Structural basis of viral RNA-dependent RNA polymerase catalysis and translocation

Abstract

Viral RNA-dependent RNA polymerases (RdRPs) play essential roles in viral genome replication and transcription. We previously reported several structural states of the poliovirus RdRP nucleotide addition cycle (NAC) that revealed a unique palm domain-based active site closure mechanism and proposed a six-state NAC model including a hypothetical state representing translocation intermediates. Using the RdRP from another human enterovirus, enterovirus 71, here we report seven RdRP elongation complex structures derived from a crystal lattice that allows three NAC events. These structures suggested a key order of events in initial NTP binding and NTPinduced active site closure and revealed a bona fide translocation intermediate featuring asymmetric movement of the templateproduct duplex. Our work provides essential missing links in understanding NTP recognition and translocation mechanisms in viral RdRPs and emphasizes the uniqueness of the viral RdRPs compared with other processive polymerases.RNA-dependent RNA polymerase | nucleotide addition cycle | translocation intermediate | enterovirus 71 | crystal structure I n recent years, several notable emerging infectious diseases have been caused by RNA viruses, including highly pathogenic avian influenza viruses, Ebola virus, and Middle East respiratory syndrome coronavirus. RNA viruses are quite diverse in virus particle and genome structure and in virus entry and assembly mechanisms. However, they do share fundamental features in their genome replication and transcription, using a virally encoded RNAdependent RNA polymerase (RdRP) to carry out the biosynthesis of an RNA product directed by an RNA template. Although the genome replication machinery often requires the participation of other factors, typically at the initiation phase of synthesis, the RdRP governs the elongation phase of synthesis that includes thousands of efficient nucleotide addition cycles (NACs). Viral RdRPs vary greatly in size and structural organization, from the ∼50-kDa picornavirus 3D pol (1, 2), to the ∼100-kDa flavivirus NS5 that contains a naturally fused methyltransferase domain (3), to the ∼250-kDa nonsegmented negative-strand RNA virus L protein harboring at least three enzyme modules (4) and the ∼260-kDa three-subunit PA-PB1-PB2 influenza virus replicase complex (5). On the other hand, all RdRPs share a 50-to 70-kDa polymerase core that forms a unique encircled right-hand structure with palm, fingers, and thumb domains. Among the seven classic RdRP catalytic motifs, A-E are within the most conserved palm domain, and F and G are located in the fingers; they are all arranged similarly around the active site (6-9). The structural conservation of the RdRP polymerase core and the seven motifs form the basis for understanding the common features in viral RdRP catalytic mechanism and for finding intervention strategies targeting these enzymes with possible broad-spectrum potential.As with other classes of nucleic acid polymerases, the viral RdRP elongation NAC comprises sequential steps of in...

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

“…4C and D). These data, in general, suggest that both SUMOylation sites in 3D may modulate EV71 polymerase activity, despite their differences in structural properties based on available crystallographic data (56,64).…”

“…The plasmids for the expression of WT 3D and its variants were transformed into Escherichia coli strain BL21(DE3)(pCG1) (kindly supplied by Craig Cameron) for expression (54). 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).…”

“…Of the two SUMOylation sites in 3D, K159 is a highly conserved active-site residue in RNA-dependent RNA polymerase (RdRp) motif F and within hydrogen-bonding distance from the edge of the nascent base pair of the RdRp elongation complex, while the 150-to-152 site is on the surface of 3D and does not directly participate in RNA binding, NTP entry, and catalysis ( Fig. 4B) (56). 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.…”

Triphosphate Reorientation of the Incoming Nucleotide as a Fidelity Checkpoint in Viral RNA-dependent RNA Polymerases

“…2). Crystal structures of nucleic acid polymerase superfamily members have indicated that there is a reorientation of the triphosphate of the incoming nucleotide following initial binding to properly align the ␣-phosphate for nucleophilic attack by the primer 3Ј-OH (10,48) (Fig. 12).…”

“…There is then a re-alignment of the NTP triphosphate and conserved residue Asp-233 around the two metal ions (10). Residues in motif F, including Lys-167 and Arg-174, are also involved in binding the triphosphate.…”