The F1 Motif of Dengue Virus Polymerase NS5 Is Involved in Promoter-Dependent RNA Synthesis

Abstract

The mechanism by which viral RNA-dependent RNA polymerases (RdRp) specifically amplify viral genomes is still unclear. In the case of flaviviruses, a model has been proposed that involves the recognition of an RNA element present at the viral 5 untranslated region, stem-loop A (SLA), that serves as a promoter for NS5 polymerase binding and activity. Here, we investigated requirements for specific promoter-dependent RNA synthesis of the dengue virus NS5 protein. Using mutated purified NS5 recombinant proteins and infectious viral RNAs, we analyzed the requirement of specific amino acids of the RdRp domain on polymerase activity and viral replication. A battery of 19 mutants was designed and analyzed. By measuring polymerase activity using nonspecific poly(rC) templates or specific viral RNA molecules, we identified four mutants with impaired polymerase activity. Viral full-length RNAs carrying these mutations were found to be unable to replicate in cell culture. Interestingly, one recombinant NS5 protein carrying the mutations K456A and K457A located in the F1 motif lacked RNA synthesis dependent on the SLA promoter but displayed high activity using a poly(rC) template. Promoter RNA binding of this NS5 mutant was unaffected while de novo RNA synthesis was abolished. Furthermore, the mutant maintained RNA elongation activity, indicating a role of the F1 region in promoter-dependent initiation. In addition, four NS5 mutants were selected to have polymerase activity in the recombinant protein but delayed or impaired virus replication when introduced into an infectious clone, suggesting a role of these amino acids in other functions of NS5. This work provides new molecular insights on the specific RNA synthesis activity of the dengue virus NS5 polymerase.Dengue virus (DENV) is the single most significant arthropod-borne virus pathogen in humans. It belongs to the Flaviviridae family together with other important pathogens such as yellow fever virus (YFV), West Nile virus (WNV), Saint Louis encephalitis virus (SLEV), and Japanese encephalitis virus (JEV). The World Health Organization continues reporting dengue outbreaks every year in the Americas and Asia. In spite of the urgent medical need to control DENV infections, vaccines and antivirals are still unavailable. Although a model for DENV RNA synthesis was previously proposed (17), molecular aspects of the mechanism by which the polymerase specifically amplifies the viral genome are still unclear for DENV and other flaviviruses. To further understand this viral process, we investigated functional properties of the viral polymerase NS5.NS5 is the largest of the flavivirus proteins (105 kDa); it contains an N-terminal methyltransferase domain (MTase) and a C-terminal RNA-dependent RNA polymerase (RdRp) domain. The MTase is responsible for methylation of the cap structure present at the 5Ј end of the viral genome. This process involves methylation in two positions, guanine N-7 and ribose 2Ј-O (12, 13, 28, 38). The RdRp domain has primer independent (de novo) RNA synth...

Nuclear Localization of Dengue Virus Nonstructural Protein 5 Does Not Strictly Correlate with Efficient Viral RNA Replication and Inhibition of Type I Interferon Signaling

“…Such a crucial function would be underscored by the fact that K402 is strictly conserved across the Flavivirus genus (18). Moreover, our results obtained with mutant 401-402 are consistent with a previous study (39) showing that mutations of the corresponding NS5 residues (RK 400-401) of DENV-2 strain 16681 abrogate RdRp activity.…”

Uncoupling cis -Acting RNA Elements from Coding Sequences Revealed a Requirement of the N-Terminal Region of Dengue Virus Capsid Protein in Virus Particle Formation

“…In recent years, a number of cis-acting RNA elements that modulate viral RNA amplification have been identified in the viral genome (for a review, see reference 38). Viral RNA synthesis is catalyzed by the RNA-dependent RNA-polymerase activity of the viral protein NS5 and requires interaction with a 5=-end promoter element (9,10,13). During flavivirus morphogenesis, the newly synthesized viral RNA is recruited by the capsid protein (C).…”

Residues Arg283, Arg285, and Ile287 in the Nucleotide Binding Pocket of Bovine Viral Diarrhea Virus NS5B RNA Polymerase Affect Catalysis and Fidelity

“…We used sitedirected mutagenesis and kinetic assays to examine more closely the functional role of motif F residues in BVDV NS5B with respect to the formation of the preinitiation complex and the remainder of the RdRp catalytic cycle. We also compared and contrasted these data with similar studies carried out with DFV and FMDV RdRps (35). R283, R285, and I287, which are located above the rNTP binding pocket in the BVDV NS5B fingertips (Fig.…”

“…Similar results have been observed with Ala substitutions at the equivalent positions of DFV NS5. For example, the motif F mutants M10 (K456/R457 ¡ A) and M11 (K456/ R457/K459/K460 ¡ A) displayed Ͻ10% of wild-type DFV NS5 activity on the heteropolymeric 5= DFV template RNA (35). As a hydrophobic residue, L461 (BVDV I287) (Fig.…”

“…As a hydrophobic residue, L461 (BVDV I287) (Fig. 1) was not considered in this otherwise wide-ranging and complete study of motif F residues in DFV NS5 (35).…”