Perturbation in the Conserved Methyltransferase-Polymerase Interface of Flavivirus NS5 Differentially Affects Polymerase Initiation and Elongation

Abstract

The flavivirus NS5 is a natural fusion of a methyltransferase (MTase) and an RNA-dependent RNA polymerase (RdRP). Analogous to DNA-dependent RNA polymerases, the NS5 polymerase initiates RNA synthesis through a de novo mechanism and then makes a transition to a processive elongation phase. However, whether and how the MTase affects polymerase activities through intramolecular interactions remain elusive. By solving the crystal structure of the Japanese encephalitis virus (JEV) NS5, we recently identified an MTase-RdRP interface containing a set of six hydrophobic residues highly conserved among flaviviruses. To dissect the functional relevance of this interface, we made a series of JEV NS5 constructs with mutations of these hydrophobic residues and/or with the N-terminal first 261 residues and other residues up to the first 303 residues deleted. Compared to the wild-type (WT) NS5, full-length NS5 variants exhibited consistent up-or downregulation of the initiation activities in two types of polymerase assays. Five representative full-length NS5 constructs were then tested in an elongation assay, from which the apparent single-nucleotide incorporation rate constant was estimated. Interestingly, two constructs exhibited different elongation kinetics from the WT NS5, with an effect rather opposite to what was observed at initiation. Moreover, constructs with MTase and/or the linker region (residues 266 to 275) removed still retained polymerase activities, albeit at overall lower levels. However, further removal of the N-terminal extension (residues 276 to 303) abolished regular template-directed synthesis. Together, our data showed that the MTase-RdRP interface is relevant in both polymerase initiation and elongation, likely with different regulation mechanisms in these two major phases of RNA synthesis. IMPORTANCEThe flavivirus NS5 is very unique in having a methyltransferase (MTase) placed on the immediate N terminus of its RNA-dependent RNA polymerase (RdRP). We recently solved the crystal structure of the full-length NS5, which revealed a conserved interface between MTase and RdRP. Building on this discovery, here we carried out in vitro polymerase assays to address the functional relevance of the interface interactions. By explicitly probing polymerase initiation and elongation activities, we found that perturbation in the MTase-RdRP interface had different impacts on different phases of synthesis, suggesting that the roles and contribution of the interface interactions may change upon phase transitions. By comparing the N-terminal-truncated enzymes with the full-length NS5, we collected data to indicate the indispensability to regular polymerase activities of a region that was functionally unclarified previously. Taken together, we provide biochemical evidence and mechanistic insights for the cross talk between the two enzyme modules of flavivirus NS5. The Flavivirus genus of family Flaviviridae includes more than 70 viruses which often cause human encephalitis and hemorrhagic diseases. The majority o...

A nucleobase-binding pocket in a viral RNA-dependent RNA polymerase contributes to elongation complex stability

“…Examples of the first type include the regulation of flavivirus NS5 by viral protein NS3 and viral RNA elements within the 5 -untranslated region (UTR) (14)(15)(16), picornavirus 3D pol by viral protein 3CD and host SUMOylation (SUMO stands for small ubiquitinlike modifier) machineries (17,18), and coronavirus nsp12 by nsp7 and nsp8 (19). Representative cases of the second type have been identified in the Flaviviridae RdRPs, with the methyltransferase (MTase, part of the flavivirus NS5) and the N-terminal domain (NTD, part of the pestivirus NS5B) regulating the corresponding polymerases, respectively, through intra-molecular interactions (20)(21)(22)(23)(24)(25).…”

SUMO Modification Stabilizes Enterovirus 71 Polymerase 3D To Facilitate Viral Replication

“…With an aim of assessing the relevance of both SUMOylation sites to the polymerase function, we conducted primer-dependent polymerase assays (58,64) for WT 3D and the K159R, K159A, and L150A/D151A mutants. With the T33/P10 RNA construct and GTP/ATP as the only substrates, the 10-nucleotide primer (P10) was expected to be elongated by 4 nucleotides, producing a 14-nucleotide product (58). For the WT enzyme, the majority of the primer was converted to the product within a 60-min incubation time (Fig.…”

“…The bacteria were cultured and purified as described previously, except that the induction condition before harvesting was 11 h at 25°C (56). The preparation of the 33-nucleotide RNA template (T33) was as described previously (57,58). The 10-nucleotide RNA primer (P10) was purchased from Integrated DNA Technologies.…”

“…Color images obtained by scanning the stained gels were converted to grayscale images prior to quantification by Image J (http://imageJ.nih.gov/ij/). The Stains-All-based quantification method was used previously to assess the amounts of RNAs with similar lengths (58).…”

Uncoupling of Protease trans -Cleavage and Helicase Activities in Pestivirus NS3

“…Protein purification. Purification of NS3 S163A /NS4A PCS and its variants and NS3Hel was performed according to methods for purifying flavivirus replication proteins (64,65). The cells were resuspended in lysis buffer (150 mM Na 2 SO 4 , 50 mM Tris [pH 8.0], 10 mM imidazole, 0.02% [wt/vol] NaN 3 , 20% [vol/vol] glycerol) and were lysed by passage through an AH-2010 homogenizer (ATS Engineering Ltd.) at 14,500 lb/in 2 .…”