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Structural Basis of Viral RNA-Dependent RNA Polymerase

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Structural Basis of Viral RNA-Dependent RNA Polymerase Structural basis of viral RNA-dependent RNA PNAS PLUS polymerase catalysis and translocation Bo Shua,b and Peng Gonga,1 aKey Laboratory of Special Pathogens and Biosafety, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, Hubei 430071, China; and bUniversity of Chinese Academy of Sciences, Beijing 100049, China Edited by Thomas A. Steitz, Howard Hughes Medical Institute, Yale University, New Haven, CT, and approved May 19, 2016 (received for review February 15, 2016) Viral RNA-dependent RNA polymerases (RdRPs) play essential roles NAC model featuring six reference states (10). The model starts with in viral genome replication and transcription. We previously reported astate1(S1) complex with a vacant active site that is in the catalyt- several structural states of the poliovirus RdRP nucleotide addition ically open conformation and upon NTP binding proceeds to a state 2 cycle (NAC) that revealed a unique palm domain-based active site (S2) complex with the active site still in the open conformation. An closure mechanism and proposed a six-state NAC model including a important conformational change then takes place to position key hypothetical state representing translocation intermediates. Using catalytic residues and two magnesium ions around the priming nu- the RdRP from another human enterovirus, enterovirus 71, here cleotide and the substrate NTP to achieve proper geometry of a we report seven RdRP elongation complex structures derived from closed active site for catalysis, yielding state 3 (S3) immediately before a crystal lattice that allows three NAC events. These structures and state 4 (S4) immediately after the phosphoryl transfer reaction. suggested a key order of events in initial NTP binding and NTP- As the catalytic geometry starts to disintegrate, the structural changes induced active site closure and revealed a bona fide translocation in the palm domain result in state 5 (S5) with an open conformation – intermediate featuring asymmetric movement of the template active site. State 6 (S6) is then a hypothetical translocation in- product duplex. Our work provides essential missing links in under- termediate state that bridges the pretranslocation S5 and the post- standing NTP recognition and translocation mechanisms in viral RdRPs translocation S1 in the next NAC. and emphasizes the uniqueness of the viral RdRPs compared with The six-state model has provided a framework for understanding other processive polymerases. the molecular details and the unique features of the viral RdRP elongation NAC. In the active site closure step, the notable RNA-dependent RNA polymerase | nucleotide addition cycle | translocation backbone conformational changes are limited to motifs A and D intermediate | enterovirus 71 | crystal structure in the palm domain. These conformational changes are in drastic contrast to those taken by the well-characterized A-family poly- – n recent years, several notable emerging infectious diseases have merases that use a large-scale rotational movement of the O-helix Ibeen caused by RNA viruses, including highly pathogenic avian containing fingers domain to achieve the same closure step (11, 12). influenza viruses, Ebola virus, and Middle East respiratory syn- One implication behind this apparent difference in the active site drome coronavirus. RNA viruses are quite diverse in virus particle closure mode is that the mechanisms by which the polymerase se- BIOCHEMISTRY and genome structure and in virus entry and assembly mechanisms. lects the correct NTP substrate and induces active site reorganiza- However, they do share fundamental features in their genome tion for catalysis are also quite different. In A-family polymerases, replication and transcription, using a virally encoded RNA- the translocation step is coupled to the postcatalysis reopen- ing of the active site when a conserved tyrosine residue in the dependent RNA polymerase (RdRP) to carry out the biosynthesis of “ ” an RNA product directed by an RNA template. Although the ge- O-helix pushes the nascent base pair upstream in a motion that is the reverse of that observed during the active site closure nome replication machinery often requires the participation of other (11, 13). However, no intermediate structure between the pre- 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 Significance and structural organization, from the ∼50-kDa picornavirus 3Dpol (1, 2), to the ∼100-kDa flavivirus NS5 that contains a naturally fused RNA viruses encode a unique class of RNA-dependent RNA methyltransferase domain (3), to the ∼250-kDa nonsegmented neg- polymerases (RdRPs) to carry out their fully RNA-based genome ative-strand RNA virus L protein harboring at least three enzyme replication and transcription. Although the chemical nature of modules (4) and the ∼260-kDa three-subunit PA-PB1-PB2 influenza nucleotide addition is essentially shared by all nucleic acid poly- virus replicase complex (5). On the other hand, all RdRPs share a 50- merases, the structural and mechanistic details taken by each to 70-kDa polymerase core that forms a unique encircled right-hand polymerase class differ to various extents. Here we report seven structure with palm, fingers, and thumb domains. Among the seven crystal structures of enterovirus 71 RdRP elongation complex at – classic RdRP catalytic motifs, A–E are within the most conserved 2.5 2.8 Å resolution. In these structures the polymerases are palm domain, and F and G are located in the fingers; they are all poised at various distinct stages to reveal mechanistic details of arranged similarly around the active site (6–9). The structural con- initial NTP binding, key amino acid side-chain conformational servation of the RdRP polymerase core and the seven motifs form the switches during active site closure, and in particular the post- catalysis movement of the RNA duplex on the way to vacate the basis for understanding the common features in viral RdRP catalytic active site for the next nucleotide addition cycle. mechanism and for finding intervention strategies targeting these enzymes with possible broad-spectrum potential. Author contributions: B.S. and P.G. designed research; B.S. performed research; B.S. and As with other classes of nucleic acid polymerases, the viral RdRP P.G. analyzed data; and B.S. and P.G. wrote the paper. elongation NAC comprises sequential steps of initial NTP binding, The authors declare no conflict of interest. active site closure, catalysis, and translocation. In a recent study using This article is a PNAS Direct Submission. CTP and deoxy-CTP analogs in the poliovirus (PV) RdRP elongation Data deposition: Crystallography, atomic coordinates, and structure factors reported in complex (EC) crystal-soaking experiments, the polymerase having a this paper have been deposited in the Protein Data Bank, www.pdb.org (PDB ID codes guanine base at the +1 template position was successfully trapped at 5F8G–5F8J, 5F8L, 5F8M, and 5F8N). different stages of a single NAC, leading to the proposal of a working 1To whom correspondence should be addressed. Email: [email protected]. www.pnas.org/cgi/doi/10.1073/pnas.1602591113 PNAS | Published online June 23, 2016 | E4005–E4014 Downloaded by guest on September 29, 2021 and posttranslocation states to refine the translocation process otides (i.e., CCU) at most, indicating a threshold for the crystal lat- further has been captured in A-family polymerases by crystallogra- tice to accommodate growth of the template–product duplex. We phy. Without an O-helix counterpart, viral RdRPs likely have then explored using this crystal lattice as a platform to capturing established unique conserved components to control translocation, important EC states within three consecutive NACs. By combining and their ECs may experience metastable intermediates that provide an incubation time scanning strategy (15, 16) and the use of different valuable details for identifying translocation-related protein com- NTP combinations in the native EC-soaking trials, we obtained seven ponents and the details of RNA movement during translocation. representative EC structures distributed in the first and the third In this study, we obtained a crystal form of the enterovirus 71 NACs (Fig. 1B and Tables 1 and 2). The resulting crystal lattices (EV71) RdRP EC that allows multiple nucleotide incorporations were nearly isomorphous based on unit cell dimensions and inter- in NTP-soaking trials. By using natural NTP substrate combina- complex packing modes (Tables 1 and 2 and Fig. 1C). This obser- tions and controlling the incubation time, we were able to capture vation is drastically different from the output of the soaking EC species that provide previously unidentified mechanistic details experiments using a PV RdRP EC crystal that also allows multiple for initial NTP binding, active site closure, and, in particular the nucleotide incorporation (14); in that lattice (named “PV_r5”)every RNA motion during translocation that shows an asymmetric translocation event resulted in obvious translational movement be- movement of the two strands in the template–product duplex. tween RdRPs within the aforementioned EC dimer as the growing RNA duplexes collided with each other. Results A Unique RdRP EC Lattice That Is Capable of in Situ Elongation for A Plausible Order of Events During Initial NTP Binding and Active Site Multiple
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