key: cord-0861020-cnwfvtfo
authors: McDonald, Sarah M.
title: RNA synthetic mechanisms employed by diverse families of RNA viruses
date: 2013-04-18
journal: Wiley Interdiscip Rev RNA
DOI: 10.1002/wrna.1164
sha: aebdcb037a55fd1a79328a876202f2b0505d9442
doc_id: 861020
cord_uid: cnwfvtfo

RNA viruses are ubiquitous in nature, infecting every known organism on the planet. These viruses can also be notorious human pathogens with significant medical and economic burdens. Central to the lifecycle of an RNA virus is the synthesis of new RNA molecules, a process that is mediated by specialized virally encoded enzymes called RNA‐dependent RNA polymerases (RdRps). RdRps directly catalyze phosphodiester bond formation between nucleoside triphosphates in an RNA‐templated manner. These enzymes are strikingly conserved in their structural and functional features, even among diverse RNA viruses belonging to different families. During host cell infection, the activities of viral RdRps are often regulated by viral cofactor proteins. Cofactors can modulate the type and timing of RNA synthesis by directly engaging the RdRp and/or by indirectly affecting its capacity to recognize template RNA. High‐resolution structures of RdRps as apoenzymes, bound to RNA templates, in the midst of catalysis, and/or interacting with regulatory cofactor proteins, have dramatically increased our understanding of viral RNA synthetic mechanisms. Combined with elegant biochemical studies, such structures are providing a scientific platform for the rational design of antiviral agents aimed at preventing and treating RNA virus‐induced diseases. WIREs RNA 2013, 4:351–367. doi: 10.1002/wrna.1164 1.. RNA Interactions with Proteins and Other Molecules > RNA–Protein Complexes; 2.. RNA in Disease and Development > RNA in Disease.

V iruses with RNA genomes comprise a vast and assorted group, whose members can differ in their particle architecture, host tropism, and pathological manifestations. RNA viruses infect nearly every type of living organism-from archea to unicellular bacteria and fungi to multicellular plants and animals. Many RNA viruses asymptomatically replicate within their hosts, inducing no obvious disease. Others lead to severe morbidity and/or lifecycle-how they synthesize new RNA molecules. During infection, an RNA virus must mediate at least two RNA synthetic processes: (1) transcription of message-sense viral RNAs (i.e., mRNAs) that can serve as templates for protein synthesis and (2) replication of viral genomic RNAs for their ultimate encapsidation into nascent virion particles. Both transcription and genome replication are mediated by specialized, virally encoded enzymes called RNA-dependent RNA polymerases (RdRps). 1 RdRps are quite conserved among RNA viral families, and they share distant structural and functional homology to cellular DNA and RNA polymerases. However, these viral enzymes have no counterpart in the host cell, making them ideal targets for antiviral drug development. 2, 3 Although RdRps are the sole polypeptides that catalyze phosphodiester bond formation between nucleoside triphosphates (NTPs) in an RNA-templated manner, these enzymes almost never function alone in the context of an infected cell. Instead, they are usually found along with other viral and/or cellular proteins in higher ordered, multisubunit polymerase complexes. Interactions between the RdRp and cofactor proteins in the polymerase complex allow RNA viruses to precisely regulate the type and timing of viral RNA synthesis during infection and coordinate it with other lifecycle events. In this review, we explore the formation and function of polymerase complexes from several diverse RNA viruses. Particular attention will be paid to summarizing the results of highresolution structural studies of RdRps and regulatory cofactor proteins from six different RNA viral families (Caliciviridae, Flaviviridae, Picornaviridae, Rhabdoviridae, Orthomyxoviridae, and Reoviridae) ( Table 1 ). This review is meant to serve as a general introduction into the complex world of RNA virus transcription and genome replication. Where appropriate, detailed review articles are referenced to provide readers enhanced insight into specific RNA synthetic mechanisms.

RNA viruses encode within their genomes many of the proteins necessary for their replication, particularly those proteins that are involved in viral RNA synthesis. However, they lack the key machinery to carry out protein synthesis. Thus, viruses are parasites of host cell ribosomes, and they must express their genes as functional mRNA molecules early in infection. The pathways leading from genome to mRNA can vary widely among the different RNA viral families. Nonetheless, the transcription strategy (and thus, the genome replication strategy) of an RNA virus generally depends upon the polarity of its genome ( Figure 1 ).

Viruses with positive-strand RNA [(+)RNA] genomes are abundant in nature, and they are particularly common in plants (http://ictvonline.org). The Caliciviridae, Flaviviridae, and Picornaviridae families are well characterized because they include several notable human (+)RNA viral pathogens (Table 1) . A shared feature of (+)RNA viruses is that their genomes are inherently message-sense. In fact, the genomes of these viruses often contain 5 7-methylguanosine (m 7 G) caps and/or 3 poly-A tails much like those of cellular mRNAs, allowing them to be efficiently recognized by the host protein synthesis machinery. Members of the Picornaviridae family, such as poliovirus, rhinovirus, hepatitis A virus, and foot-and-mouth disease virus, do not have 5 capped (+)RNA genomes, but instead they employ RNA stem-loop elements as an internal ribosomal entry sites. 4 Moreover, translation of the Caliciviridae genome is dependent on the presence of a small, virally encoded protein (VPg), which is attached to the 5 end of the incoming (+)RNA and interacts with components of the host protein genomes of (+)RNA viruses are message-sense (green), and they often contain a 5 m 7 G cap (purple circle) and 3 poly-A tail (AAAAA). Host cell ribosomes translate the genome into one or more polyproteins, which are cotranslationally and posttranslationally processed by virally encoded proteases. Some of the mature polyprotein processing precursors and products include the RNA-dependent RNA polymerase (RdRp; blue rectangle) and cofactors (blue squares) that mediate viral RNA synthesis in association with cellular membranes. Other proteins made by the virus include those that will assemble into viral particles (gray squares). The RdRp mediates the synthesis of negative-strand RNA [(−)RNA] antigenome (red) using the genome as template. The antigenome is then converted into new (+)RNA genome by the RdRp and then packaged into nascent virion particles (gray hexagon). (b) Negative-strand RNA [(−)RNA] viruses. The genomes of (−)RNA viruses are organized as ribonucleoproteins (RNPs) with their RNA molecules (red) bound by the viral polymerase complex (blue rectangle) and nucleocapsid proteins (N; grey square). Transcription is mediated by the RdRp and cofactor proteins, resulting in the synthesis of 5 capped (purple circle), 3 polyadenylated (AAAAA) mRNAs (green). The mRNAs are translated by host ribosomes into polymerase complex proteins (blue) and virion structural components (gray). Genome replication occurs when the RdRp produces a full-length, antigenome (+)RNA (green) bound by N. This antigenome is then converted into (−)RNA genome (red) and is packaged as a RNP complex into nascent virion particles (gray hexagon). (c) Double-stranded (ds) RNA viruses. The genomes of dsRNA viruses are maintained inside of particles. The RdRp (blue rectangle) will transcribe (+)RNAs (green) that contain a 5 cap (purple circle) and serve as protein synthesis templates (mRNAs) or will be packaged into assembling subviral particles (gray hexagon). Once inside the particle, the RdRp will perform (−)RNA synthesis (red) on the (+)RNS template (green) to create the dsRNA genome. synthesis machinery. 5, 6 As described below, VPg also serves an important role in initiation of RNA synthesis for Caliciviridae. Given the inherent translatability of their genomes, (+)RNA viruses do not need to encapsidate RdRps into their virions. Instead, these viruses utilize host ribosomes to make their RdRps and polymerase complex cofactors once inside the cell (Figure 1(a) ). A characteristic trait of (+)RNA viruses is the manner in which their polymerase complexes are formed early in infection. Following entry of a (+)RNA virus into the host cell, the genome is translated into at least one large polyprotein, which is cotranslationally and posttranslationally cleaved by viral proteases into smaller, mature, nonstructural protein products (Figure 1(a) ). One of these products is the actual RdRp polypeptide, whereas the other products (and oftentimes precursors) represent regulatory cofactors that play numerous roles during viral RNA synthesis. Most often, the RdRp-containing polyprotein of (+)RNA viruses is intimately associated with membranes of intracellular organelles (e.g., endoplasmic reticulum, mitochondria, endosomes, and lysosomes). 7 In fact, (+)RNA viruses are known to hijack and rearrange cellular membranes in order to fashion scaffolds on which (or within which) the RdRp mediates transcription and genome replication.

Following its synthesis and assembly, the membrane-bound polymerase complexes of (+)RNA viruses will copy genome (+)RNA into full-length, antigenome (−)RNA (Figure 1(a) ). Biochemical studies indicate that, for many (+)RNA viruses, the antigenome might remain hybridized to the genome, forming a transient double-stranded RNA (dsRNA) molecule. [8] [9] [10] The (−)RNA itself or that of the dsRNA is then copied by the polymerase complex into new, genome (+)RNA that can be packaged into nascent virions (Figure 1(a) ). For some (+)RNA viruses, the (−)RNA strand can serve as template for more than one RdRp at a time. As such, numerous (+)RNAs can be simultaneously transcribed from a single (−)RNA. [11] [12] [13] Because the (+)RNA genome of these viruses is also a functional mRNA molecule, the processes of transcription and genome replication can be essentially the same (Figure 1(a) ). In some cases, however, subgenomic (i.e., smaller than genome) mRNAs are generated, thereby allowing (+)RNA viruses to express multiple genes/proteins from a single polycistronic template. 14

Viruses with negative-strand RNA [(−)RNA] genomes are found in several different families (http://ictvonline.org). Those commonly known to cause disease in humans are either members of the order Mononegavirales (which includes families Bornaviridae, Filoviridae, Paramyxoviridae, and Rhabdoviridae) or are members of the family Orthomyxoviridae (Table 1) . Unlike the genomes of (+)RNA viruses, those of (−)RNA viruses cannot be directly translated into protein. Instead, the genomic (−)RNA must first be copied into mRNA prior to being used as templates by host ribosomes (Figure 1(b) ). Thus, all known (−)RNA viruses will package their polymerase complex, including their RdRp polypeptide, inside of the virion particle. Viruses in the Rhabdoviridae and Orthomyxoviridae families are the most well studied in terms of RNA synthetic strategies. These viruses have single-stranded, (−)RNA genomes that are directly bound by a viral polymerase complex. The genomes of Rhabdoviridae and Orthomyxoviridae members are also coated with a nucleocapsid protein, forming a tightly compact ribonucleoprotein (RNP) structure. [15] [16] [17] The RdRps of (−)RNA viruses will only catalyze RNA synthesis using RNP templates. 15, 18 Soon after entry of a (−)RNA virus into the host cell, the polymerase complex will commence viral transcription on the bound RNP 18 (Figure 1(b) ). The mRNAs generated via this process are not encased by nucleocapsid protein, and they usually contain a 5 m 7 G cap structures and 3 poly-A tails. As such, the viral mRNAs are translated very efficiently by host ribosomes, generating virion structural proteins and new polymerase complex components. For the Rhabdoviridae member, vesicular stomatitis virus, transcription occurs via a start-and-stop mechanism, whereby the RdRp pauses at regulatory signals in the RNA and then reinitiates at the next gene start. 19 Termination of the RdRp at intergenic regions also includes a slippage on a poly-U tract to generate the poly-A tails of the mRNAs. 19 Genome replication for vesicular stomatitis virus then occurs when the polymerase complex creates a full-length, antigenome (+)RNA by ignoring the transcriptional signals ( Figure 1(b) ). This antigenome is organized as an RNP, allowing it to be effectively copied into a (−)RNA genome molecule (Figure 1(b) ). While influenza A virus and other members of Orthomyxoviridae exhibit a similar RNP-dependent transcription and replication strategy, they do not employ a start-and-stop mechanism. Instead, the influenza A virus polymerase complex creates nearly fulllength mRNA copies of each of the eight (−)RNA genome segments. 20, 21 Replication of the viral genome is templated by full-length antigenome (+)RNA intermediates, which are copied into (−)RNA strands and encapsidated into RNPs. 18 Unlike (+)RNA viruses, which perform transcription and genome replication in intimate association with cellular membranes, RNA synthesis for (−)RNA viruses occurs either in cytosolic inclusions (e.g., Rhabdoviridae) or in the host nucleus (e.g., Orthomyxoviridae).

Viruses with genomes comprised of dsRNA are more limited in diversity compared with those having (+)RNA or (−)RNA genomes. Rotaviruses are members of the Reoviridae family and are the most pathogenic dsRNA virus of humans, causing acute gastroenteritis in children 22 (Table 1) . Rotaviruses contain 11 dsRNA genome segments and virionassociated polymerase complexes. A unique characteristic of rotaviruses and other Reoviridae members is that they perform all stages of RNA synthesis within a subviral particle. 23 More specifically, upon entry of the dsRNA virus into the host cell cytoplasm, the genome is maintained inside an icosahedral capsid; it is not released into the cytosol like those of (+)RNA and (−)RNA viruses (Figure 1(c) ). The RdRps within the capsid transcribe mRNAs using the (−)RNA strands of the dsRNA as templates. A single RdRp is thought to be dedicated to each of the dsRNAs, and it is tethered to the inner surface of the capsid, near the icosahedral vertices. 24 New (+)RNAs acquire a 5 m 7 G cap via viral enzymes as they exit the capsid through aqueous channels. Viral (+)RNAs lack poly-A tails, but nonetheless serve as mRNAs to direct efficient protein synthesis. Two of the rotavirus-encoded proteins induce the formation of inclusions in the host cell cytosol where early particle assembly and genome replication occur. 25 These inclusions are not necessarily membrane-associated, but have been shown to contain markers of autophagosomes and lipid droplets. 26, 27 Within viroplasms, (+)RNAs made by transcriptionally active subviral particles are packaged into assembling viral cores along with several copies of the viral RdRp 28 (Figure 1(c) ). Inside a fully or partially assembled core particle, the RdRps perform minusstrand synthesis to convert the packaged (+)RNAs into dsRNA genome segments 29 (Figure 1 (c)). A single RdRp is dedicated to each of the packaged (+)RNA segments, but acts in synchrony with the others to simultaneously replicate the dsRNA genome. 30 Thus, at its most minimal, the polymerase complex of a dsRNA virus is comprised of a capsid scaffold upon which the RNA-template-bound RdRps function. 31

All RNA viruses, regardless of their genome structure or polarity, encode an RdRp polypeptide that catalyzes phosphodiester bond formation between NTPs in an RNA-template-directed manner. 1 To date, there are more than 60 high-resolution X-ray crystal structures of viral RdRps available in the Protein Data Bank (http://www.rcsb.org/). Although RdRps from different RNA viral families can vary tremendously in their amino acid sequences, they are strikingly similar in their three-dimensional organization. For example, the RdRps of poliovirus, Norwalk virus, hepatitis C virus, and rotavirus (residues 333-778) all exhibit a central cupped, right-handed architecture with fingers, palm, and thumb subdomains [31] [32] [33] [34] [35] (Figure 2 ). This general design is also shared with the distantly related DNA-dependent DNA polymerases, DNA-dependent RNA polymerases, and reverse transcriptases. 1 Unlike these other polymerases, however, which open and close upon templates, RdRps adopt closed structures mainly because of intramolecular interactions between the fingers and thumb subdomains. For (+)RNA viruses in the Caliciviridae, Flaviviridae, and Picornaviridae families, the closed conformation of their RdRps creates three distinct tunnels from the surface of the molecules into the catalytic centers, allowing for (1) the entry of the RNA template, (2) the entry of NTPs and exit of pyrophosphates, and (3) the exit of a transient dsRNA duplex. 36 Indeed, RdRps of (+)RNA viruses have been extensively studied structurally and functionally, followed closely by those of dsRNA viruses. RdRp polypeptides of (−)RNA viruses, such as those in the Rhabdoviridae and Orthomyxoviridae families, have been difficult to express and purify. 17 As such, little high-resolution structural information exists for the RdRps of (−)RNA viruses (see also below). 18, 37 Six highly conserved sequence and structural motifs (A-F) are shared among RdRps and confer specific functions during viral RNA synthesis 38 (Figure 3) . Several of these motifs (A-D) are also seen in cellular polymerases and reverse transcriptases, suggesting that they are critical for the nucleotidyltransferase reaction. Motifs A and C are the most well characterized for RdRps, as they contain the aspartic acid residues that coordinate the two metal ions required for phosphodiester bond formation. 39 Specifically, the carboxylate groups of these amino acids anchor a pair of divalent metal ions (usually Mg 2+ ), which play the major role in catalysis. One metal ion promotes the deprotonation of the 3 -OH of the nascent primer, whereas the other ion facilitates the formation of the pentacovalent transition state at the α-phosphate of the NTP and the exit of the inorganic pyrophosphate group. 40 For the poliovirus RdRp, other motifs are While only some of these motifs have been experimentally confirmed for other RdRps, such as those in rotavirus, they are expected to play analogous roles 35, 42 (Figure 3 ). Finally, some argue for the existence of a seventh RdRp motif (i.e., motif G), which is located partially within the fingers subdomain and is implicated in primer and template binding. 43 The RdRps of viruses from different families can also show unique structural elements in addition to their shared, canonical right-handed architecture. in RNA template interactions. 35, 45 The C-terminal domain resembles the sliding clamps of cellular RNA polymerases, which encircle DNA templates and aid in processivity. 46 While the precise functions of these additional domains are not understood, they are known to be critical for the catalytic activity of the enzyme. In fact, their presence creates a caged enzyme with a central hollow active site. The C-terminal bracelet helps create a fourth tunnel (along with the fingers and palm subdomains) in the Reoviridae RdRps. Unlike other known RdRps, which only contain template entry, NTP entry/pyrophosphate exit, and dsRNA exit tunnels, those of Reoviridae have a fourth tunnel that likely serves as an exit site for (+)RNA transcripts. 35

RNA synthesis by viral RdRps always occurs in a 5 to 3 direction, and it can be separated into two distinct steps: initiation and elongation. does not serve as the initiating NTP, but instead plays a key structural role supporting the initiating NTP. 49, 50 The GTP-binding site that provides a de novo initiation platform is best described for the bovine viral diarrheal virus RdRp. 51 The GTP is bound inside the template entry tunnel, 6Å from the catalytic site, and makes contacts with fingers, palm, and thumb subdomains ( Figure 5(a) ). While there is currently no high-resolution structure of a Flaviviridae RdRp in the midst of initiation (i.e., with divalent ions, GTP, RNA template, and an initiating NTP), it is suggested that the special GTP may position the 3 -OH of the first NTP for nucleophilic attack. 51 The stabilizing GTP is not incorporated into the nascent RNA strand, but rather is thought to be released from the active site during RNA elongation. In addition to this special GTP, a C-terminal loop element is implicated in de novo initiation for Flaviviridae RdRps 52 ( Figure 5(a) ). This loop likely supports the special GTP, but it must move out of the way during elongation so as not to obstruct the egress of the nascent dsRNA. Reoviridae RdRps also initiate RNA synthesis using a primer-independent mechanism. However, rather than utilizing a GTP molecule and C-terminal loop to support the initiating NTP, these RdRps employ an internal loop, which is located in between the fingers and palm subdomains 35, 44 (Figure 5(b) ). In fact, both the first and the second NTPs are stacked together by the priming loop from the bottom and by residues of motif F from the top. 35, 44 For the reovirus RdRp, the first two NTPs are further supported by base-pair interactions with the RNA template. Following phosphodiester bond formation between these NTPs, a stable dinucleotide is formed, and the RdRp transitions to elongation mode. 44 Interestingly, in the rotavirus RdRp apoenzyme structure, this loop is in a retracted state, incapable of NTP binding. 35 It is hypothesized that engagement of the rotavirus RdRp by its cofactor protein induces a structural change in the position of the priming loop, thereby allowing for NTP binding and, thus, the initiation of RNA synthesis (see also below). 35 In contrast to de novo initiating enzymes, RdRps that employ a primer-dependent mechanism of initiation will use either a protein or an oligonucleotide of defined origin. Members of the Caliciviridae and Picornaviridae families, such as Norwalk virus, poliovirus, rhinovirus, hepatitis A virus, and foot-andmouth disease virus, employ VPg, a small (∼20-100 amino acids in length), virally encoded protein, to prime RNA synthesis. [53] [54] [55] VPg is covalently bound to the 5 end of the (+)RNA genome, but it must be modified by nucleotide addition and transferred to the 3 end of the template prior to its use as a primer. For Picornaviridae members, the VPg protein is uridylated (i.e., several UMP molecules are attached). This uridylation reaction is catalyzed de novo by the viral RdRp and is templated by an internal stem-loop element in the genomic (+)RNA. Much of our understanding of VPg-dependent initiation of RNA synthesis is based on structural studies of the Picornaviridae member foot-and-mouth disease virus. In particular, the structures of the foot-and-mouth disease virus RdRp (1) in complex with uridylated VPg and (2) in complex with a template-primer duplex have provided remarkable insight into this initiation mechanism. [56] [57] [58] Together, these structures reveal how VPg accesses the RdRp catalytic site from the front part of the molecule and how the first five nucleotides of the oligo (A) template traverse the template entry tunnel ( Figure 5(c) ). The final base of the RNA template points toward the catalytic site and is in a correct position to be paired with the UMP molecule that is covalently attached to VPg. Essentially, the 3 -OH of the UMP serves as a molecular mimic of a nucleic acid primer. Moreover, it is important to note that, for the foot-and-mouth disease virus RdRp, highresolution structures of the enzyme in different states of RNA elongation also exist, providing exquisite insight into the molecular basis for the lack of RdRp fidelity. 59 The influenza A virus RdRp utilizes different initiation mechanisms for transcription versus genome replication. 21 Specifically, transcriptional initiation is primed by small pieces of m 7 G-capped oligonucleotides that are stolen from cellular pre-mRNAs. 60 The 10-13 nucleotides of these primers do not basepair with sequences at the 3 ends of influenza A virus (−)RNAs. Instead, a GTP is added to the 3 end of the capped primer (by the RdRp), and this GTP base-pairs with the penultimate C residue at the 3 end of the viral genome. 61 The viral polymerase complex then elongates the primer in a template-directed manner until it reaches a poly-U tract, on which it stutters to form the poly-A tail of the nascent mRNA. 21 In contrast to the initiation of mRNA synthesis, initiation of both (+)RNA antigenome and (−)RNA genome synthesis by the influenza A virus RdRp does not require a cellular cap, but instead occurs de novo. For (−)RNA synthesis, the de novo initiation mode occurs at the terminal template residue, whereas that for (+)RNA antigenome synthesis occurs via an internal residue, requiring template realignment. 62 How the influenza RdRp makes the switch from transcription to genome replication is unclear; however, it has been suggested that the virus creates structurally distinct RdRp complexes (see also below). 63

Many viral RdRps are capable of performing transcription and genome replication in vitro as single polypeptides. However, in the context of a virusinfected cell, this is rarely the case. More often, viral RdRps are found along with other viral and/or cellular proteins in large, multisubunit complexes. Cofactors within these polymerase complexes modulate the type and timing of RNA synthesis by directly engaging the RdRp or by indirectly affecting its capacity to recognize template RNA. While far from inclusive, the examples below show how the high-resolution structures of RdRps and their cofactor proteins have increased our understanding of RNA virus transcription and genome replication regulation.

As mentioned previously, (+)RNA viruses share the characteristic features of (1) expressing their polymerase complex proteins via a polyprotein cleavage strategy and (2) using cellular membranes as scaffolds for polymerase complex function. One of the simplest human (+)RNA viruses in terms of polymerase complexes is poliovirus, a prototypic Picornaviridae family member. The 7.5-kb message-sense (+)RNA genome molecule encodes a single polyprotein, which is cleaved into three precursors P1, P2, and P3. The majority of products processed from P2 and P3 are polymerase complex proteins, including 2A, 2B, 2C, 2BC, 3AB, 3B, 3C, 3CD, and 3D (Figure 6(a) ).

Protein 3AB is believed to be a main anchor for the poliovirus polymerase complex in endoplasmic reticulum-derived, double-membrane vesicles. 64 This protein also has nucleic acid chaperone activity and increases the binding of the viral polymerase to template. 66, 67 The 3AB membrane association occurs via a hydrophobic region in the C-terminus of 3A. 64 Additional poliovirus proteins (e.g., 3A, 2B, 2C, and 2BC) are also thought to aid in membrane rearrangements/polymerase complex anchoring in the infected cell. [68] [69] [70] [71] The viral protein 3B (alone or possibly in the context of a precursor) represents VPg, the protein primer required for initiation of RNA synthesis (see also above). 55, 71, 72 Polymerase complex proteins 2C, 3CD, and 3D all interact with viral RNA and with each other in some manner. Moreover, Volume 4, July/August 2013 cellular proteins such as poly-rC-and poly-A-binding proteins (PCBP and PABP, respectively) are also important components of the poliovirus polymerase complex. 73 Poliovirus 3CD is a multifunctional protein and an essential component of the poliovirus polymerase complex (Figure 6(a) and (b) ). The 3CD is an active protease, but it lacks polymerase activity. As such, 3D only becomes an active RdRp following cleavage from 3C. High-resolution X-ray crystal structures of 3D versus 3CD have provided insight into proteolysisdependent activation of the RdRp. 33, 74, 75 Initial X-ray crystal structures of 3D contained packing artifacts, because of the inherent (albeit possibly biologically relevant) capacity of the polymerase to oligomerize. 74, 75 These 3D-3D interactions obscured the location of the extreme N-terminus of the protein. 74 More accurate structures of 3D were generated using a mutant protein that cannot form oligomers. 76 In that later structure, the N-terminus of 3D is buried in a pocket at the base of the fingers subdomain, where it helps position the catalytic-site aspartate residues to select the incoming 3 -OH group of NTPs. In the structure of the RdRp-inactive 3CD precursor, the 3C and 3D domains do not make any direct contacts with each other and are connected by a seven-residue polypeptide linker 76 (Figure 6(b) ). The N-terminal residues of the 3D domain that comprises the linker are relocated in the context of 3CD, thereby preventing catalysis ( Figure 6(b) ). Thus, the lack of polymerase activity in 3CD is thought to be associated with the preclusion of the N-terminus from the 3D domain. Moreover, in the 3CD structure, the 3D domain makes extensive contacts with the 3C and 3D domains of neighboring molecules. 76 The 3C N-terminus is proximal to the VPg-binding site in 3CD, an arrangement that is consistent with a possible role for these contacts in forming and regulating the poliovirus VPg uridylation complex. Essentially, polyprotein processing allows poliovirus to tightly regulate the stoichiometry of 3CD and 3D in the polymerase complex, and therefore to maximize the functions of these proteins during VPg uridylation, initiation, and elongation.

The most well-studied members of the Rhabdoviridae family are rabies virus and vesicular stomatitis virus. The 11-to 12-kb (−)RNA genomes of these viruses are encased with nucleocapsid protein (N), forming an RNP structure, and are bound at their 3 ends with the viral polymerase complex 13 (Figure 7(a) ). The polymerase complex is composed of two proteins: a single molecule of the large (L) protein, which has RdRp and 5 RNA capping activities, and a dimer of the small viral phosphoprotein (P). During both transcription and genome replication, P serves as a noncatalytic cofactor that simultaneously engages L and N (bound to RNA), allowing the RdRp to gain access to the RNA template. 77 While the precise mechanism(s) of P function are not fully understood, it is thought that P directly binds to L and thereby increases its affinity for RNA template. P is a modular protein comprised of three distinct ordered domains: (1) an N-terminal domain, (2) a central dimerization domain, and (3) a C-terminal domain 79 (Figure 7(b) ). The ordered domains are interspersed by disordered regions, causing the protein to behave as a nonglobular molecule. 80 The P protein forms parallel dimers as a result of interactions between the central domains of individual monomers. 80 The C-terminal domain of P binds to the N protein, only when it is in complex with RNA (i.e., N-RNA). 81, 82 In contrast, the N-terminal domain of P binds to free N protein (N 0 ), possibly preventing it from inappropriately binding to viral or cellular mRNAs. 83 For vesicular stomatitis virus, the N-terminal domain and the central dimerization domain are thought to engage L, the viral RdRp 78 (Figure 7(b) ). However, the structure of the N-terminal domain of the P protein has not been determined for any Rhabdoviridae family member. Also, while no X-ray crystal structures of L have been determined to date, recent electron microscopic analyses of the protein show that it contains two domains: (1) a ring-like RdRp domain and (2) a globular appendage domain that harbors the enzymatic activities associated with capping 37 (Figure 7(c) ). Interestingly, after complex formation with P, the appendage domain of L rearranges into a more compact structure 78 (Figure 7(c) ).

It is not clear exactly how P binding to L and to the N-RNA complex keeps L attached to the template. It is also not understood how the polymerase moves across its RNA template. Studies of closely related Rhabdoviridae members suggest that P might progress along the template in a type of cart-wheeling motion, in which it alternatively associates and disassociates from N. 84 Other studies suggest that P molecules remain bound at regular intervals along the N-RNA and that L jumps between bound P proteins. 85 In this model, the binding of one P dimer to every five N monomers is sufficient to allow one P dimer to 'catch' the viral RdRp and 'transfer' it to the next P dimer. Additional roles of the P protein in regulation of the vesicular stomatitis virus RdRp have been implicated by biochemical studies. Specifically, the same region of P that induces a structural rearrangement (i.e., the N-terminal domain and the central domain) also stimulates the initiation of RNA synthesis and processivity of L in vitro. 78 Therefore, (−)RNA viruses of the Rhabdoviridae family employ a multifunctional cofactor to modulate the template binding and enzymatic activities of its RdRp. 

Influenza A virus and other Orthomyxoviridae family members maintain their (−)RNA genomes as eight separate segments that are bound by the viral nucleoprotein (NP) and a heterotrimeric polymerase complex (Figure 8(a) ). Unlike Rhabdoviridae members, whose RNP is single-stranded with a 3 end-bound polymerase complex, the 5 and 3 ends of Orthomyxoviridae RNPs are base-paired, forming a folded corkscrew-like structure. 16, 21 The virus polymerase complex is bound to the double-stranded RNP terminus, and it consists of three proteins: PB1 (RdRp), PB2 (cap-binding protein), and PA (endonuclease), which interact head-to-tail in the order PA-PB1-PB2. 16, 20, 21 While most RNA viruses transcribe and replicate their RNA in the cytoplasm, the influenza virus RdRp complex only functions within the nucleus of a host cell. The location of RNA synthesis for this virus makes sense in light of its mechanism of transcriptional initiation using 5 m 7 G-capped oligonucleotides stolen from cellular pre-mRNAs. The PB2 subunit is responsible for binding to the 5 m 7 G cap of a cellular pre-mRNA molecule, while the PA subunit will cleave the structure. Then, PB1 will utilize the stolen capped oligonucleotide as a primer for viral RNA synthesis (see also above).

The influenza A virus polymerase complex has been relatively intractable to high-resolution structural analysis. Of the three subunits, only PA has been expressed as a soluble, recombinant protein. This protein was cleaved into N-terminal and C-terminal domains using trypsin, and the two individual structures were solved by X-ray crystallography. The N-terminal domain of PA has a fold similar to other type II restriction endonucleases. 86, 87 In fact, the isolated PA N-terminal domain shows endonuclease activity on single-stranded nucleic acid, suggesting that this region is sufficient for the cap-snatching activities of the influenza A virus polymerase complex. 86 The C-terminal domain of PA has only been crystallized in complex with a short N-terminal peptide of PB1, essentially revealing the interface between these two proteins [88] [89] [90] (Figure 8(b) ). Biochemical assays have shown that a small peptide corresponding to the N-terminus of PB1 can competitively inhibit polymerase activity, suggesting that the insertion of this region into the PA cavity is necessary for catalysis. 91, 92 Aside from interacting with PB1, the function of the C-terminal domain of PA remains to be determined.

Compared to PA, the influenza virus PB2 protein has been even more difficult to express and purify in a manner suitable for structural analysis. However, three small regions of PB2 have been crystallized, revealing the structures of: (1) a nuclear localization region, (2) an independently folded central region containing the 5 mRNA cap-binding site required for cap snatching, and (3) a putative pre-mRNA-binding region involved in influenza host specificity. [93] [94] [95] [96] Moreover, the first 35 N-terminal residues of PB2 have been resolved via a cocrystal with a C-terminal portion of PB1 97 (Figure 8(b) ).

To date, no high-resolution structure is available for PB1, the influenza A virus RdRp subunit. Only the subunit interaction interfaces of the PB1 protein are known in atomic detail (i.e., PA-PB1 and PB1-PB2) 89, 97 (Figures 6(c) and 8(b) ). These subunit interfaces are essential for polymerase function, as mutations in them severely affect transcription and genome replication. Thus, PA and PB2 are essential cofactors that engage PB1 and allow the RdRp to mediate catalysis. This heterotrimeric polymerase complex ensures that PB1 will only elongate viral templates that have been primed with m 7 G-capped RNAs via the coordinated activities of PA and PB2.

Like other segmented, dsRNA viruses of the family Reoviridae, rotaviruses perform all stages of viral RNA synthesis within the confines of proteinaceous particles. A single RdRp monomer (VP1) in complex with the RNA capping enzyme is dedicated to transcribing and replicating an associated genome , which is thought to engage VP1 and aid in enzymatic activation, is shown in gold. No structure yet exists for VP2 N-terminal residues 1-77, but they, as well as regions of the VP2 principal domain, are also important for VP1 activation. segment, presumably while tethered to the interior of the core shell protein (VP2). However, the role of VP2 extends beyond serving as a scaffold for the viral polymerase. It is also a critical cofactor that causes VP1 to initiate genome replication. 98 High-resolution structures of the VP2 core shell in the context of a double-layered particle or mature rotavirus virion have been determined by using cryoelectron microscopy or X-ray crystallography. [99] [100] [101] [102] In these structures, the VP2 core shell is seen as a smooth icosahedron that is organized into 12 decameric units (Figure 9(a) ). Each VP2 decamer is comprised of two chemically identical VP2 conformers (A-VP2 and B-VP2). Five A-VP2 conformers encircle the icosahedral fivefold axes, whereas five B-VP2 confers sit further back from the fivefold axis in between A-VP2 units (Figure 9 (a)). A monomer of VP2 is defined into two separate domains: (1) the amino-terminal domain, made up of residues ∼1-100 and (2) the principal scaffold domain, made up of residues ∼101-880. The VP2 N-terminal domain cannot be fully resolved in the known structures, most likely because it forms a flexible protein region. However, the N-termini of neighboring monomers have been suggested to multimerize beneath the icosahedral fivefold axes. 100 Recent analysis of the X-ray crystallographic data of rotavirus double-layered particles indicates that VP1 is localized to the inner face of the VP2 principal scaffold domain, just off center from the fivefold axis, with a binding footprint that overlaps the apical subdomains of neighboring A-B VP2 conformers in a decamer 24 (Figure 9 (b)). Portions of the VP2 N-terminal domain are also detected near VP1, perhaps forming tethers that support the polymerase. Thus, in the context of a static structure, both the N-terminal domain and the apical subdomains of the VP2 core shell directly bind VP1. These same regions have been shown to be involved in VP1 enzymatic activation in vitro, suggesting that they may trigger the capacity of the enzyme to mediate RNA synthesis. 103 It is hypothesized that engagement by VP2 causes structural rearrangements in VP1 that may include lifting the priming loop to allow for NTP binding. The VP2-dependent mechanism of RNA synthesis for rotavirus ensures that dsRNA genome is only made within a particle that is the morphogenic precursor to a mature virion. Interestingly, the RdRps of several Caliciviridae members, Norwalk virus and murine norovirus, also show enhanced enzymatic activity in the presence of their cognate capsid proteins. 104 This observation suggests that coordination of RNA synthesis with virion assembly via polymerase regulation may occur for other families of RNA viruses in addition to Reoviridae.

This review summarizes the RNA synthetic mechanisms employed by diverse RNA viruses of the families Caliciviridae, Flaviviridae, Picornaviridae, Rhabdoviridae, Orthomyxoviridae, and Reoviridae. While far from inclusive, the examples provided here underscore the various shared and virus-specific features of transcription and genome replication. It is clear from these examples that RNA viruses have evolved distinctive strategies to control the activity of their RdRps and to orchestrate the kinetics of their lifecycle. Indeed, RNA viral RdRps are almost always found along with other viral (and oftentimes cellular) proteins in higher ordered, multisubunit polymerase complexes. In combination with biochemical studies, high-resolution structures are revealing the details of functional protein-protein interactions between the polymerase complex components. Moreover, newer imaging technologies are providing unprecedented views of RNA virus polymerase complexes within or isolated from infected host cells. [105] [106] [107] Studies aimed at elucidating the molecular dynamics of these complexes in their most native state during RNA synthesis are expected to provide the 'missing link' between structure and function. 108 The knowledge gained from these studies not only contributes to our general understanding of RNA virus biology but also enhances our ability to develop pharmacological inhibitors of viral RNA synthesis.

Structure-function relationships among RNA-dependent RNA polymerases

Potential targets and their relevant inhibitors in anti-influenza fields

The genome-linked protein VPg of the Norwalk virus binds eIF3, suggesting its role in translation initiation complex recruitment

Calicivirus translation initiation requires an interaction between VPg and eIF4E

Cytoplasmic viral replication complexes

Comparative studies on the genomes of some picornaviruses: denaturation mapping of replicative form RNA and electron microscopy of heteroduplex RNA

Flavivirus replication strategy

Replication strategy of kunjin virus: evidence for recycling role of replicative form RNA as template in semiconservative and asymmetric replication

Structure of the poliovirus replicative intermediate RNA

Architecture of the poliovirus replicative intermediate RNA

Identification and characterization of type 2 dengue virus replicative intermediate and replicative form RNAs

A contemporary view of coronavirus transcription

Nucleoproteins and nucleocapsids of negative-strand RNA viruses

Towards an atomic resolution understanding of the influenza virus replication machinery

Structural insights into the rhabdovirus transcription/replication complex

Architecture and regulation of negative-strand viral enzymatic machinery

Polymerase slippage at vesicular stomatitis virus gene junctions to generate poly-(A) is regulated by the upstream 3'-AUAC-5' tetranucleotide: implications for the mechanism of transcription termination

Influenza A virus polymerase: structural insights into replication and host adaptation mechanisms

Orthomyxoviridae: the viruses and their replication

estimate of worldwide rotavirusassociated mortality in children younger than 5 years before the introduction of universal rotavirus vaccination programmes: a systematic review and metaanalysis

Mechanism of intraparticle synthesis of the rotavirus double-stranded RNA genome

Location of the dsRNA-dependent polymerase, VP1, in rotavirus particles

Nonstructural proteins involved in genome packaging and replication of rotaviruses and other members of the Reoviridae

Autophagy hijacked through viroporin-activated calcium/calmodulin-dependent kinase kinase-β signaling is required for rotavirus replication

Rotaviruses associate with cellular lipid droplet components to replicate in viroplasms, and compounds disrupting or blocking lipid droplets inhibit viroplasm formation and viral replication

Assortment and packaging of the segmented rotavirus genome

Rotavirus RNA polymerase requires the core shell protein to synthesize the double-stranded RNA genome

Rotavirus RNA replication: single-stranded RNA extends from the replicase particle

Bluetongue virus: dissection of the polymerase complex

Crystal structure of the RNAdependent RNA polymerase from hepatitis C virus reveals a fully encircled active site

Structural basis for proteolysis-dependent activation of the poliovirus RNA-dependent RNA polymerase

Crystal structure of norwalk virus polymerase reveals the carboxyl terminus in the active site cleft

Mechanism for coordinated RNA packaging and genome replication by rotavirus polymerase VP1

The ins and outs of four-tunneled reoviridae RNA-dependent RNA polymerases

Molecular architecture of the vesicular stomatitis virus RNA polymerase

Analysis of RNA-dependent RNA polymerase structure and function as guided by known polymerase structures and computer predictions of secondary structure

A mechanism for all polymerases

What is the role of motif D in the nucleotide incorporation catalyzed by the RNAdependent RNA polymerase from poliovirus?

Poliovirus RNA-dependent RNA polymerase (3Dpol): kinetic, thermodynamic, and structural analysis of ribonucleotide selection

Mutational analysis of residues involved in nucleotide and divalent cation stabilization in the rotavirus RNA-dependent RNA polymerase catalytic pocket

The palm subdomain-based active site is internally permuted in viral RNA-dependent RNA polymerases of an ancient lineage

RNA synthesis in a cage-structural studies of reovirus polymerase lambda3

Residues of the rotavirus RNA-dependent RNA polymerase template entry tunnel that mediate RNA recognition and genome replication

DNA replication. A familiar ring to DNA polymerase processivity

De novo initiation of RNA synthesis by the RNA-dependent RNA polymerase (NS5B) of hepatitis C virus

De novo initiation of RNA synthesis by a recombinant flaviviridae RNA-dependent RNA polymerase

Comparative mechanistic studies of de novo RNA synthesis by flavivirus RNA-dependent RNA polymerases

Requirements for de novo initiation of RNA synthesis by recombinant flaviviral RNA-dependent RNA polymerases

The structure of the RNA-dependent RNA polymerase from bovine viral diarrhea virus establishes the role of GTP in de novo initiation

Structural analysis of the hepatitis C virus RNA polymerase in complex with ribonucleotides

The genome-linked protein VPg of vertebrate viruses-a multifaceted protein

VPg-primed RNA synthesis of norovirus RNA-dependent RNA polymerases by using a novel cell-based assay

Expanding knowledge of P3 proteins in the poliovirus lifecycle

Structure of foot-andmouth disease virus RNA-dependent RNA polymerase and its complex with a template-primer RNA

The structure of a protein primer-polymerase complex in the initiation of genome replication

Structural insights into replication initiation and elongation processes by the FMDV RNA-dependent RNA polymerase

Sequential structures provide insights into the fidelity of RNA replication

A unique cap(m7Gpppxm)-dependent influenza virion endonuclease cleaves capped RNAs to generate the primers that initiate viral RNA transcription

Selected host cell capped RNA fragments prime influenza A viral transcription in vivo

Different de novo initiation strategies are used by influenza virus RNA polymerase on its cRNA and viral RNA promoters during viral RNA replication

Distinct regions of the influenza virus PB1 polymerase subunit recognize vRNA and cRNA templates

Determinants of membrane association for poliovirus protein 3ab

Remodeling the endoplasmic reticulum by poliovirus infection and by individual viral proteins: an autophagylike origin for virus-induced vesicles

Poliovirus protein 3AB displays nucleic acid chaperone and helix-destabilizing activities

The twenty-nine amino acid C-terminal cytoplasmic domain of poliovirus 3AB is critical for nucleic acid chaperone activity

Membrane integration of poliovirus 2B viroporin

Evidence for functional protein interactions required for poliovirus RNA replication

Initiation of poliovirus negative-strand RNA synthesis requires precursor forms of P2 proteins

Similar structural basis for membrane localization and protein priming by an RNA-dependent RNA polymerase

Picornavirus genome replication: roles of precursor proteins and rate-limiting steps in oriIdependent VPg uridylylation

The dependence of viral RNA replication on co-opted host factors

Structure of the RNA-dependent RNA polymerase of poliovirus

Visualization and functional analysis of RNA-dependent RNA polymerase lattices

Crystal structure of poliovirus 3CD protein: virally encoded protease and precursor to the RNA-dependent RNA polymerase

Location of the binding domains for the RNA polymerase l and the ribonucleocapsid template within different halves of the NS phosphoprotein of vesicular stomatitis virus

Critical phosphoprotein elements that regulate polymerase architecture and function in vesicular stomatitis virus

Structure of the vesicular stomatitis virus N 0 -P complex

Structure of the dimerization domain of the rabies virus phosphoprotein

Structure of the vesicular stomatitis virus nucleocapsid in complex with the nucleocapsidbinding domain of the small polymerase cofactor

Structure and function of the C-terminal domain of the polymerase cofactor of rabies virus

Interaction of vesicular stomatitis virus P and N proteins: identification of two overlapping domains at the N terminus of P that are involved in N 0 -P complex formation and encapsidation of viral genome RNA

Viral DNA polymerase scanning and the gymnastics of sendai virus RNA synthesis

Binding of rabies virus polymerase cofactor to recombinant circular nucleoprotein-RNA complexes

The cap-snatching endonuclease of influenza virus polymerase resides in the PA subunit

Crystal structure of an avian influenza polymerase PA(N) reveals an endonuclease active site

Amino acid residues in the N-terminal region of the PA subunit of influenza A virus RNA polymerase play a critical role in protein stability, endonuclease activity, cap binding, and virion RNA promoter binding

Crystal structure of the polymerase PA(C)-PB1(N) complex from an avian influenza H5N1 virus

The structural basis for an essential subunit interaction in influenza virus RNA polymerase

Peptidemediated interference with influenza A virus polymerase

Identification of a PA-binding peptide with inhibitory activity against influenza A and B virus replication

Structure and nuclear import function of the C-terminal domain of influenza virus polymerase PB2 subunit

Host determinant residue lysine 627 lies on the surface of a discrete, folded domain of influenza virus polymerase PB2 subunit

The structural basis for cap binding by influenza virus polymerase subunit PB2

Structural basis of the influenza a virus RNA polymerase PB2 RNA-binding domain containing the pathogenicity-determinant lysine 627 residue

Structural insight into the essential PB1-PB2 subunit contact of the influenza virus RNA polymerase

Rotavirus VP1 alone specifically binds to the 3' end of viral mRNA, but the interaction is not sufficient to initiate minus-strand synthesis

Rotavirus architecture at subnanometer resolution

X-ray crystal structure of the rotavirus inner capsid particle at 3.8Åresolution

Atomic model of an infectious rotavirus particle

Near-atomic resolution using electron cryomicroscopy and single-particle reconstruction

Rotavirus VP2 core shell regions critical for viral polymerase activation

Norovirus RNA synthesis is modulated by an interaction between the viral RNA-dependent RNA polymerase and the major capsid protein, VP1

The structure of native influenza virion ribonucleoproteins

Organization of the influenza virus replication machinery

Unraveling the structure of viral replication complexes at super-resolution