Single-molecule FRET reveals a corkscrew RNA PNAS PLUS structure for the polymerase-bound virus promoter

Alexandra I. Tomescua,1, Nicole C. Robba,1, Narin Hengrungb,c, Ervin Fodorb,2, and Achillefs N. Kapanidisa,2

aBiological Physics Research Group, Clarendon Laboratory, Department of Physics, , Oxford OX1 3PU, United Kingdom; bSir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, United Kingdom; and cDivision of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford OX3 7BN, United Kingdom

Edited by Robert A. Lamb, Northwestern University, Evanston, IL, and approved July 8, 2014 (received for review April 1, 2014) The influenza virus is a major human and animal pathogen re- development of specific antiviral agents, such as decoy sponsible for seasonal epidemics and occasional pandemics. The (10). Despite this, little structural information is available re- genome of the influenza A virus comprises eight segments of single- garding the mechanisms of RNA promoter recognition and stranded, negative-sense RNA with highly conserved 5′ and 3′ ter- binding by the influenza virus RNAP. mini. These termini interact to form a double-stranded promoter During the influenza virus life cycle, the viral RNAP tran- structure that is recognized and bound by the viral RNA-dependent scribes the vRNA genome into capped and polyadenylated RNA polymerase (RNAP); however, no 3D structural information for mRNAs using short primers containing a 5′ 7-methyl-guanosine the influenza polymerase-bound promoter exists. Functional studies cap structure derived from host cell pre-mRNAs. The vRNA have led to the proposal of several 2D models for the secondary segments are also replicated by the RNAP via cRNA inter- structure of the bound promoter, including a corkscrew model in mediates, which in turn are used as templates to make more ′ ′ which the 5 and 3 termini form short hairpins. We have taken vRNA (reviewed in ref. 9). To date, it remains unclear how the advantage of an insect-cell system to prepare large amounts of ac- polymerase commits to either transcription or replication using tive recombinant influenza virus RNAP, and used this to develop the same vRNA template. Current models invoke differential a highly sensitive single-molecule FRET assay to measure distances binding of RNAP either to the individual terminal ends of the between fluorescent dyes located on the promoter and map its struc- vRNA (or cRNA) or to the dsRNA promoter at different stages ture both with and without the polymerase bound. These advances of replication and transcription. Although several studies have enabled the direct analysis of the influenza promoter structure in addressed binding of the influenza virus RNAP to the dsRNA complex with the viral RNAP, and provided 3D structural information promoter or to the individual terminal sequences (11–18), no that is in agreement with the corkscrew model for the influenza virus quantitative analyses have been performed to measure the af- promoter RNA. Our data provide insights into the mechanisms of K promoter binding by the influenza RNAP and have implications for finity (i.e., the dissociation constant d) of the trimeric viral the understanding of the regulatory mechanisms involved in the polymerase for the individual termini of the vRNA and dsRNA transcription of viral genes and replication of the viral RNA genome. promoter structure. NMR structures of the unbound dsRNA In addition, the simplicity of this system should translate readily to promoter show that it resembles a double-stranded helix, with A the study of any virus polymerase–promoter interaction. base-pairing between complementary residues (Fig. 1 ) (19, 20). Structural information on the vRNA promoter in complex with nfluenza A virus is an Orthomyxovirus that causes seasonal Irespiratory infections and occasional severe pandemics and is Significance responsible for substantial morbidity and mortality worldwide. The influenza genome consists of eight single-stranded, negative- The genome of the influenza virus consists of eight single- sense viral RNA (vRNA) segments encoding 10 major and sev- stranded segments of RNA with highly conserved 5′ and 3′ eral auxiliary polypeptides (1–4). The conserved 13 termini. These termini associate to form double-stranded at the 5′ end and 12 nucleotides at the 3′ end of each RNA structures that act as promoters for viral transcription and segment display partial and inverted complementarity and form replication. Structural information on the polymerase-bound a double-stranded promoter structure created by base-pairing promoter currently does not exist, so to address this we de- between the two ends (5, 6). Similar promoter structures based veloped a sensitive single-molecule FRET assay that allowed us on complementarity also may be found at the genomic ends of to measure distances between fluorescent dyes located on the other single-stranded, negative-sense viruses, such as the arena- promoter and map its structure. The distances obtained are and bunyaviruses (7). Comparisons likewise may be drawn with consistent with the polymerase-bound RNA promoter being in “ ” ′ ′ single-stranded, positive-sense viruses, such as flaviviruses, in a corkscrew conformation, in which the 5 and 3 termini form short hairpins. This work has implications for the de- which polymerase recruitment and binding are guided by circu- – larization of the genome due to interaction between the 5′ and velopment of inhibitors that target polymerase promoter the 3′ untranslated regions (8). interactions in this important group of pathogens. In the influenza virus, the double-stranded RNA promoter Author contributions: A.I.T., N.C.R., E.F., and A.N.K. designed research; A.I.T., N.C.R., and MICROBIOLOGY is bound by the viral polymerase, which is a versatile RNA- N.H. performed research; A.I.T. and N.H. contributed new reagents/analytic tools; A.I.T. dependent RNA polymerase (RNAP) capable of functioning as and N.C.R. analyzed data; and A.I.T., N.C.R., E.F., and A.N.K. wrote the paper. a transcriptase, a replicase, and a poly(A) polymerase (reviewed The authors declare no conflict of interest. in ref. 9). Three subunits associate to form the polymerase: PB1, This article is a PNAS Direct Submission. the actual polymerase; PB2, shown to have a cap-binding function; 1A.I.T. and N.C.R. contributed equally to this work. and PA, which acts as an endonuclease. Unlike DNA-dependent 2To whom correspondence may be addressed. Email: [email protected] or RNAPs, RNA-dependent RNAPs generally are viral-encoded [email protected]. polymerases, without cellular counterparts, and therefore their This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. interaction with the RNA promoter is a potential target for the 1073/pnas.1406056111/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1406056111 PNAS | Published online July 28, 2014 | E3335–E3342 Downloaded by guest on September 27, 2021 Results Characterizing Interactions Between Influenza RNAP and Single- Stranded RNA. Recombinant RNAP from influenza A/North- ernTerritories/60/68 (H3N2) virus was expressed using the MultiBac system in Sf9 insect cells and affinity purified using IgG-Sepharose followed by size-exclusion chromatography (29). The polymerase was assayed for purity (Fig. S1A) and activity (Fig. S1B) before being used to study binding to short, fluo- rescently labeled RNAs corresponding to the conserved 5′ and 3′ termini by using an electrophoretic mobility shift assay (Fig. 2A). Polymerase binding to 20 nM wild-type (WT) 5′ vRNA was observed, and was reduced significantly when the G2A, A7U, and C9A were introduced into the RNA sequence (30, 31). The polymerase bound less well to a WT 3′ vRNA, and this binding was reduced by introducing mutations (C2U, U7A,

Fig. 1. Two-dimensional models of the vRNA promoter structure. (A) The unbound promoter is largely double-stranded, with base-pairing between the residues that are complementary. The proximal region is defined as residues 1–9, whereas the distal region is defined as residues 11–16 on the 5′ end and residues 10–15 on the 3′ end. (B) The corkscrew model of the vRNA promoter upon polymerase binding. Residues 2 and 3 base pair with residues 9 and 8, respectively, on both the 5′ and 3′ ends, forming two tetraloops.

the viral RNAP is not available; however, functional studies led to the proposal of a corkscrew model, in which residues 2 and 3 base pair with residues 9 and 8, respectively, on both the 5′ and 3′ ends, forming two tetraloops (Fig. 1B) (21). Supportive evidence for the corkscrew derives from studies showing that the hairpin loops are essential for the endonuclease activity of RNAP (22, 23), that the 5′ strand loop is required for polyadenylation of the viral mRNA (24), and that the polymerase is stabilized through interactions with the promoter in the corkscrew conformation (25). However, the proposed corkscrew structure has never been observed directly. Here, we have taken advantage of an insect-cell system to obtain highly purified influenza RNAP free from bound vRNA and used it to quantitatively assess binding of the polymerase to single-stranded genomic terminal sequences using fluorescence- based assays. A single-molecule Förster resonance energy transfer (smFRET) assay with alternating-laser excitation (ALEX) (26–28) allowed quantitation of binding to the dsRNA promoter by dis- tinguishing between double-stranded or single-stranded RNA species. The quantitative information obtained provides in- sight into the molecular mechanisms of influenza transcrip- tion and replication. We also extended our assay to measure FRET between fluorescent dyes located at different positions on the dsRNA promoter to map its structure both with and without the polymerase bound; we confirmed that the un- Fig. 2. Binding of the viral polymerase to single-stranded RNA. (A) Gel-shift bound RNA is a double-stranded helix, and provided direct assay showing binding of the viral polymerase to vRNA templates labeled with ATTO647N. Twenty nanomolars of RNA was incubated with or without structural information on conformational changes in the 20 nM polymerase for 15 min at 28 °C before being analyzed by non-

promoter upon binding. The distances obtained for denaturing electrophoresis and visualized at λex = 640 nm. Sequences of the the RNAP-bound influenza promoter support a 3D model of the RNA templates used are given in the figure; mutations are underlined. (B) dsRNA in a corkscrew conformation. This work has implications Anisotropy curves of the viral polymerase binding to the vRNA templates. for the mechanisms of promoter binding by single-stranded seg- Increasing concentrations of polymerase were incubated with 1 nM ATTO647N-labeled RNA for 15 min at 28 °C, and anisotropy was measured in mented RNA viruses, as well as the development of inhibitors that a scanning fluorimeter (Photon Technology International) with λex = 640 nm. target RNAP functions in this important group of pathogens. Error bars represent the SD from three independent repeats.

E3336 | www.pnas.org/cgi/doi/10.1073/pnas.1406056111 Tomescu et al. Downloaded by guest on September 27, 2021 and G9C) into the template (30, 31). To measure the affinity of concentrations led to formation of only a small amount of the PNAS PLUS binding accurately, we used a homogenous binding assay based RNA–RNAP complex (E* ∼0.8) (Fig. 3 L–N), suggesting that on fluorescence anisotropy, which monitors the degree by which the affinity of the polymerase for this mutant promoter is much light emitted by a fluorophore on the RNA is polarized (32). lower than that for the WT promoter. Titrating increasing amounts of the polymerase while the WT 5′ We note that affinity of the polymerase for the WT dsRNA end promoter strand was kept at a constant concentration led to construct is high, with the complex being clearly detectable even an initial increase in anisotropy corresponding to the formation at protein concentrations as low as 2 nM. Using these binding of the RNA–protein complex, until saturation was reached at data, we quantified the percentage of bound RNA (relative to higher protein concentrations (Fig. 2B). The data were fitted to the total RNA available) at each protein concentration and fitted K ± Materials and Methods give a d of 2.2 0.6 nM ( ). The poly- these values to determine the dissociation constant. A Kd value merase showed a lower affinity for the 3′ end of the viral pro- of 0.4 nM for the 5′ U18(Cy3)/3′ U4(A647N) promoter was obtained moter and did not reach saturation within the range of protein (Fig. 3O); a similar value (0.7 nM) was obtained by using an (Cy3) (A647N) concentrations used, indicating a Kd greater than 1 μM. The poor alternative 5′ U18 /3′ U1 fluorophore labeling scheme binding exhibited by the WT 3′ vRNA was not a result of the (Fig. S5C). presence of the dye, as we obtained similar behavior for a 3′ RNA with a dye placed further downstream (Fig. S2). Weak Analysis of Binding to a dsRNA Promoter Using Single-Molecule binding also was observed during binding studies of the poly- Quenchable FRET. The increase in FRET upon polymerase bind- merase to the mutated 5′ and 3′ vRNAs. As binding to only the ing demonstrates that the polymerase causes a structural change 5′ vRNA reached saturation, we conclude that the RNAP binds in the promoter, which moves the two fluorophores closer to- the 5′ end of the vRNA with significantly higher affinity than the gether. This, however, does not allow us to distinguish between 3′ end. a structural change in the proximal promoter region (residues 1– 9) or in the distal promoter region (residues 11–18). To address The Influenza Polymerase Binds a dsRNA Promoter with High Affinity. this, we used quenchable FRET (quFRET), a fluorescence assay Having characterized the interaction between the RNAP and the that we recently introduced to study bacterial promoter opening individual terminal sequences, we extended our analysis to the (33). When two reporting fluorophores are attached close to double-stranded promoter by using a highly sensitive smFRET each other on opposite strands of a dsRNA (a distance of ∼2nm assay. Here, FRET was measured between a donor Cy3 dye at or less, taking into account the diameter of the RNA helix and position U18 on the 5′ strand and an acceptor ATTO647N dye at the linker lengths of the dyes), they undergo contact-induced position U4 on the 3′ strand of dsRNA molecules diffusing in quenching, which suppresses fluorescence emission. When the solution (Fig. 3A). We used ALEX to allow detection of distinct two strands separate (such as in promoter opening), the emission signatures for all diffusing species (26–28), and calcu- quenching is lifted, energy transfer between the donor and ac- lated two fluorescence ratios: the uncorrected FRET efficiency ceptor is re-established, and a FRET signal is detected. We (E*), which reports on the donor–acceptor distance, and the therefore have used quFRET to study whether the observed ratio S*, which reports on the stoichiometry of donor–acceptor structural change brought about by the polymerase binding to species (Fig. S3). This scheme carries the advantage that doubly the vRNA promoter occurs in the proximal region. labeled dsRNA species can be distinguished from single-stranded We modified the WT dsRNA promoter so that the Cy3 donor donor-only– or acceptor-only–labeled RNAs, thus allowing the dye on the 5′ end was attached to the residue at position 3, specific analysis of polymerase binding to dsRNA rather than to whereas the ATTO647N acceptor dye remained on 3′ U4 (Fig. dissociated single-stranded 5′ or 3′ RNA. The FRET efficiency 4A). No fluorescence was detected using the WT RNA only (Fig. values (E*) obtained for the dsRNA species were depicted as 4B). Upon addition of 2 nM RNAP, a high-FRET population histograms and fitted with Gaussian functions to determine the (E* ∼0.84) was observed (Fig. 4C). Addition of higher concen- mean of the distributions (Fig. 3). trations of protein increased this population, suggesting that A WT, doubly labeled promoter gave a single FRET pop- more complex was formed (Fig. 4 D–G). The loss of quenching ulation with a mean E* of 0.57 (Fig. 3B). Titration with between the two fluorophores when polymerase was added increasing amounts of polymerase (2–50 nM) gave rise to a bi- suggests opening of the dsRNA in the proximal region of the modal FRET distribution (Fig. 3 C–G), indicating the presence promoter. A mutant dsRNA promoter (also 5′ U3(Cy3)/3′ of two species. The first population was attributed to the un- U4(A647N)) (Fig. 4H) was used in a similar experiment; this bound RNA (mean E* ∼0.57), and the second population to promoter showed significantly reduced complex formation dsRNA bound to the polymerase (E* ∼0.79). It is unlikely that compared with WT (Fig. 4 M and N). To address the question of formation of the higher FRET population is the result of whether a structural change occurs in the distal region of the a change in quantum yields of the dyes, because the mean promoter, we also analyzed a WT dsRNA with a donor dye at positions of the stoichiometry histograms do not change as position 18 on the 5′ end and an acceptor dye at position 13 on a function of increasing polymerase concentration (Fig. S4). The the 3′ end (Fig. S6A). The RNA-only population gave a mean increase in FRET therefore demonstrates that a structural FRET value of E* ∼0.82, and addition of 50 nM RNAP did change in the promoter takes place upon polymerase binding, not result in a significant change in the FRET distribution bringing the two fluorophores closer together in space and (E* ∼0.83) (Fig. S6B). Taken together, these data demonstrate therefore resulting in increased energy transfer. A similar con- that the conformational change induced by protein binding to struct carrying the ATTO647N dye at position 1 on the 3′ end the dsRNA promoter occurs in the proximal promoter region. A

(rather than at position 4; Fig. S5 ) showed similar behavior MICROBIOLOGY (Fig. S5B), indicating that the fluorescent dyes do not interfere Distance Determination and 3D Modeling of the Influenza Promoter. with polymerase binding. FRET histograms are commonly depicted in an uncorrected We also tested a polymerase-binding mutant of the dsRNA format, as this represents a minimally processed form of the data promoter (containing the mutations discussed in the previous (refs. 26 and 27 and this paper); however, it previously was section), labeled at position U18 on the 5′ strand and U4 on the shown that FRET data acquired using ALEX can be corrected to 3′ strand (Fig. 3H). The RNA-only population corresponded to determine distances between the reporting fluorophores, which a mean FRET value of E* = 0.59 (Fig. 3I). Addition of either in turn may generate structural information (34). To obtain 2 nM (Fig. 3J) or 5 nM RNAP (Fig. 3K) did not result in any structural information for the influenza promoter structure in change in the FRET distribution, whereas addition of higher the absence and presence of the polymerase, we converted our

Tomescu et al. PNAS | Published online July 28, 2014 | E3337 Downloaded by guest on September 27, 2021 apparent FRET values (E*) into accurate FRET values by correcting for background, cross-talk, and γ-factor effects (the latter accounting for differences in quantum yield and detection efficiency of the fluorophores), and then calculated donor–ac- ceptor distances (Materials and Methods) (reviewed in ref. 35). The distances between the fluorophores located 18 bp (5′ U18 and 3′ U1), 14 bp (5′ U18 and 3′ U4), and 5 bp (5′ U18 and 3′ U13) apart on the free dsRNA were ∼6.3, ∼5.7, and ∼3.7 nm, respectively, whereas the distance between the fluorophores lo- cated at positions 5′ U3 and 3′ U4 was presumed to be ∼2nmor less, because no FRET signal could be detected as the result of contact-mediated quenching (Table 1). The distances obtained for the polymerase-bound dsRNA were ∼5.2, ∼4.6, ∼4.6, and ∼3.0 nm for the dyes located at positions 5′ U18 and 3′ U1, 5′ U18 and 3′ U4, 5′ U3 and 3′ U4, and 5′ U18 and 3′ U13, respectively (Table 1). These experimentally determined distances were compared with 3D models of the labeled dsRNA promoter in either the bound or unbound form. Because no 3D representations of the dsRNA promoter exist, we modeled the structures ab initio. We based our models on 2D secondary structures proposed from functional studies for the unbound promoter (17) (Fig. 5A) and corkscrew (21) (Fig. 5C), which then were translated into 3D space (Materials and Methods). The 3D models for the dsRNA were used as scaffolds for the dyes, and the mean position of each dye was determined using FRET-restrained positioning and screening (FPS) (36, 37). For the free promoter RNA, the donor–acceptor distances based on the 3D model were ∼6.1 nm (5′ U18 and 3′ U1), ∼5.2 nm (5′ U18 and 3′ U4), ∼1.1 nm (5′ U3 and 3′ U4), and ∼3.0 nm (5′ U18 and 3′ U13) (Fig. 5B). These values are consistent with the ones determined experimentally using our corrected FRET efficiencies (Table 1). As a control, we superimposed our 3D structural model with a published NMR structure of the dsRNA promoter (19), and the two were in good agreement (Fig. S7). Having verified that the values obtained from the 3D model of the unbound promoter RNA closely matched our experimentally determined distances, we generated a 3D representation of the polymerase-bound corkscrew promoter structure (Fig. 5D). This was used to measure the distances between the FRET pairs: 5′ U18 and 3′ U1 are located ∼4.7 nm apart, whereas 5′ U18 and 3′ U4, 5′ U3 and 3′ U4, and 5′ U18 and 3′ U13 are located ∼4.6 nm, ∼4.8 nm, and ∼3.2 nm apart, respectively (Fig.5D). These values also are in good agreement with the distances determined ex- perimentally using smFRET (Table 1), strongly supporting the proposal that the promoter is forming a corkscrew structure when bound by the viral polymerase. Discussion Polymerase–Promoter Interactions. In this work, we present in- sights into the mechanisms of promoter binding by the influenza RNAP. No previous studies quantitatively measured the affinity of the individual 5′ or 3′ ends of the influenza A virus vRNA promoter for the trimeric viral RNAP. Here, we show that the RNAP has a significantly higher affinity for the 5′ end than the 3′ end; we also detect an even higher affinity for the dsRNA promoter. Our affinity value is significantly higher than the one reported for the PB1 subunit binding to dsRNA (Kd = 20 nM) (13), likely because of PB1 alone exhibiting different binding Fig. 3. Detecting binding of the viral polymerase to a double-stranded vRNA promoter by using smFRET. WT (A–G) and mutant (H–N) dsRNAs were labeled with donor and acceptor fluorophores at positions 4 and 18 and incubated with increasing concentrations of polymerase at 28 °C for 15 min from fitting of the single peaks obtained for the “RNA only” samples), before smFRET spectroscopy combined with ALEX on diffusing molecules whereas the peaks assigned to the bound fractions were fitted freely. (O) was carried out. Ratio E* represents the uncorrected FRET efficiency, and Peaks assigned to bound WT RNA were used to calculate the percentage curves were fitted with Gaussian functions to determine the center of the bound at each concentration, plotted against polymerase concentration, distributions. Peaks assigned to free WT RNA and free mutant RNA were and fitted with a hyperbolic curve to calculate the dissociation constant. fixed at mean E* = 0.57 and E* = 0.59, respectively (using values obtained Error bars represent the SD from three independent repeats.

E3338 | www.pnas.org/cgi/doi/10.1073/pnas.1406056111 Tomescu et al. Downloaded by guest on September 27, 2021 preinitiation complex. The PB2 subunit of the RNA-bound poly- PNAS PLUS merase binds the 5′ 7-methyl-guanosine cap structure of a cellular mRNA (12, 15); subsequently, an endonuclease activity associated with the PA subunit (38, 39) cleaves the cellular mRNA 10–15 nucleotides downstream of the cap. This generates a short capped RNA fragment that serves as a primer to initiate mRNA synthesis on the vRNA template. At this stage, the 3′ end of vRNA pre- sumably has to transfer into the active site of PB1, where it interacts with the mRNA primer for transcription initiation. Thus, during initiation, the 5′ and 3′ ends of the promoter separate to allow copying of the 3′ template; however, once the 3′ end has been copied, it likely reassociates with the 5′ end, restoring the dsRNA promoter structure. In fact, it has been proposed that the poly- merase association with the 5′ end of the vRNA promoter is the major factor in allowing polyadenylation of viral mRNA (24). Stable association of the polymerase with the 5′ end of the vRNA template while the template is threaded through the active site of the same polymerase inevitably leads to steric constraints. These constraints result in repeated copying of a sequence of five to seven uridine residues close to the 5′ terminus, giving rise to the poly(A) tail of the viral mRNA molecule (reviewed in ref. 9). Our data offer intriguing insights into these processes; the high affinity of the polymerase for the dsRNA promoter may ensure that both ends of the RNA remain bound during transcription elongation so that the poly(A) tail is produced. The low affinity of the 3′ end of the vRNA for the polymerase may allow this end to form multiple low-affinity interactions with the polymerase at distinct sites, i.e., the promoter binding site and polymerization active site. The low affinity between the 3′ end and the poly- merase also may allow read-through during transcription elon- gation, whereas the significantly higher affinity between the 5′ end of the RNA and the polymerase ensures that this end remains bound during this time. This is supported by suggestions that the presence of the 5′ end increases the affinity for the 3′ terminal sequence (13, 14), which may explain energetically why the two ends reassociate. Several models have been proposed for viral replication, during which the negative-sense vRNA is copied into full-length, nonpolyadenylated, positive-sense cRNA which, after assembly into a complementary RNP (cRNP) complex, acts as a template for genomic vRNA synthesis (reviewed in ref. 9). It has been shown that replication of cRNA into vRNA requires additional polymerase molecules (29, 40). According to one model, this additional, “trans-acting” polymerase performs vRNA synthesis, whereas another model proposes that the trans-activating poly- merase performs a nonenzymatic role while replication is carried out by the resident polymerase of the cRNP. It is unclear whether replication of vRNA to cRNA also requires an addi- tional, nonresident polymerase. Common to all replication Fig. 4. Analysis of binding of viral polymerase to a double-stranded vRNA models is the requirement that during the initiation of cRNA – – promoter by using single-molecule quFRET. WT (A G) and mutant (H N) synthesis, the 5′ and 3′ ends of the vRNA promoter must sepa- dsRNAs were labeled with donor and acceptor fluorophores at positions 3 ′ and 4 on opposite strands and incubated with increasing concentrations of rate, and the 5 end of the vRNA template must be released by polymerase at 28 °C for 15 min before smFRET spectroscopy combined with the resident polymerase, independent of whether replication is ALEX on diffusing molecules was carried out. Ratio E* represents the un- performed by the resident or nonresident polymerase. This corrected FRET efficiency. No FRET events were detected with RNA only ensures that the replicating polymerase does not encounter steric because of quenching of the fluorophores (33), whereas the addition of restraints and copies through the sequence of uridine residues polymerase resulted in high FRET peaks centered at E* = 0.84. located near the 5′ terminus. Although these models are im- portant steps forward in our understanding of viral replication,

many questions remain unanswered. For example, the model MICROBIOLOGY behavior to the trimeric polymerase; in support of this, we note that proposes that replication is performed by a trans-acting that the PB2 and PA subunits also have been directly implicated polymerase requires the initial release of the 3′ end by the res- in promoter binding (30, 31). ident polymerase and subsequent binding by a second poly- Current models of influenza virus transcription propose that the merase (reviewed in ref. 9). It is difficult to reconcile our data, in RNAP that is bound to the dsRNA promoter within viral ribonu- which the 3′ end of the vRNA alone has an extremely low affinity cleoprotein (RNP) complexes (introduced into the cell by infecting for the polymerase, with this idea. Our data are more consistent virions) is responsible for carrying out viral mRNA synthesis with a model in which the 3′ end of the template is replicated by (reviewed in ref. 9). The complex of this so-called resident or cis- the resident polymerase, whereas the additional polymerase acting polymerase and dsRNA promoter presumably represents a would be fulfilling a nonenzymatic role, in agreement with the

Tomescu et al. PNAS | Published online July 28, 2014 | E3339 Downloaded by guest on September 27, 2021 Fig. 5. smFRET distances support a corkscrew model of promoter structure upon RNAP binding. (A) Two-dimensional cartoon of the vRNA promoter in the duplex panhandle conformation, showing the fluorophore labeling schemes used to determine accurate FRET and distances. A donor dye was attached to residues 5′ U3 or 5′ U18, and an acceptor dye was attached to residues 3′ U1, 3′ U4, or 3′ U13. (B) Three-dimensional modeled structure of the vRNA promoter in the duplex panhandle conformation. Distances in nanometers between the dyes are in boldface. (C) Two-dimensional cartoon of the vRNA promoter in the corkscrew conformation assumed upon polymerase binding. (D) Three-dimensional modeled structure of the vRNA promoter in the corkscrew conformation (same notation as in B).

trans-activating polymerase model (29). However, it is possible proximal promoter region (residues 1–9), suggesting that the that in vivo other factors also play a role; for example, the in- promoter might be in the corkscrew conformation. teraction of polymerase with the 3′ end of the vRNA may be Investigating the FRET changes by using four distinct labeling stabilized by the presence of the initiating or small schemes allowed us to determine the distances between the vRNAs that correspond to the 5′ end of each of the vRNA fluorophores in our assays. The distances obtained characterized segments and are expressed at high levels in infected cells (41). an RNA molecule in 3D space; however, no 3D structure is currently available for the influenza promoter in the corkscrew Promoter Structure. Functional studies have demonstrated that conformation. The unbound promoter was investigated pre- secondary structure, as well as sequence, of the vRNA promoter viously using NMR (19); however, this structure (Protein Data is crucial for recognition, binding, and function of the influenza Bank ID code 1J07) differs from our construct, as the RNA used polymerase. The promoter is thought to be a largely base-paired was shorter and formed a hairpin. To overcome these limi- helix in the absence of polymerase (42), whereas several 2D tations, we 3D modeled the RNA in both the bound and un- models for the secondary structure of the promoter in the bound bound conformations. Our experimentally determined distances state have been proposed, including a duplex (unchanged from were in good agreement with those derived from our models and the unbound state) (17), fork (31), and corkscrew model (21). support the base-paired helix structure for the free RNA and the Our FRET results are consistent with the unbound dsRNA being corkscrew structure for the bound RNA. In obtaining molecular a base-paired helix and show that a polymerase-dependent distances from accurate FRET measurements, we have made conformational change in the promoter occurs. Using quFRET, several assumptions, including that the R0 value for our dye pair we could show that the conformational change occurs in the is not affected by the microenvironment of the fluorophores, that

E3340 | www.pnas.org/cgi/doi/10.1073/pnas.1406056111 Tomescu et al. Downloaded by guest on September 27, 2021 Table 1. Comparison of the distances between dye pairs on the vRNA promoter determined PNAS PLUS experimentally (from accurate FRET calculations) and through modeling Observed Fluorophore Corrected FRET Distance from corrected Modeled species labeling scheme efficiency FRET efficiency, nm distance, nm

RNA only 5′ U18(Cy3),3′ U1(A647N) 0.42 6.25 6.14 5′ U18(Cy3),3′ U4(A647N) 0.55 5.69 5.17 5′ U3(Cy3),3′ U4(A647N) n/a n/a 1.14 5′ U18(Cy3),3′ U13(A647N) 0.95 3.67 2.98 RNA + RNAP 5′ U18(Cy3),3′ U1(A647N) 0.68 5.19 4.74 5′ U18(Cy3),3′ U4(A647N) 0.82 4.55 4.61 5′ U3(Cy3),3′ U4(A647N) 0.82 4.57 4.76 5′ U18(Cy3),3′ U13(A647N) 0.98 3.03 3.24

n/a, not applicable.

the orientational freedom of the dyes is not affected by the being analyzed by nondenaturing agarose gel electrophoresis and visualized λ = presence of the polymerase (hence, we have used a mean dye- at ex 640 nm. orientation factor of 2/3 in all cases), and that our mean FRET efficiencies represent single-state populations rather than fast- Fluorescence Anisotropy. Increasing concentrations of polymerase were in- interconverting populations with an averaged-out FRET value. It cubated with 1 nM ATTO647N-labeled RNA for 15 min at 28 °C in 50 mM Tris·HCl (pH 8), 500 mM NaCl, 10 mM MgCl2, 100 μg/μL BSA, 1 mM DTT, and should be noted that all these factors may introduce some un- 5% glycerol before being placed in a quartz cuvette. Anisotropy was mea- certainty into our distance assessments, and this may be responsible sured in a scanning fluorimeter (Photon Technology International) with λex = for the small differences between our modeled distances and those 640 nm. The data were plotted in OriginPro 8, and a Kd value was extracted obtained experimentally. by fitting with the following function, where A0 is the initial anisotropy In our experiments using doubly labeled promoter RNA, we value, Af is the final anisotropy value, D is the RNA concentration, and KD is obtained a bimodal FRET distribution (Fig. 3). The low-FRET the binding constant (43): ∼ population (E* 0.57) was attributed to unbound RNA, as it had 0 qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi1 an E* similar to that obtained from fitting of the single peak of ð + + Þ − ð + + Þ2 − @ x D KD x D KD 4DxA the “RNA-only” sample, whereas the higher-FRET population y = A + ðA − A Þ : 0 f 0 2D (E* ∼0.79) was attributed to RNA in the bound state. We note, however, that although the high-FRET population saturates with increasing polymerase concentrations (at ∼70% of the total), the In Vitro Transcription Assays. Transcription was carried out using dsRNA low-FRET population remains. A possible explanation for this is templates in the presence of ApG or ApA as a primer, as previously described that the low-FRET population represents RNA that is bound by (44). Sequences of the RNA templates used are described in the figure leg- the polymerase but has not opened into a corkscrew structure. A end. Reactions were incubated for 2 h at 30 °C before being stopped by addition of loading dye [90% (vol/vol) formamide, 10 mM EDTA, bromo- dynamic equilibrium therefore might exist between the fully phenol blue, and xylene cyanol]; analyzed on a 6-M urea, 20% (vol/vol) duplexed RNA and the corkscrew conformation while the RNA polyacrylamide sequencing gel; and visualized by autoradiography. is bound by the polymerase. The main breakthrough of our study is to provide direct Single-Molecule Fluorescence Spectroscopy. A custom-built confocal micro- structural information on the polymerase-bound RNA structure, scope was used for smFRET experiments as previously described (45–47), and rather than extrapolating from loss-of-function experiments us- the setup was modified to allow ALEX of donor and acceptor fluorophores ing mutagenesis. This work has implications for the development (26, 48). Increasing concentrations of viral RNAP were incubated with 1 nM of specific antiviral inhibitors that target the RNAP interaction ATTO647N-labeled RNA for 15 min at 28 °C in 50 mM Tris·HCl (pH 8), 500 mM μ μ with vRNA in this important group of pathogens. In addition, the NaCl, 10 mM MgCl2, 100 g/ L BSA, 1 mM DTT, and 5% glycerol before being diluted in a buffer comprising 50 mM Tris·HCl (pH 8), 100 mM KCl, 10 mM major advantage of our solution-based FRET measurements is μ μ that it allows the conformational changes of RNA (as well as any MgCl2,100 g/ L BSA, 1 mM DTT, and 5% glycerol to a final RNA concentration of 100 pM for confocal analysis. In a typical experiment, the average excitation dynamics) to be mapped directly in solution, and these tech- μ μ – intensities were 250 Wat532nmand60 W at 635 nm. Custom-written niques might be extended to the study of any virus polymerase LabVIEW software was used to register and evaluate the detected signal. promoter interaction. Data Analysis. Fluorescence photons were assigned to either donor- or ac- Materials and Methods ceptor-based excitation with respect to their photon arrival time. Fluores- RNA and Protein. The RNA strands corresponding to the 3′ and 5′ conserved cence bursts were identified by using a search algorithm that uses a minimum ends of the single-stranded viral RNA segment encoding for the neuramin- of 12 successive photons, with at least 7 neighboring photons within a time idase were custom synthesized by IBA and were labeled with the fluo- window length of 500 μs centered on their arrival time. We then calculated rophores Cy3 (GE Healthcare) and ATTO647N (Sigma), as previously two characteristic ratios, the fluorophore stoichiometry S and apparent FRET described (33), before being purified by gel electrophoresis. Single-stranded efficiency E* for each fluorescent burst, yielding a 2D histogram (26, 33, 48). RNAs were annealed in hybridization buffer (50 mM Tris·HCl, pH 8.0; 1 mM One-dimensional E* distributions for donor–acceptor species were obtained EDTA; 500 mM NaCl). WT and mutant RNA sequences are described in the by using a 0.4 < S < 0.8 threshold. These E* distributions were fitted using MICROBIOLOGY text and figures. Recombinant A/NorthernTerritories/60/68 (H3N2) viral Gaussian functions, yielding the mean E* value for a certain distribution and polymerase with a protein-A tag on the PB2 subunit was expressed using the an associated SD. Mean E* values were converted into accurate FRET effi- MultiBac system in Sf9 insect cells and affinity purified using IgG-Sepharose ciencies (E) as previously described (35); each collected burst was corrected followed by size exclusion chromatography as previously described (29). for background (calculated by subtracting the background count rate in each channel multiplied by the length of the burst), cross-talk (leakage of Electrophoretic Mobility Shift Assay. Twenty nanomolars of single-stranded donor-emission into the acceptor-detection channel and direct excitation of ATTO647N-labeled RNA was incubated with or without 20 nM viral poly- the acceptor dye by donor excitation), and differences in quantum yields merase for 15 min at 28 °C in 50 mM Tris·HCl (pH 8), 500 mM NaCl, 10 mM and detection efficiency for the donor and acceptor (compounded in the

MgCl2, 100 μg/μL BSA, 1 mM DTT, and 5% (vol/vol) glycerol. Reactions were γ-correction factor; Tables S1 and S2). The corrected bursts were collected stopped by addition of 1× reaction volume of 50% (vol/vol) glycerol before into histograms that were fitted to give accurate FRET values (E). Accurate

Tomescu et al. PNAS | Published online July 28, 2014 | E3341 Downloaded by guest on September 27, 2021 FRET efficiencies were converted into molecular distances with the FRET accessible volumes of the dyes and the average dye positions (represented as 6 efficiency equation (E = 1/([1+[(r/R0) ]) using a Förster radius (R0)of5.9nm a sphere) (36, 51). Distances between the average positions of the FRET dye (ATTO-TEC) and assuming a κ2 = 2/3. pairs were determined by using the “measure distance” function available in PyMOL. Three-Dimensional Modeling of the Influenza Virus RNA. Secondary structures for each of the constructs were determined by using the DuplexFold module ACKNOWLEDGMENTS. We thank Mónica Martínez Alonso (University of of the RNAstructure web server (49). Secondary structure information was Oxford) for reagents, and Thorben Cordes (University of Groningen), used in the Chimera plugin Assemble2 (50) to construct 3D models. These Timothy Craggs, George Brownlee, and Frank Vreede (University of structures were exported from Assemble2/Chimera and opened in PyMOL. Oxford) for helpful discussions. This work was supported by European The attachment point for each dye was identified (on the C5′ of the uracil Commission Seventh Framework Program Grant FP7/2007-2013 HEALTH- F4-2008-201418 and Biotechnology and Biological Sciences Research Council base), and information on the dye characteristics was inserted. The Cy3 dye Grants BB/J001694/1 and DKRVYA0/AK (to A.N.K.), by Medical Research was characterized by a linker length of 14.2 Å, a linker width of 4.5 Å, and Council (MRC) Grants G0700848 and MR/K000241/1 (to E.F.), by an MRC dye radii of 8.2, 3.3, and 2.2 Å (x, y, and z, respectively). The ATTO647N dye Doctoral Training Award (to A.I.T.), by Wellcome Trust Studentship was characterized by a linker length of 17.8 Å, a linker width of 4.5 Å, and 092931/Z/10/Z (to N.H.), and by a Junior Research Fellowship from Linacre dye radii of 7.4, 4.8, and 2.6 Å. FPS software was used to calculate the College, Oxford (to N.C.R.).

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