nonstructural protein 5 (NS5) contains an N-terminal methyltransferase (MTase) domain and a C-terminal polymerase (RNA-dependent RNA polymerase [RdRp]) domain fused through a 9-amino-acid linker. While the individual NS5 domains are structurally conserved, in the full-length protein, their relative orientations fall into two classes: the NS5 proteins from Japanese encephalitis virus (JEV) and Zika virus (ZIKV) adopt one conformation, while the NS5 protein from dengue virus serotype 3 (DENV3) adopts another. Here, we report a crystallographic structure of NS5 from DENV2 in a conformation similar to the extended one seen in JEV and ZIKV NS5 crystal structures. Replacement of the DENV2 NS5 linker with DENV1, DENV3, DENV4, JEV, and ZIKV NS5 linkers had modest or minimal effects on in vitro DENV2 MTase and RdRp activities. Heterotypic DENV NS5 linkers attenuated DENV2 replicon growth in cells, while the JEV and ZIKV NS5 linkers abolished replication. Thus, the JEV and ZIKV linkers likely hindered essential DENV2 NS5 interactions with other viral or host proteins within the virus replicative complex. Overall, this work sheds light on the dynamics of the multifunctional flavivirus NS5 protein and its interdomain linker. Targeting the NS5 linker is a possible strategy for producing attenuated flavivirus strains for vaccine design.NRF (Natl Research Foundation, S’pore)Published versio

Molecular basis for specific viral RNA recognition and 2’-O ribose methylation by the dengue virus NS5 protein.

Dengue virus (DENV) causes several hundred million human infections and more than 20,000 deaths annually. Neither an efficacious vaccine conferring immunity against all four circulating serotypes nor specific drugs are currently available to treat this emerging global disease. Capping of the DENV RNA genome is an essential structural modification that protects the RNA from degradation by 5’ exo-ribonucleases, ensures efficient expression of viral proteins and allows escape from the host innate immune response. The large flavivirus NS5 protein (105 kDa) has RNA methyl-transferase activities at its N terminal region, which is responsible for capping the virus RNA genome. The methyl transfer reactions are thought to occur sequentially using the strictly conserved flavivirus 5’ RNA sequence as substrate (GpppAG-RNA), leading to the formation of the 5’ RNA cap: G0pppAGRNA →m7G0pppAG-RNA (named “cap-0”) →m7G0pppAm2’-OG-RNA (named “cap-1”). To elucidate how viral RNA is specifically recognized and methylated, we determined the crystal structure of a ternary complex between the full-length NS5 protein from dengue virus, an octameric cap-0 viral RNA substrate bearing the authentic DENV genomic sequence (5’- m7G0pppA1G2U3U4G5U6U7-3’) and S-adenosyl-L-homocysteine (SAH), the by-product of the methylation reaction. The structure provides for the first time a molecular basis for specific adenosine 2’-O methylation, rationalizes mutagenesis studies targeting the K61-D146-K180- E216 enzymatic tetrad as well as residues lining the RNA binding groove and offers novel mechanistic and evolutionary insights into cap-1 formation by NS5, which underlies innate immunity evasion by flaviviruses

Molecular basis for specific viral RNA recognition and 2' -O-ribose methylation by the dengue virus nonstructural protein 5 (NS5)

Dengue virus (DENV) causes several hundred million human infections and more than 20,000 deaths annually. Neither an efficacious vaccine conferring immunity against all four circulating serotypes nor specific drugs are currently available to treat this emerging global disease. Capping of the DENV RNA genome is an essential structural modification that protects the RNA from degradation by 5′ exoribonucleases, ensures efficient expression of viral proteins, and allows escape from the host innate immune response. The large flavivirus nonstructural protein 5 (NS5) (105 kDa) has RNA methyltransferase activities at its N-terminal region, which is responsible for capping the virus RNA genome. The methyl transfer reactions are thought to occur sequentially using the strictly conserved flavivirus 5′ RNA sequence as substrate (GpppAG-RNA), leading to the formation of the 5′ RNA cap: G0pppAG-RNA→m7G0pppAG-RNA (“cap-0”)→m7G0pppAm2′-O-G-RNA (“cap-1”). To elucidate how viral RNA is specifically recognized and methylated, we determined the crystal structure of a ternary complex between the full-length NS5 protein from dengue virus, an octameric cap-0 viral RNA substrate bearing the authentic DENV genomic sequence (5′-m7G0pppA1G2U3U4G5U6U7-3′), and S-adenosyl-l-homocysteine (SAH), the by-product of the methylation reaction. The structure provides for the first time, to our knowledge, a molecular basis for specific adenosine 2′-O-methylation, rationalizes mutagenesis studies targeting the K61-D146-K180-E216 enzymatic tetrad as well as residues lining the RNA binding groove, and offers previously unidentified mechanistic and evolutionary insights into cap-1 formation by NS5, which underlies innate immunity evasion by flaviviruses.NMRC (Natl Medical Research Council, S’pore)Accepted Versio

Molecular basis for specific viral RNA recognition and 2′-O-ribose methylation by the dengue virus nonstructural protein 5 (NS5)

Dengue virus (DENV) causes several hundred million human infections and more than 20,000 deaths annually. Neither an efficacious vaccine conferring immunity against all four circulating serotypes nor specific drugs are currently available to treat this emerging global disease. Capping of the DENV RNA genome is an essential structural modification that protects the RNA from degradation by 5′ exoribonucleases, ensures efficient expression of viral proteins, and allows escape from the host innate immune response. The large flavivirus nonstructural protein 5 (NS5) (105 kDa) has RNA methyltransferase activities at its N-terminal region, which is responsible for capping the virus RNA genome. The methyl transfer reactions are thought to occur sequentially using the strictly conserved flavivirus 5′ RNA sequence as substrate (G(ppp)AG-RNA), leading to the formation of the 5′ RNA cap: G(0ppp)AG-RNA→(m7)G(0ppp)AG-RNA (“cap-0”)→(m7)G(0ppp)A(m2′-O-)G-RNA (“cap-1”). To elucidate how viral RNA is specifically recognized and methylated, we determined the crystal structure of a ternary complex between the full-length NS5 protein from dengue virus, an octameric cap-0 viral RNA substrate bearing the authentic DENV genomic sequence (5′-(m7)G(0ppp)A(1)G(2)U(3)U(4)G(5)U(6)U(7)-3′), and S-adenosyl-l-homocysteine (SAH), the by-product of the methylation reaction. The structure provides for the first time, to our knowledge, a molecular basis for specific adenosine 2′-O-methylation, rationalizes mutagenesis studies targeting the K61-D146-K180-E216 enzymatic tetrad as well as residues lining the RNA binding groove, and offers previously unidentified mechanistic and evolutionary insights into cap-1 formation by NS5, which underlies innate immunity evasion by flaviviruses

A crystal structure of the dengue virus NS5 protein reveals a novel inter-domain interface essential for protein flexibility and virus replication

Flavivirus RNA replication occurs within a replication complex (RC) that assembles on ER membranes and comprises both non-structural (NS) viral proteins and host cofactors. As the largest protein component within flavivirus RC, NS5 plays key enzymatic roles through its N-terminal methyltransferase (MTase) and C-terminal RNA-dependent-RNA polymerase (RdRp) domains and constitutes a major target for antivirals. We determined a crystal structure of the full-length NS5 protein (NS5FL) from Dengue virus serotype 3 (DENV3) at a resolution of 2.3 Å in the presence of bound SAH and GTP. Although the overall molecular shape of NS5FL from DENV3 resembles that of NS5FL from Japanese Encephalitis Virus (JEV), the relative orientation between the MTase and RdRp domains differs between the two structures, providing direct evidence for the existence of a set of discrete stable molecular conformations. While the inter-domain region is mostly disordered in NS5FL from JEV, the NS5FL structure from DENV3 reveals a well-ordered linker region comprising a short 310 helix that is likely to act as a conformational switch. Solution Hydrogen/Deuterium Exchange Mass Spectrometry (HDX-MS) analysis showed that the thumb subdomain of RdRp is more dynamic in NS5FL compared to NS5-RdRp, suggesting that the MTase domain regulates the structural dynamics of the RdRp domain. Site-directed mutagenesis targeting the mostly polar interface between the MTase and RdRp domains identified several evolutionarily conserved residues that are important for viral replication in a sub-genomic replicon, suggesting an inter-domain cross-talk. A picture for the molecular origin of NS5 flexibility now emerges which has profound implications for flavivirus replication and for the developing therapeutics targeting NS5

Flexibility of NS5 methyltransferase-polymerase linker region is essential for dengue virus replication

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

Potent allosteric dengue virus NS5 polymerase inhibitors: mechanism of action and resistance profiling.

Flaviviruses comprise major emerging pathogens such as dengue virus (DENV) or Zika virus (ZIKV). The flavivirus RNA genome is replicated by the RNA-dependent-RNA polymerase (RdRp) domain of non-structural protein 5 (NS5). This essential enzymatic activity renders the RdRp attractive for antiviral therapy. NS5 synthesizes viral RNA via a “de novo” initiation mechanism. Crystal structures of the flavivirus RdRp revealed a “closed” conformation reminiscent of a pre-initiation state, with a well ordered priming loop that extrudes from the thumb subdomain into the dsRNA exit tunnel, close to the “GDD” active site. To-date, no allosteric pockets have been identified for the RdRp, and compound screening campaigns did not yield suitable drug candidates. Using fragment-based screening via X-ray crystallography, we found a fragment that bound to a pocket of the apo-DENV RdRp close to its active site (termed “N pocket”). Structure-guided improvements yielded DENV pan-serotype inhibitors of the RdRp de novo initiation activity with nano-molar potency that also impeded elongation activity at micro-molar concentrations. Inhibitors exhibited mixed inhibition kinetics with respect to competition with the RNA or GTP substrate. The best compounds have EC50 values of 1-2 M against all four DENV serotypes in cell culture assays. Genome-sequencing of compound-resistant DENV, identified amino acid changes that mapped to the N pocket. Since inhibitors bind at the thumb/palm interface of the RdRp, this class of compounds is proposed to hinder RdRp conformational changes during its transition from initiation to elongation. This is the first report of a class of pan-serotype and cell-active DENV RdRp inhibitors. Given the evolutionary conservation of residues lining the N pocket, these molecules offer insights to treat other serious conditions caused by flaviviruses

A Crystal Structure of the Dengue Virus NS5 Protein Reveals a Novel Inter-domain Interface Essential for Protein Flexibility and Virus Replication

International audienceFlavivirus RNA replication occurs within a replication complex (RC) that assembles on ER membranes and comprises both non-structural (NS) viral proteins and host cofactors. As the largest protein component within the flavivirus RC, NS5 plays key enzymatic roles through its N-terminal methyltransferase (MTase) and C-terminal RNA-dependent-RNA polymerase (RdRp) domains, and constitutes a major target for antivirals. We determined a crystal structure of the full-length NS5 protein from Dengue virus serotype 3 (DENV3) at a resolution of 2.3 Å in the presence of bound SAH and GTP. Although the overall molecular shape of NS5 from DENV3 resembles that of NS5 from Japanese Encephalitis Virus (JEV), the relative orientation between the MTase and RdRp domains differs between the two structures, providing direct evidence for the existence of a set of discrete stable molecular conformations that may be required for its function. While the inter-domain region is mostly disordered in NS5 from JEV, the NS5 structure from DENV3 reveals a well-ordered linker region comprising a short 310 helix that may act as a swivel. Solution Hydrogen/Deuterium Exchange Mass Spectrometry (HDX-MS) analysis reveals an increased mobility of the thumb subdomain of RdRp in the context of the full length NS5 protein which correlates well with the analysis of the crystallographic temperature factors. Site-directed mutagenesis targeting the mostly polar interface between the MTase and RdRp domains identified several evolutionarily conserved residues that are important for viral replication, suggesting that inter-domain cross-talk in NS5 regulates virus replication. Collectively, a picture for the molecular origin of NS5 flexibility is emerging with profound implications for flavivirus replication and for the development of therapeutics targeting NS5

Potent Allosteric Dengue Virus NS5 Polymerase Inhibitors: Mechanism of Action and Resistance Profiling

International audienceFlaviviruses comprise major emerging pathogens such as dengue virus (DENV) or Zika virus (ZIKV). The flavivirus RNA genome is replicated by the RNA-dependent-RNA poly-merase (RdRp) domain of non-structural protein 5 (NS5). This essential enzymatic activity renders the RdRp attractive for antiviral therapy. NS5 synthesizes viral RNA via a " de novo " initiation mechanism. Crystal structures of the flavivirus RdRp revealed a " closed " confor-mation reminiscent of a pre-initiation state, with a well ordered priming loop that extrudes from the thumb subdomain into the dsRNA exit tunnel, close to the " GDD " active site. To-date, no allosteric pockets have been identified for the RdRp, and compound screening campaigns did not yield suitable drug candidates. Using fragment-based screening via X-ray crystallography, we found a fragment that bound to a pocket of the apo-DENV RdRp close to its active site (termed " N pocket "). Structure-guided improvements yielded DENV pan-serotype inhibitors of the RdRp de novo initiation activity with nano-molar potency that also impeded elongation activity at micro-molar concentrations. Inhibitors exhibited mixed inhibition kinetics with respect to competition with the RNA or GTP substrate. The best compounds have EC 50 values of 1–2 μM against all four DENV serotypes in cell culture assays. Genome-sequencing of compound-resistant DENV replicons, identified amino acid changes that mapped to the N pocket. Since inhibitors bind at the thumb/palm interface of the RdRp, this class of compounds is proposed to hinder RdRp conformational changes during its transition from initiation to elongation. This is the first report of a class of pan-sero-type and cell-active DENV RdRp inhibitors. Given the evolutionary conservation of residues lining the N pocket, these molecules offer insights to treat other serious conditions caused by flaviviruses