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A conformation-based intra-molecular initiation factor identified in the flavivirus RNA-dependent RNA polymerase.

ABSTRACT: The flaviviruses pose serious threats to human health. Being a natural fusion of a methyltransferase (MTase) and an RNA-dependent RNA polymerase (RdRP), NS5 is the most conserved flavivirus protein and an important antiviral target. Previously reported NS5 structures represented by those from the Japanese encephalitis virus (JEV) and Dengue virus serotype 3 (DENV3) exhibit two apparently different global conformations, defining two sets of intra-molecular MTase-RdRP interactions. However, whether these NS5 conformations are conserved in flaviviruses and their specific functions remain elusive. Here we report two forms of DENV serotype 2 (DENV2) NS5 crystal structures representing two conformational states with defined analogies to the JEV-mode and DENV3-mode conformations, respectively, demonstrating the conservation of both conformation modes and providing clues for how different conformational states may be interconnected. Data from in vitro polymerase assays further demonstrate that perturbing the JEV-mode but not the DENV3-mode intra-molecular interactions inhibits catalysis only at initiation, while the cell-based virological analysis suggests that both modes of interactions are important for virus proliferation. Our work highlights the role of MTase as a unique intra-molecular initiation factor specifically only through the JEV-mode conformation, providing an example of conformation-based crosstalk between naturally fused protein functional modules.

SUBMITTER: Wu J

PROVIDER: S-EPMC7219791 | biostudies-literature | 2020 May

REPOSITORIES: biostudies-literature

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A conformation-based intra-molecular initiation factor identified in the flavivirus RNA-dependent RNA polymerase.

Wu Jiqin J Ye Han-Qing HQ Zhang Qiu-Yan QY Lu Guoliang G Zhang Bo B Gong Peng P

PLoS pathogens 20200501 5

The flaviviruses pose serious threats to human health. Being a natural fusion of a methyltransferase (MTase) and an RNA-dependent RNA polymerase (RdRP), NS5 is the most conserved flavivirus protein and an important antiviral target. Previously reported NS5 structures represented by those from the Japanese encephalitis virus (JEV) and Dengue virus serotype 3 (DENV3) exhibit two apparently different global conformations, defining two sets of intra-molecular MTase-RdRP interactions. However, whethe ...[more]

PMID: 32357182

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Project description:Encephalomyocarditis virus (EMCV) is a member of the Cardiovirus genus within the large Picornaviridae family, which includes a number of important human and animal pathogens. The RNA-dependent RNA polymerase (RdRp) 3Dpol is a key enzyme for viral genome replication. In this study, we report the X-ray structures of two different crystal forms of the EMCV RdRp determined at 2.8- and 2.15-? resolution. The in vitro elongation and VPg uridylylation activities of the purified enzyme have also been demonstrated. Although the overall structure of EMCV 3Dpol is shown to be similar to that of the known RdRps of other members of the Picornaviridae family, structural comparisons show a large reorganization of the active-site cavity in one of the crystal forms. The rearrangement affects mainly motif A, where the conserved residue Asp240, involved in ribonucleoside triphosphate (rNTP) selection, and its neighbor residue, Phe239, move about 10 ? from their expected positions within the ribose binding pocket toward the entrance of the rNTP tunnel. This altered conformation of motif A is stabilized by a cation-? interaction established between the aromatic ring of Phe239 and the side chain of Lys56 within the finger domain. Other contacts, involving Phe239 and different residues of motif F, are also observed. The movement of motif A is connected with important conformational changes in the finger region flanked by residues 54 to 63, harboring Lys56, and in the polymerase N terminus. The structures determined in this work provide essential information for studies on the cardiovirus RNA replication process and may have important implications for the development of new antivirals targeting the altered conformation of motif A.The Picornaviridae family is one of the largest virus families known, including many important human and animal pathogens. The RNA-dependent RNA polymerase (RdRp) 3Dpol is a key enzyme for picornavirus genome replication and a validated target for the development of antiviral therapies. Solving the X-ray structure of the first cardiovirus RdRp, EMCV 3Dpol, we captured an altered conformation of a conserved motif in the polymerase active site (motif A) containing the aspartic acid residue involved in rNTP selection and binding. This altered conformation of motif A, which interferes with the correct positioning of the rNTP substrate in the active site, is stabilized by a number of residues strictly conserved among picornaviruses. The rearrangements observed suggest that this motif A segment is a dynamic element that can be modulated by external effectors, either activating or inhibiting enzyme activity, and this type of modulation appears to be general to all picornaviruses.

Dataset Information

A conformation-based intra-molecular initiation factor identified in the flavivirus RNA-dependent RNA polymerase.

Publications

A conformation-based intra-molecular initiation factor identified in the flavivirus RNA-dependent RNA polymerase.

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