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ScienceDirect Exploring the architecture of viral RNA genomes Beth L Nicholson and K Andrew White The genomes of RNA viruses contain local structural elements and long-range interactions that control various steps in virus replication. While many individual RNA elements have been characterized, it remains less clear how the structure and activity of such elements are integrated and regulated within the complex context of complete viral genomes. Recent technical advances, particularly the development of highthroughput solution structure mapping methods, have made secondary structural analysis of entire viral RNA genomes feasible. As a consequence, whole-genome structural models have been deduced for a number of plus-strand RNA viruses and retroviruses and these structures have provided intriguing functional and evolutionary insights into global genome architecture. Address Department of Biology, York University, Toronto, Ontario M3J 1P3, Canada Corresponding author: White, K Andrew ([email protected])
Current Opinion in Virology 2015, 12:66–74 This review comes from a themed issue on Virus structure and expression Edited by Jane Tao and Eric Jan
http://dx.doi.org/10.1016/j.coviro.2015.03.018 1879-6257/# 2015 Elsevier B.V. All rights reserved.
Introduction The genomes of RNA viruses serve many roles during viral reproduction and the complexity of the structural basis of these functions is becoming increasingly apparent. At a basic level, RNA genomes serve as repositories for the genetic information needed for virus reproduction. Coding information is accompanied by an assortment of regulatory RNA elements (RREs) that contribute to controlling and coordinating the different functions that the genome must perform. These RREs can modulate a variety of processes in viruses, including, for example, translation of viral proteins, transcription of viral subgenomic mRNAs, replication of viral genomes, and virion assembly [1–3]. Some RREs operate at the primary sequence level, while others mediate their activities via secondary and tertiary RNA structures. Both coding and non-coding regions can harbor RREs and many of them act by recruiting viral or host proteins. In addition to local Current Opinion in Virology 2015, 12:66–74
RREs, intra-genomic long-range RNA base pairing interactions, which can span thousands of nucleotides, have also been reported to be functionally relevant in a variety of viruses [4]. This latter observation underscores the importance of employing a ‘wide-angle’ view when studying the activities of viral RREs. Indeed, the emerging field of whole-genome structural analysis has led to new levels of understanding of the functional complexity of viral genomes and has revealed important roles for global RNA conformation. In this review, we discuss the methodologies that have enabled insightful structural analyses of entire viral RNA genomes and highlight the novel features uncovered from the whole-genome secondary structure models generated for plus-strand RNA viruses and retroviruses.
Rationale and methods for investigating whole-genome architecture The diverse functions that are performed by plus-strand viral RNA genomes require spatial and temporal coordination during infections. For example, translation of viral proteins must precede genome replication, which in turn must occur prior to packaging. Some of these genomic processes are mutually antagonistic, for example, translation in the 50 to 30 direction and minus-strand RNA synthesis in the 30 to 50 direction [5]. Consequently, translation of viral proteins needs to be suppressed before genome replication begins. Similarly, genome replication needs to be temporally coordinated with subgenomic mRNA transcription, as both utilize the same genomic template for synthesis of these distinct classes of viral RNA [6]. Undoubtedly, viral and host proteins play critical roles in facilitating and regulating many of these processes [7], however, RREs also contribute significantly to the harmonious management of viral events [1–3]. To fully understand the cooperative and antagonistic activities of the RREs in a virus, these elements need to be studied in their natural context; that is, within a complete viral genome. This setting is important for uncovering framework-dependent RNA-based modes of regulation, such as alternative local conformations and global rearrangements, which would be missed if isolated elements were analyzed. Accordingly, methodologies that allow for the generation of structural models that describe RNA organization on a whole-genome scale are important for accurately deducing RRE functions and inter-relationships. Atomic force microscopy (AFM) is a technique widely used to study physical and structural features of viral particles [8] and it has been gaining popularity for the visualization of viral RNA genomes [9–12,13,14,15]. www.sciencedirect.com
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In this technique, a probe scans over the surface of a sample and the data gathered are used to generate a topographical image [8]. Thus, this type of analysis can yield useful information on the overall architecture of biological molecules. Topographical information on the structure of genomic RNAs obtained by AFM has been used for comparisons with genomic conformations predicted by alternative means. For example, the existence of distinct ‘open’ and ‘closed’ conformational states of the Satellite tobacco mosaic virus (STMV) genome, which were suggested by biochemical analyses, were confirmed by AFM micrographs [10,11]. AFM analysis was also used to reveal a compact structure for the Hepatitis C virus (HCV) RNA genome and a more extended structure for that of poliovirus (Figure 1), both of which correlated well with the predicted level of genome-scale ordered RNA structure (GORS) determined by computational approaches (13). Similarly, AFM images of the RNA genomes of Hepatitis G virus (HGV), Tomato bushy stunt virus (TBSV), and STMV were consistent with the overall compact structures that were predicted using bioinformatic, biochemical, and/or genetic approaches [13,14,15]. For Dengue virus, AFM provided direct visualization of a circularized RNA conformation, which is formed by long-range base pairing interactions that are essential for genome replication of this plus-strand RNA virus [12]. However, while AFM can provide information on the overall organizational features of a viral genome, image resolution is limited and dependent on the physical features of the probe used [10]; thus, a more detailed map of genome structure requires other approaches that
are able to define secondary and tertiary RNA structures within these molecules. Secondary structure analyses of RNA elements have traditionally relied on thermodynamics-based computer-aided structural predictions, with the potential for further refinement using data from phylogenetic comparisons or enzymatic/chemical structure-probing experiments [16,17]. Early studies of viral genomic architecture used such methods to predict overall secondary structure of relatively large viral RNA domains (i.e., up to 1200 nt) in the bacteriophages MS2 and Qb [18,19,20]. Unfortunately, the accuracy of thermodynamics-based structure prediction decreases as the RNA under study gets larger [16]. Moreover, the acquisition of corresponding probing information from long RNAs can be both tedious and laborious [21]. Fortunately, there has been a recent innovation in solution structure analysis of large RNAs, termed high-throughput selective 20 -hydroxyl acylation analyzed by primer extension (SHAPE) [22,23]. This method employs reagents that modify the 20 -hydroxyl ribose group of the RNA backbone to generate covalent 20 -O-ribose adducts in structurally flexible regions, while nucleotides that are base paired or in other structurally inflexible conformations show reduced reactivity. Unlike conventional chemical probing techniques, SHAPE-based modifications are largely insensitive to base identity, thus providing structural information at every nucleotide using a single chemical reagent (such as, N-methylisatoic anhydride). Acylated positions are identified by primer extension because reverse transcriptase stalls at modified nucleotides. In the high-throughput SHAPE
Figure 1
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Analysis of the RNA genomes of (a) Hepatitis C virus (HCV) and (b) Poliovirus by atomic force microscopy. Scale bars represent 200 nm. Regions in the upper panels (boxed) are magnified in the lower panels.Adapted from Davis et al. [13]. www.sciencedirect.com
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method, fluorescently labeled primers are used, which allow the resulting cDNAs to be analyzed by capillary electrophoresis. The electropherograms generated are then quantitatively analyzed using available software; ShapeFinder [24] or QuShape [25]. For structure prediction, the normalized reactivity values generated for each nucleotide in the sequence are then added as constraints to a thermodynamics-based RNA folding program [23]. Recently, an alternative method for detecting positions modified by SHAPE reagents was developed which employs next-generation sequencing. SHAPE-mutational profiling (SHAPE-MaP) uses massively parallel sequencing to identify sites of nucleotide misincorporation, which correspond to sites of 20 -hydroxyl acylation [26]. The substitutions are introduced during the reverse transcription step that generates the templates for sequencing; that is, a mismatched deoxyribonucleotide is inserted opposite to the modified base in the RNA when the reaction is performed in the presence of Mn2+. SHAPE-based technical advances have now made the analysis of large RNAs readily feasible, and have allowed for the generation of secondary structural models of entire viral RNA genomes at single-nucleotide resolution. To date, genomic models using SHAPE methodology have been deduced for the animal viruses Human immunodeficiency virus-1 (HIV-1) [26,27], Simian immunodeficiency virus (SIV) [28], poliovirus [29], and HCV [30], as well as the plant viruses TBSV [14] and STMV [15,31]. The structural features of these models and the functional insights gained from them are discussed below.
Global architectural features of RNA virus genomes Currently, the global genomic architectures defined by AFM and/or SHAPE tend to fall into two broad categories: those with compact structures, including HCV, HGV [13], TBSV [14], and STMV [15,31], and those with elongated structures, including HIV [26,27], SIV [28], Rubella virus [13], and poliovirus [29]. Figure 1 provides examples of AFM images depicting viral RNA genomes structures that are largely compact (i.e. HCV) or more extended (i.e. poliovirus). Compact conformations are the result of multiple long-range interactions within
genomes that act to cluster individual domains. For example, SHAPE-based structural mapping of the 4.8 kb long TBSV genome revealed a genomic organization consisting of variable-sized RNA domains arising from a central core (Figure 2b) [14]. The core region is formed by a localized grouping of long-range interactions, some of which are known to function as RREs in this virus. Interestingly, the largest domains encompassed the majority of the protein-coding regions, while the smaller domains were positioned either within coding regions proximal to a translational readthrough site or in the 50 and 30 untranslated regions (UTRs) [14] (Figure 2a). A similar compact type of organization was reported for the smaller STMV genome (1.1 kb), in which longdistance interactions enclosed three domains corresponding roughly to the 50 UTR, capsid coding region, and 30 UTR [15,31]. In contrast to those just described, the more elongated types of genomic conformations tend to be formed by locally folded and unstructured regions interspersed along the RNA. This is the case for the HIV and SIV genomes, which contain differently sized and variably spaced RNA domains arranged consecutively along their lengths [26,27,28]. Examples of the diverse RNA secondary and tertiary structures identified within the HIV-1 genome are presented in Figure 3 [27]. Interestingly, for poliovirus, a consistent global structure could not be deduced from SHAPE analysis [29], which is consistent with the AFM analysis of this genome indicating a pleomorphic appearance [13]. Accordingly, some viruses may not form distinct genomic configurations, suggesting that global conformation is less relevant in such cases.
Functional insights from whole-genome structural models There are clear benefits to obtaining genome-wide structural information for an RNA virus. Particularly for poorly studied viruses, a secondary structure map of the genome can identify novel RNA elements and provide a basis for detailed functional analyses. In addition, an overall picture of genome configuration allows for observations that are impossible when studying smaller portions of the
(Figure 2 Legend) (a) Linear representation of the TBSV RNA genome. Encoded proteins are shown as boxes with molecular masses (in thousands). Shown below are the locations of the initiation sites for transcription of subgenomic mRNAs (open arrows labeled sg1 and sg2). The relative locations of intra-genomic long-range RNA–RNA interactions are shown by the labeled double-headed arrows above the genome. Each double-headed arrow connects a complementary cognate sequence pair identified by their acronyms (see key below). The genomic regions corresponding to defined domains are labeled and represented by color-coded bars below the genome (blue, small; red, medium; green, large). Above the genome, the partner sequences in long-range interactions are color-coded according to the domain in which they reside. (b) Simplified cartoon of the TBSV genome secondary structure. Each dot represents a nucleotide and domains are labeled and color-coded as in panel (a). Inter-domain regions are depicted in black. The regions containing complementary segments known to form functional long-range RNA–RNA interactions are labeled. Only the AS1–RS1 and DE–CE interactions involved in subgenomic mRNA transcription are base paired in this structure (gold ovals), while the other four interactions, AS2–RS2, UL–DL, PRTE–DRTE, and 30 CITE–50 UTR, are not paired. Transcription-related RNA elements: AS1 or 2, activator sequence 1 or 2; RS1 or 2, receptor sequence 1 or 2; DE, distal element; CE, core element. Replication-related RNA elements: UL, upstream linker; DL, downstream linker. Translation-related RNA elements: 30 CITE, 30 cap-independent translation enhancer; 50 UTR. Translational readthrough-related elements: PRTE, proximal readthrough element; DRTE, distal readthrough element.Adapted from Wu et al. [14]. www.sciencedirect.com
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SHAPE-MaP analysis and motif discovery in the HIV-1 NL4-3 genome. (a) SHAPE reactivities are shown as the centered 55-nt median window, relative to the global median; regions above or below the line are more flexible or constrained than the median, respectively. (b) Gene organization and RNA regions identified as having biological functions. (c) Models for RNA genome regions with well-determined secondary structures. Base pairs and validated pseudoknots (PK) are shown. Adapted from Siegfried et al. [27].
molecule and can provide insights into the dynamic integration of viral processes regulated at the RNA level. SHAPE analysis of the poliovirus genome identified several novel RNA structures, and their possible functional Current Opinion in Virology 2015, 12:66–74
relevance was further assessed by evolutionary conservation analysis across genome isolates [29]. Among the newly identified structures was an element within the RNAdependent RNA polymerase coding region that showed extremely high conservation, termed 3D-7000. Mutational www.sciencedirect.com
Architecture of viral RNA genomes Nicholson and White 71
analysis uncovered a role for 3D-7000 in viral replication and infectivity, and suggested that this function might be mediated by an interaction with the viral protein 3CPro [29]. For TBSV, several novel RNA structures were predicted to form in the SHAPE-guided genome model that were not predicted when using a thermodynamics-only approach [14]. Two of these elements, termed SL27 and S31, are highly conserved within the genus Tombusvirus and were functionally validated by mutational analysis [14]. For HCV, four previously uncharacterized RNA elements with well-determined stable structures and strong evidence of evolutionary maintenance were uncovered by a genomewide structural analysis of three diverse genotypes [30]. Three of these four elements were experimentally confirmed to be important for viral fitness, demonstrating the utility of applying a structure-first comparative approach to the analysis of viral cis-regulatory elements [30]. The importance of analyzing local structures in the context of a full-length genome was also reinforced by studies on the HIV-1 frameshift signal, which supported an alternate structure for this RNA element that extended beyond the boundaries of the conventional domain [32]. Nonetheless, not all highly-conserved RNA structures in the HIV-1 genome are necessarily functionally relevant, as was demonstrated by the dispensability of several such structures for virus replication [33].
and disease progression [34,35]. Remarkably, the RNA regions encoding these hypervariable domains were found to be segregated from the rest of the genome in unstructured regions flanked by conserved stable secondary structures, termed insulator helices [26,27]. Theoretically, this organizational feature would allow for relatively large insertions and deletions in the sequences enclosed by the insulator helices without affecting the overall structure of the genome [26,27].
In addition to identifying novel local structures, genomewide structural models provide a unique opportunity to observe functions served at the level of global organization. Indeed, SHAPE-directed modeling of the HIV-1 genome uncovered previously unknown functions for RNA structure [26,27]. In this virus, highly structured regions of the viral RNA were found to correspond to sequences encoding flexible inter-domain loops in viral proteins and inter-protein peptide linkers in viral polyproteins [26,27]. Based on these observations, it was proposed and validated that the progress of elongating ribosomes is slowed by the highly structured RNA regions of the HIV genome, thereby providing time for independent co-translational folding of the encoded protein domains [26,27]. Accordingly, this analysis unveiled a new level of the genetic code that assists in proper HIV1 protein biosynthesis [26,27]. Although this RNA structure-mediated mechanism could represent a generic means to assist proper protein folding during translation of polyproteins, similar analysis of the poliovirus genome suggested that RNA structure does not significantly correlate with protein structure in this virus [29].
Whole-genome structural models of RNA viruses have also generated insights into RNA-based regulation of the viral replication cycle at the global level. Several viruses have been shown to contain multiple long-range intra-genomic interactions required for different viral processes, suggesting that the formation and activities of these elements must be highly integrated and regulated in the viral genome [4]. For example, in addition to multiple local RREs, TBSV is known to harbor six different sets of long-range interactions that function in translation initiation, translational readthrough, genome replication, and subgenomic mRNA transcription (Figure 2a) [14]. Interestingly, only two of these six long-range interactions were predicted to form in the TBSV genome secondary structure model [14], suggesting that the genome must undergo dynamic structural rearrangements to adopt the other conformations required for additional viral processes (Figure 2b). The two interactions that were present in the structural model involved sequences located in the closing helices of two large domains, suggesting that formation of these domains acts to bring the partner sequences together. Interestingly, these AS1–RS1 and DE–CE interactions are both involved in the viral process of subgenomic mRNA transcription, implying a transcription-ready genomic conformation. For the four interactions that did not form, their partner sequences were relatively close to each other in the structure model, thus their formation may not require major global rearrangements (Figure 2b) [14]. The absence of certain interactions in this model may be the result of different effects; for example, the formation of these
The HIV-1 whole-genome structural model also highlighted another novel role for RNA architecture: sequestration of RNA sequences encoding highly variable protein regions [26,27]. Five domains in the HIV envelope protein gp120 are highly variable and exhibit length polymorphisms, and this variability contributes to HIV cell tropism, sensitivity to neutralizing antibodies, www.sciencedirect.com
A further advantage of whole-genome structural analysis is the ability to investigate evolutionary conservation and drift of structural elements at a global level by comparing the structural models of related viruses. A comparison of SHAPE-guided structural models of two retroviruses in the genus Lentivirus, HIV and SIV, revealed a remarkably low level of base pair conservation, even in regions of structural similarity [28]. Overall, the study found significant rearrangement of the RNA secondary structures in these two viral genomes, despite corresponding functional requirements for these RNA elements. Indeed, as more genomic structures become available, particularly those of related viruses, comparative structural analyses will reveal important new information on the diversification and evolution of viral genomes at various levels of RNA structural hierarchy.
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interactions may be less thermodynamically favorable, proteins may be required to promote and/or stabilize the interactions, the interactions may be mutually exclusive, and/or the conditions under which the analysis was performed did not favor their formation. Indeed, the accurate modeling of a defined genome structure via SHAPE analysis requires a dominant structure, which would preclude the detection of less abundant structural sub-populations that may also be biologically relevant. Nonetheless, such dominant structures provide useful starting points for pursuing alternative functional structures and the factors that govern their formation and stability.
Figure 4
STMV RNA
Perspectives and future directions The advent of new investigative tools has allowed for comprehensive and detailed analyses of the structures of complete viral RNA genomes. This has permitted the discovery of new local RREs and provided a context to understand how these elements, as well as active longrange interactions, are functionally integrated within these molecules. Nonetheless, alternative biologicallyactive RNA conformations can be missed in these studies and not all RNA structures identified will prove to be functionally relevant. Accordingly, the activities of deduced RNA structures must be validated experimentally and the concept of RNA conformational flexibility should be entertained when building structural models. Indeed, recent cryo-electron microscopic analysis of the STMV RNA genome revealed that it exists as an ensemble of conformations (Figure 4) [36]. The viral genomic models generated thus far have been restricted primarily to the level of RNA secondary structure, due in part to limitations in the algorithms used to generate them that exclude predictions of tertiary interactions or noncanonical base pairing. However, there does exist a variation of the SHAPE method that can detect these types of interactions and, though more challenging for large RNAs, such higher level structure could be integrated into viral secondary structure models [37]. Solving a three-dimensional structure of a viral genome at the atomic level would be ideal; however, the large size and/or dynamics of these RNAs makes them poor candidates for methods such as nuclear magnetic resonance spectroscopy, X-ray crystallography, and small-angle Xray scattering [38]. Nonetheless, a lot can still be learned from investigating these molecules with lower resolution techniques. For instance, performing SHAPE analysis under different conditions, such as using different in vitro RNA refolding protocols or allowing the genome to fold co-transcriptionally, could assist in identifying alternative conformations. Also, the inclusion of viral proteins that interact with the genome, such as those with RNA chaperone activity, could lead to genome remodeling into other configurations [39–41]. Certain viral processes, such as ribosomes traversing coding regions, could also direct structural rearrangements. These translational events and Current Opinion in Virology 2015, 12:66–74
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Skeleton representation of a diverse collection of one hundred individual STMV genomes imaged by cryo-electron microscopy. The scale bar is equivalent to 60 nm. Adapted from Garmann et al. [36].
their consequences could potentially be analyzed using in vitro systems; however, discerning only the RNAs that were engaged by ribosomes could prove difficult. Monitoring viral genome structure and transitions during infections of cells represents an ideal approach, but it too poses significant challenges. A method for RNA SHAPE analysis in living cells does exist [42]; however, a major drawback to utilizing this technique is the dynamic nature of viral infections, which makes it difficult to precisely monitor individual viral events and their corresponding genomic structures, synchronously. Inhibiting a particular step in the infectious cycle could be one way to help align a particular viral event, but this approach remains untested and its unnatural nature could potentially introduce artifacts. The field therefore awaits new methods and technologies that will assist in monitoring the genomic structures and conformational rearrangements that occur during viral infections of cells. Research in viral RNA structure would also benefit from advances in the computational analysis of large RNAs that include prediction of tertiary and noncanonical base pairing. Although such new technologies would indeed propelling the field forward, the existing methodology is still very valuable and has uncovered many novel aspects of these multifunctional and dynamic molecules. Undoubtedly, both current and future research will lead to new discoveries related to this intriguing class of regulatory RNA www.sciencedirect.com
Architecture of viral RNA genomes Nicholson and White 73
that will help to identify unique and unifying principles that govern viral genome architecture. Such information should ultimately prove to be useful in designing antiviral drugs that target structural components in viral RNA genomes [43].
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Acknowledgments
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Research in the author’s laboratory was supported by the Natural Sciences and Engineering Research Council of Canada.
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34. Wei X, Decker JM, Wang S, Hui H, Kappes JC, Wu X, SalazarGonzalez JF, Salazar MG, Kilby JM, Saag MS et al.: Antibody neutralization and escape by HIV-1. Nature 2003, 422:307-312. 35. Curlin ME, Zioni R, Hawes SE, Liu Y, Deng W, Gottlieb GS, Zhu T, Mullins TI: HIV-1 envelope subregion length variation during disease progression. PLoS Pathog 2010, 6:e1001228. 36. Garmann RF, Gopal A, Athavale SS, Knobler CM, Gelbart WM, Harvey SC: Visualizing the global secondary structure of a viral RNA genome with cryo-electron microscopy. RNA 2015. in press, PMID: 25752599. Demonstration of viral genome conformational diversity using cryo-EM. 37. Steen KA, Rice GM, Weeks KM: Fingerprinting noncanonical and tertiary RNA structures by differential SHAPE reactivity. J Am Chem Soc 2012, 134:13160-13163. A modified SHAPE protocol for identifying higher-order RNA structures.
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40. Ivanyi-Nagy R, Darlix JL: Core protein-mediated 50 –30 annealing of the West Nile virus genomic RNA in vitro. Virus Res 2012, 167:226-235. 41. Gebhard LG, Kaufman SB, Gamarnik AV: Novel ATPindependent RNA annealing activity of the dengue virus NS3 helicase. PLoS One 2012, 7:e36244. 42. Spitale RC, Crisalli P, Flynn RA, Torre EA, Kool ET, Chang HY: RNA SHAPE analysis in living cells. Nat Chem Biol 2013, 9:18-20. SHAPE applied to investigating the structures of RNAs in living cells. 43. Le Grice SF: Targeting the HIV RNA genome: high-hanging fruit only needs a longer ladder. Curr Top Microbiol Immunol 2015. in press, PMID: 25735922.
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ScienceDirect Exploring the architecture of viral RNA genomes Beth L Nicholson and K Andrew White The genomes of RNA viruses contain local structural elements and long-range interactions that control various steps in virus replication. While many individual RNA elements have been characterized, it remains less clear how the structure and activity of such elements are integrated and regulated within the complex context of complete viral genomes. Recent technical advances, particularly the development of highthroughput solution structure mapping methods, have made secondary structural analysis of entire viral RNA genomes feasible. As a consequence, whole-genome structural models have been deduced for a number of plus-strand RNA viruses and retroviruses and these structures have provided intriguing functional and evolutionary insights into global genome architecture. Address Department of Biology, York University, Toronto, Ontario M3J 1P3, Canada Corresponding author: White, K Andrew ([email protected])
Current Opinion in Virology 2015, 12:66–74 This review comes from a themed issue on Virus structure and expression Edited by Jane Tao and Eric Jan
http://dx.doi.org/10.1016/j.coviro.2015.03.018 1879-6257/# 2015 Elsevier B.V. All rights reserved.
Introduction The genomes of RNA viruses serve many roles during viral reproduction and the complexity of the structural basis of these functions is becoming increasingly apparent. At a basic level, RNA genomes serve as repositories for the genetic information needed for virus reproduction. Coding information is accompanied by an assortment of regulatory RNA elements (RREs) that contribute to controlling and coordinating the different functions that the genome must perform. These RREs can modulate a variety of processes in viruses, including, for example, translation of viral proteins, transcription of viral subgenomic mRNAs, replication of viral genomes, and virion assembly [1–3]. Some RREs operate at the primary sequence level, while others mediate their activities via secondary and tertiary RNA structures. Both coding and non-coding regions can harbor RREs and many of them act by recruiting viral or host proteins. In addition to local Current Opinion in Virology 2015, 12:66–74
RREs, intra-genomic long-range RNA base pairing interactions, which can span thousands of nucleotides, have also been reported to be functionally relevant in a variety of viruses [4]. This latter observation underscores the importance of employing a ‘wide-angle’ view when studying the activities of viral RREs. Indeed, the emerging field of whole-genome structural analysis has led to new levels of understanding of the functional complexity of viral genomes and has revealed important roles for global RNA conformation. In this review, we discuss the methodologies that have enabled insightful structural analyses of entire viral RNA genomes and highlight the novel features uncovered from the whole-genome secondary structure models generated for plus-strand RNA viruses and retroviruses.
Rationale and methods for investigating whole-genome architecture The diverse functions that are performed by plus-strand viral RNA genomes require spatial and temporal coordination during infections. For example, translation of viral proteins must precede genome replication, which in turn must occur prior to packaging. Some of these genomic processes are mutually antagonistic, for example, translation in the 50 to 30 direction and minus-strand RNA synthesis in the 30 to 50 direction [5]. Consequently, translation of viral proteins needs to be suppressed before genome replication begins. Similarly, genome replication needs to be temporally coordinated with subgenomic mRNA transcription, as both utilize the same genomic template for synthesis of these distinct classes of viral RNA [6]. Undoubtedly, viral and host proteins play critical roles in facilitating and regulating many of these processes [7], however, RREs also contribute significantly to the harmonious management of viral events [1–3]. To fully understand the cooperative and antagonistic activities of the RREs in a virus, these elements need to be studied in their natural context; that is, within a complete viral genome. This setting is important for uncovering framework-dependent RNA-based modes of regulation, such as alternative local conformations and global rearrangements, which would be missed if isolated elements were analyzed. Accordingly, methodologies that allow for the generation of structural models that describe RNA organization on a whole-genome scale are important for accurately deducing RRE functions and inter-relationships. Atomic force microscopy (AFM) is a technique widely used to study physical and structural features of viral particles [8] and it has been gaining popularity for the visualization of viral RNA genomes [9–12,13,14,15]. www.sciencedirect.com
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In this technique, a probe scans over the surface of a sample and the data gathered are used to generate a topographical image [8]. Thus, this type of analysis can yield useful information on the overall architecture of biological molecules. Topographical information on the structure of genomic RNAs obtained by AFM has been used for comparisons with genomic conformations predicted by alternative means. For example, the existence of distinct ‘open’ and ‘closed’ conformational states of the Satellite tobacco mosaic virus (STMV) genome, which were suggested by biochemical analyses, were confirmed by AFM micrographs [10,11]. AFM analysis was also used to reveal a compact structure for the Hepatitis C virus (HCV) RNA genome and a more extended structure for that of poliovirus (Figure 1), both of which correlated well with the predicted level of genome-scale ordered RNA structure (GORS) determined by computational approaches (13). Similarly, AFM images of the RNA genomes of Hepatitis G virus (HGV), Tomato bushy stunt virus (TBSV), and STMV were consistent with the overall compact structures that were predicted using bioinformatic, biochemical, and/or genetic approaches [13,14,15]. For Dengue virus, AFM provided direct visualization of a circularized RNA conformation, which is formed by long-range base pairing interactions that are essential for genome replication of this plus-strand RNA virus [12]. However, while AFM can provide information on the overall organizational features of a viral genome, image resolution is limited and dependent on the physical features of the probe used [10]; thus, a more detailed map of genome structure requires other approaches that
are able to define secondary and tertiary RNA structures within these molecules. Secondary structure analyses of RNA elements have traditionally relied on thermodynamics-based computer-aided structural predictions, with the potential for further refinement using data from phylogenetic comparisons or enzymatic/chemical structure-probing experiments [16,17]. Early studies of viral genomic architecture used such methods to predict overall secondary structure of relatively large viral RNA domains (i.e., up to 1200 nt) in the bacteriophages MS2 and Qb [18,19,20]. Unfortunately, the accuracy of thermodynamics-based structure prediction decreases as the RNA under study gets larger [16]. Moreover, the acquisition of corresponding probing information from long RNAs can be both tedious and laborious [21]. Fortunately, there has been a recent innovation in solution structure analysis of large RNAs, termed high-throughput selective 20 -hydroxyl acylation analyzed by primer extension (SHAPE) [22,23]. This method employs reagents that modify the 20 -hydroxyl ribose group of the RNA backbone to generate covalent 20 -O-ribose adducts in structurally flexible regions, while nucleotides that are base paired or in other structurally inflexible conformations show reduced reactivity. Unlike conventional chemical probing techniques, SHAPE-based modifications are largely insensitive to base identity, thus providing structural information at every nucleotide using a single chemical reagent (such as, N-methylisatoic anhydride). Acylated positions are identified by primer extension because reverse transcriptase stalls at modified nucleotides. In the high-throughput SHAPE
Figure 1
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Analysis of the RNA genomes of (a) Hepatitis C virus (HCV) and (b) Poliovirus by atomic force microscopy. Scale bars represent 200 nm. Regions in the upper panels (boxed) are magnified in the lower panels.Adapted from Davis et al. [13]. www.sciencedirect.com
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Figure 2
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Architecture of viral RNA genomes Nicholson and White 69
method, fluorescently labeled primers are used, which allow the resulting cDNAs to be analyzed by capillary electrophoresis. The electropherograms generated are then quantitatively analyzed using available software; ShapeFinder [24] or QuShape [25]. For structure prediction, the normalized reactivity values generated for each nucleotide in the sequence are then added as constraints to a thermodynamics-based RNA folding program [23]. Recently, an alternative method for detecting positions modified by SHAPE reagents was developed which employs next-generation sequencing. SHAPE-mutational profiling (SHAPE-MaP) uses massively parallel sequencing to identify sites of nucleotide misincorporation, which correspond to sites of 20 -hydroxyl acylation [26]. The substitutions are introduced during the reverse transcription step that generates the templates for sequencing; that is, a mismatched deoxyribonucleotide is inserted opposite to the modified base in the RNA when the reaction is performed in the presence of Mn2+. SHAPE-based technical advances have now made the analysis of large RNAs readily feasible, and have allowed for the generation of secondary structural models of entire viral RNA genomes at single-nucleotide resolution. To date, genomic models using SHAPE methodology have been deduced for the animal viruses Human immunodeficiency virus-1 (HIV-1) [26,27], Simian immunodeficiency virus (SIV) [28], poliovirus [29], and HCV [30], as well as the plant viruses TBSV [14] and STMV [15,31]. The structural features of these models and the functional insights gained from them are discussed below.
Global architectural features of RNA virus genomes Currently, the global genomic architectures defined by AFM and/or SHAPE tend to fall into two broad categories: those with compact structures, including HCV, HGV [13], TBSV [14], and STMV [15,31], and those with elongated structures, including HIV [26,27], SIV [28], Rubella virus [13], and poliovirus [29]. Figure 1 provides examples of AFM images depicting viral RNA genomes structures that are largely compact (i.e. HCV) or more extended (i.e. poliovirus). Compact conformations are the result of multiple long-range interactions within
genomes that act to cluster individual domains. For example, SHAPE-based structural mapping of the 4.8 kb long TBSV genome revealed a genomic organization consisting of variable-sized RNA domains arising from a central core (Figure 2b) [14]. The core region is formed by a localized grouping of long-range interactions, some of which are known to function as RREs in this virus. Interestingly, the largest domains encompassed the majority of the protein-coding regions, while the smaller domains were positioned either within coding regions proximal to a translational readthrough site or in the 50 and 30 untranslated regions (UTRs) [14] (Figure 2a). A similar compact type of organization was reported for the smaller STMV genome (1.1 kb), in which longdistance interactions enclosed three domains corresponding roughly to the 50 UTR, capsid coding region, and 30 UTR [15,31]. In contrast to those just described, the more elongated types of genomic conformations tend to be formed by locally folded and unstructured regions interspersed along the RNA. This is the case for the HIV and SIV genomes, which contain differently sized and variably spaced RNA domains arranged consecutively along their lengths [26,27,28]. Examples of the diverse RNA secondary and tertiary structures identified within the HIV-1 genome are presented in Figure 3 [27]. Interestingly, for poliovirus, a consistent global structure could not be deduced from SHAPE analysis [29], which is consistent with the AFM analysis of this genome indicating a pleomorphic appearance [13]. Accordingly, some viruses may not form distinct genomic configurations, suggesting that global conformation is less relevant in such cases.
Functional insights from whole-genome structural models There are clear benefits to obtaining genome-wide structural information for an RNA virus. Particularly for poorly studied viruses, a secondary structure map of the genome can identify novel RNA elements and provide a basis for detailed functional analyses. In addition, an overall picture of genome configuration allows for observations that are impossible when studying smaller portions of the
(Figure 2 Legend) (a) Linear representation of the TBSV RNA genome. Encoded proteins are shown as boxes with molecular masses (in thousands). Shown below are the locations of the initiation sites for transcription of subgenomic mRNAs (open arrows labeled sg1 and sg2). The relative locations of intra-genomic long-range RNA–RNA interactions are shown by the labeled double-headed arrows above the genome. Each double-headed arrow connects a complementary cognate sequence pair identified by their acronyms (see key below). The genomic regions corresponding to defined domains are labeled and represented by color-coded bars below the genome (blue, small; red, medium; green, large). Above the genome, the partner sequences in long-range interactions are color-coded according to the domain in which they reside. (b) Simplified cartoon of the TBSV genome secondary structure. Each dot represents a nucleotide and domains are labeled and color-coded as in panel (a). Inter-domain regions are depicted in black. The regions containing complementary segments known to form functional long-range RNA–RNA interactions are labeled. Only the AS1–RS1 and DE–CE interactions involved in subgenomic mRNA transcription are base paired in this structure (gold ovals), while the other four interactions, AS2–RS2, UL–DL, PRTE–DRTE, and 30 CITE–50 UTR, are not paired. Transcription-related RNA elements: AS1 or 2, activator sequence 1 or 2; RS1 or 2, receptor sequence 1 or 2; DE, distal element; CE, core element. Replication-related RNA elements: UL, upstream linker; DL, downstream linker. Translation-related RNA elements: 30 CITE, 30 cap-independent translation enhancer; 50 UTR. Translational readthrough-related elements: PRTE, proximal readthrough element; DRTE, distal readthrough element.Adapted from Wu et al. [14]. www.sciencedirect.com
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SHAPE-MaP analysis and motif discovery in the HIV-1 NL4-3 genome. (a) SHAPE reactivities are shown as the centered 55-nt median window, relative to the global median; regions above or below the line are more flexible or constrained than the median, respectively. (b) Gene organization and RNA regions identified as having biological functions. (c) Models for RNA genome regions with well-determined secondary structures. Base pairs and validated pseudoknots (PK) are shown. Adapted from Siegfried et al. [27].
molecule and can provide insights into the dynamic integration of viral processes regulated at the RNA level. SHAPE analysis of the poliovirus genome identified several novel RNA structures, and their possible functional Current Opinion in Virology 2015, 12:66–74
relevance was further assessed by evolutionary conservation analysis across genome isolates [29]. Among the newly identified structures was an element within the RNAdependent RNA polymerase coding region that showed extremely high conservation, termed 3D-7000. Mutational www.sciencedirect.com
Architecture of viral RNA genomes Nicholson and White 71
analysis uncovered a role for 3D-7000 in viral replication and infectivity, and suggested that this function might be mediated by an interaction with the viral protein 3CPro [29]. For TBSV, several novel RNA structures were predicted to form in the SHAPE-guided genome model that were not predicted when using a thermodynamics-only approach [14]. Two of these elements, termed SL27 and S31, are highly conserved within the genus Tombusvirus and were functionally validated by mutational analysis [14]. For HCV, four previously uncharacterized RNA elements with well-determined stable structures and strong evidence of evolutionary maintenance were uncovered by a genomewide structural analysis of three diverse genotypes [30]. Three of these four elements were experimentally confirmed to be important for viral fitness, demonstrating the utility of applying a structure-first comparative approach to the analysis of viral cis-regulatory elements [30]. The importance of analyzing local structures in the context of a full-length genome was also reinforced by studies on the HIV-1 frameshift signal, which supported an alternate structure for this RNA element that extended beyond the boundaries of the conventional domain [32]. Nonetheless, not all highly-conserved RNA structures in the HIV-1 genome are necessarily functionally relevant, as was demonstrated by the dispensability of several such structures for virus replication [33].
and disease progression [34,35]. Remarkably, the RNA regions encoding these hypervariable domains were found to be segregated from the rest of the genome in unstructured regions flanked by conserved stable secondary structures, termed insulator helices [26,27]. Theoretically, this organizational feature would allow for relatively large insertions and deletions in the sequences enclosed by the insulator helices without affecting the overall structure of the genome [26,27].
In addition to identifying novel local structures, genomewide structural models provide a unique opportunity to observe functions served at the level of global organization. Indeed, SHAPE-directed modeling of the HIV-1 genome uncovered previously unknown functions for RNA structure [26,27]. In this virus, highly structured regions of the viral RNA were found to correspond to sequences encoding flexible inter-domain loops in viral proteins and inter-protein peptide linkers in viral polyproteins [26,27]. Based on these observations, it was proposed and validated that the progress of elongating ribosomes is slowed by the highly structured RNA regions of the HIV genome, thereby providing time for independent co-translational folding of the encoded protein domains [26,27]. Accordingly, this analysis unveiled a new level of the genetic code that assists in proper HIV1 protein biosynthesis [26,27]. Although this RNA structure-mediated mechanism could represent a generic means to assist proper protein folding during translation of polyproteins, similar analysis of the poliovirus genome suggested that RNA structure does not significantly correlate with protein structure in this virus [29].
Whole-genome structural models of RNA viruses have also generated insights into RNA-based regulation of the viral replication cycle at the global level. Several viruses have been shown to contain multiple long-range intra-genomic interactions required for different viral processes, suggesting that the formation and activities of these elements must be highly integrated and regulated in the viral genome [4]. For example, in addition to multiple local RREs, TBSV is known to harbor six different sets of long-range interactions that function in translation initiation, translational readthrough, genome replication, and subgenomic mRNA transcription (Figure 2a) [14]. Interestingly, only two of these six long-range interactions were predicted to form in the TBSV genome secondary structure model [14], suggesting that the genome must undergo dynamic structural rearrangements to adopt the other conformations required for additional viral processes (Figure 2b). The two interactions that were present in the structural model involved sequences located in the closing helices of two large domains, suggesting that formation of these domains acts to bring the partner sequences together. Interestingly, these AS1–RS1 and DE–CE interactions are both involved in the viral process of subgenomic mRNA transcription, implying a transcription-ready genomic conformation. For the four interactions that did not form, their partner sequences were relatively close to each other in the structure model, thus their formation may not require major global rearrangements (Figure 2b) [14]. The absence of certain interactions in this model may be the result of different effects; for example, the formation of these
The HIV-1 whole-genome structural model also highlighted another novel role for RNA architecture: sequestration of RNA sequences encoding highly variable protein regions [26,27]. Five domains in the HIV envelope protein gp120 are highly variable and exhibit length polymorphisms, and this variability contributes to HIV cell tropism, sensitivity to neutralizing antibodies, www.sciencedirect.com
A further advantage of whole-genome structural analysis is the ability to investigate evolutionary conservation and drift of structural elements at a global level by comparing the structural models of related viruses. A comparison of SHAPE-guided structural models of two retroviruses in the genus Lentivirus, HIV and SIV, revealed a remarkably low level of base pair conservation, even in regions of structural similarity [28]. Overall, the study found significant rearrangement of the RNA secondary structures in these two viral genomes, despite corresponding functional requirements for these RNA elements. Indeed, as more genomic structures become available, particularly those of related viruses, comparative structural analyses will reveal important new information on the diversification and evolution of viral genomes at various levels of RNA structural hierarchy.
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72 Virus structure and expression
interactions may be less thermodynamically favorable, proteins may be required to promote and/or stabilize the interactions, the interactions may be mutually exclusive, and/or the conditions under which the analysis was performed did not favor their formation. Indeed, the accurate modeling of a defined genome structure via SHAPE analysis requires a dominant structure, which would preclude the detection of less abundant structural sub-populations that may also be biologically relevant. Nonetheless, such dominant structures provide useful starting points for pursuing alternative functional structures and the factors that govern their formation and stability.
Figure 4
STMV RNA
Perspectives and future directions The advent of new investigative tools has allowed for comprehensive and detailed analyses of the structures of complete viral RNA genomes. This has permitted the discovery of new local RREs and provided a context to understand how these elements, as well as active longrange interactions, are functionally integrated within these molecules. Nonetheless, alternative biologicallyactive RNA conformations can be missed in these studies and not all RNA structures identified will prove to be functionally relevant. Accordingly, the activities of deduced RNA structures must be validated experimentally and the concept of RNA conformational flexibility should be entertained when building structural models. Indeed, recent cryo-electron microscopic analysis of the STMV RNA genome revealed that it exists as an ensemble of conformations (Figure 4) [36]. The viral genomic models generated thus far have been restricted primarily to the level of RNA secondary structure, due in part to limitations in the algorithms used to generate them that exclude predictions of tertiary interactions or noncanonical base pairing. However, there does exist a variation of the SHAPE method that can detect these types of interactions and, though more challenging for large RNAs, such higher level structure could be integrated into viral secondary structure models [37]. Solving a three-dimensional structure of a viral genome at the atomic level would be ideal; however, the large size and/or dynamics of these RNAs makes them poor candidates for methods such as nuclear magnetic resonance spectroscopy, X-ray crystallography, and small-angle Xray scattering [38]. Nonetheless, a lot can still be learned from investigating these molecules with lower resolution techniques. For instance, performing SHAPE analysis under different conditions, such as using different in vitro RNA refolding protocols or allowing the genome to fold co-transcriptionally, could assist in identifying alternative conformations. Also, the inclusion of viral proteins that interact with the genome, such as those with RNA chaperone activity, could lead to genome remodeling into other configurations [39–41]. Certain viral processes, such as ribosomes traversing coding regions, could also direct structural rearrangements. These translational events and Current Opinion in Virology 2015, 12:66–74
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Skeleton representation of a diverse collection of one hundred individual STMV genomes imaged by cryo-electron microscopy. The scale bar is equivalent to 60 nm. Adapted from Garmann et al. [36].
their consequences could potentially be analyzed using in vitro systems; however, discerning only the RNAs that were engaged by ribosomes could prove difficult. Monitoring viral genome structure and transitions during infections of cells represents an ideal approach, but it too poses significant challenges. A method for RNA SHAPE analysis in living cells does exist [42]; however, a major drawback to utilizing this technique is the dynamic nature of viral infections, which makes it difficult to precisely monitor individual viral events and their corresponding genomic structures, synchronously. Inhibiting a particular step in the infectious cycle could be one way to help align a particular viral event, but this approach remains untested and its unnatural nature could potentially introduce artifacts. The field therefore awaits new methods and technologies that will assist in monitoring the genomic structures and conformational rearrangements that occur during viral infections of cells. Research in viral RNA structure would also benefit from advances in the computational analysis of large RNAs that include prediction of tertiary and noncanonical base pairing. Although such new technologies would indeed propelling the field forward, the existing methodology is still very valuable and has uncovered many novel aspects of these multifunctional and dynamic molecules. Undoubtedly, both current and future research will lead to new discoveries related to this intriguing class of regulatory RNA www.sciencedirect.com
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that will help to identify unique and unifying principles that govern viral genome architecture. Such information should ultimately prove to be useful in designing antiviral drugs that target structural components in viral RNA genomes [43].
15. Archer EJ, Simpson MA, Watts NJ, O’Kane R, Wang B, Erie DA, McPherson A, Weeks KM: Long-range architecture in a viral RNA genome. Biochemistry 2013, 52:3182-3190. First secondary structure model for the genome of a satellite RNA virus.
Acknowledgments
17. Pace NR, Thomas BC, Woese CR: Probing RNA structure, function, and history by comparative analysis. The RNA World. edn 2. 1999:113-141:. (Chapter 4).
Research in the author’s laboratory was supported by the Natural Sciences and Engineering Research Council of Canada.
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