The Crystal Structure of the PB2 Cap-binding Domain of Influenza B Virus Reveals a Novel Cap Recognition Mechanism

Background: PB2_cap is critical for the initiation of influenza virus transcription.

Results: FluB PB2_cap binds to GDP and m⁷GDP utilizing unique structural features, which is corroborated by data from ITC.

Conclusion: FluB PB2_cap has a unique cap recognition mechanism compared with FluA PB2_cap.

Significance: We characterize the cap recognition mechanism of FluB PB2_cap, consequently providing insight into inhibitor design targeting FluB PB2_cap.

Abstract

The influenza RNA-dependent RNA polymerase is a core enzyme required for both transcription and replication of the virus RNA genome, making it a potential drug target for the influenza virus. To detect the feature of cap-dependent transcription of influenza B virus (FluB) polymerase, we determined the crystal structures of the wild-type FluB polymerase PB2 subunit cap-binding domain (PB2_cap) with bound GDP and the mutant FluB Q325F PB2_cap with bound m⁷GDP or GDP. These structures revealed that, distinct from influenza A virus (FluA) PB2_cap, the guanine and ribose moieties of substrates invert in FluB PB2_caps. Moreover, we characterized the substrate specificity and affinity of the PB2_caps using isothermal titration calorimetry. FluB PB2_cap has a weaker affinity for m⁷GDP than FluA PB2_cap. Unlike FluA PB2_cap that has a preference for m⁷GDP in comparison with GDP, FluB PB2_cap shows an analogous affinity for both substrates. Replacement of FluB PB2 Glu³²⁵ by Phe, the corresponding residue of FluA PB2, increased the binding affinity of FluB PB2_cap for m⁷GDP to a level approximate to that of FluA PB2_cap and caused a significant higher affinity to GDP. This study indicated that FluB PB2_cap has a unique cap recognition mechanism compared with FluA PB2_cap, providing molecular insight into inhibitor design targeting FluB PB2_cap.

Introduction

The influenza virus is subdivided into three different categories: influenza A virus (FluA),² influenza B virus (FluB), and influenza C virus (FluC). FluA is well studied as it frequently causes both pandemic and seasonal influenza. FluB mainly infects humans as a cause of seasonal influenza and is also detected in seals (1, 2). As an important pathogen of influenza-associated hospitalizations, FluB infection causes influenza and even mortality in humans, predominantly in children (3–5). The report for influenza-associated pediatric mortality showed that FluB, as 26% of circulating influenza viruses, caused 38% of all influenza-associated pediatric deaths in the United States during 2010–2011 (6). The latest report from the World Health Organization FluNet showed that FluB is prevalent as 24.8% of the seasonal epidemics each year and co-circulates with certain subtypes of FluA, such as FluA (H3N2) (29.5%) and FluA (H1N1) pdm09 (16.5%) viruses. In contrast, FluC has a distant evolutionary relationship with FluA and FluB and causes rare but still severe flu and sometimes local epidemics (7).

FluB is a negative-sense RNA virus, and its eight viral genome RNA segments (vRNA) encode 11 proteins. PA, PB1, and PB2 subunits form the influenza RNA-dependent RNA polymerase for both transcription and replication of vRNA. In the host nucleus, the polymerase transcribes vRNA by the “cap-snatching” mechanism (8). In transcription, the cap-binding domain of PB2 (PB2_cap) binds to the 5′ cap structures of host pre-mRNAs (9), and then the cap, together with 10–13 nucleotides downstream of the cap, is cleaved off by the N-terminal endonuclease domain of PA (10, 11). Subsequently, the generated 5′-capped RNA fragment serves as the primer to synthesize viral mRNA by the PB1 subunit. As a milestone contribution, the newest work from Cusack and co-workers (12, 13) clarified the atomic level assembly mode of the whole polymerase, including PA, PB1, and PB2 subunits and the viral RNA promoter.

The N7-methyl group of guanine in the cap structure of eukaryotic mRNA is essential for FluA to prime transcription (14). However, FluB polymerase recognizes not only the N7-methyl group of guanine (m⁷G-capped RNAs) but also the unmethylated GpppG-RNAs efficiently (15). Moreover, FluB polymerase is demonstrated to have a weaker cap binding activity than FluA polymerase (15). A study recently proved that VX-787, which is an inhibitor targeting FluA PB2_cap, is active against FluA but not against FluB (16). Taken together, these data suggest that FluB functions differently from FluA during the cap binding process.

The structures of the cap recognition mechanism for FluA PB2_cap have been extensively studied. The structure of FluA PB2_cap (amino acids 318–483) was first determined by Stephen Cusack and co-workers (9), and the cap-binding pocket complex with the cap analog m⁷GTP was characterized. Furthermore, the conservative characteristics of the cap-binding pocket were evaluated (17), and the unliganded structure was solved (17, 18). Recently, inhibitors targeting the PB2 cap-binding pocket were reported (19, 20). These efforts provided clear atomic information for FluA PB2_cap and indicated that the conserved cap-binding pocket is indeed a potential drug target. However, because several key amino acids in the cap-binding pocket are different from those of FluA, how FluB PB2_cap functions in the cap binding mechanism and whether the cap-binding pocket of FluB is also a suitable drug target still remain to be resolved. Most recently, the structure of the whole FluB polymerase without cap binding for PB2 subunit was reported (12). The substrate binding information is still necessary for understanding the cap recognition mechanism of FluB PB2_cap.

In this study, we first report the crystal structure of FluB Q325F PB2_cap with bound m⁷GDP. Compared with FluA PB2_cap, the structure reveals that the guanine and ribose moieties of m⁷GDP invert, resulting in the N7-methyl group and hydroxyl group of guanine facing outward of the binding pocket. We then solved the crystal structures of wild-type FluB PB2_cap and FluB Q325F PB2_cap with bound GDP. Here GDP shows the same cap binding pattern as m⁷GDP, and the side chain of Gln³²⁵ in FluB PB2_cap-GDP shows conformational flexibility. Moreover, we characterize the cap binding affinity and specificity of these FluB PB2 _caps and compare them with the FluA PB2_cap by isothermal titration calorimetry (ITC). Our results indicate that the FluB polymerase possesses a novel cap recognition mechanism.

MATERIALS AND METHODS

Cloning, Protein Expression, and Purification

The cDNA encoding amino acids 320–484 of FluB polymerase PB2 subunit was obtained from influenza B/Jiangxi/BV/2006.³ The Q325F, W359H, and Y434H mutations (amino acids 320–484) were constructed using a QuikChange^TM site-directed mutagenesis kit (Stratagene) and confirmed by DNA sequencing. FluB PB2_cap (amino acids 318–484) that contains two more wild-type residues Gly³¹⁸-Leu³¹⁹ at the N terminus was cloned and is designated as FluB PB2_cap in the following text. The FluA PB2_cap (amino acids 318–483) gene from influenza A/Puerto Rico/8/34 (H1N1) was cloned as described previously (17, 22). All the cDNAs were digested with NdeI and XhoI and inserted into the pET28a vector with a His₆ tag at the N terminus. The recombinant FluB PB2_cap plasmids were overexpressed in Escherichia coli strain BL21(DE3), and FluA PB2_cap was expressed in E. coli strain Rosetta(DE3). Cells were grown at 37 °C until an A₆₀₀ of 0.8–1.0 was reached, and then the protein expressions were induced with 0.3 mm isopropyl d-1-thiogalactopyranoside (Sigma) at 18 °C overnight. Cells were then harvested in a binding buffer (50 mm Tris, pH 8.0, 500 mm NaCl) and lysed by sonication. The cell lysate was centrifuged twice at 38,900 × g at 4 °C for 30 min each. The supernatant was filtered with a 0.22-μm membrane and purified by metal affinity chromatography using a HiTrap chelating HP column (GE Healthcare). The FluB PB2_cap and FluB Q325F PB2_cap proteins were subjected to thrombin digestion at 4 °C overnight, whereas FluA PB2_cap was digested at 22 °C for 1 h, and then proteins were applied to a nickel affinity column to remove the His tag and undigested protein followed by purification using Superdex 75 gel filtration chromatography (GE Healthcare). The FluB Q325F PB2_cap was further purified by ion exchange chromatography using a Q column (GE Healthcare). The protein was then dialyzed against buffer containing 10 mm Tris, pH 8.0, 200 mm NaCl for crystal screening or buffer containing 10 mm HEPES, pH 7.4, 150 mm NaCl for isothermal titration calorimetry.

Crystallization

10 mg ml⁻¹ FluB Q325F PB2_cap proteins were incubated with 5 mm m⁷GTP (Sigma) and 5 mm GDP (Sigma) separately on ice for 1 h. The crystals used for data collection were obtained via the sitting drop vapor diffusion method at 20 °C. The FluB Q325F PB2_cap with m⁷GTP grew crystal clusters from 0.2 m ammonium acetate, 0.1 m Tris, pH 8.5, 25% (w/v) PEG 3350 after approximately 2 weeks, and a single crystal was extracted for data collection. The crystal of FluB Q325F PB2_cap with GDP grew from 0.15 m dl-malic acid, pH 7.0, 20% (w/v) PEG 3350 within 2–4 days. 10–12 mg ml⁻¹ FluB PB2_cap (amino acids 318–484) was incubated with 5 mm m⁷GTP, 5 mm, and 10 mm m⁷GDP (Sigma), and 5 mm GDP separately for crystal screening purposes, and only the crystal of FluB PB2_capwith GDP was obtained from 0.2 m sodium formate, 20% (w/v) PEG 3350 within 2 days.

Data Collection and Structure Determination

All of the x-ray diffraction data were collected at Shanghai Synchrotron Radiation Facility (China) beamline BL17U. The corresponding reservoir solutions with a gradient of 5 and 10% (v/v) glycerol as a cryoprotectant for crystals of FluB Q325F PB2_cap-m⁷GDP and FluB Q325F PB2_cap-GDP and with 5% (v/v) glycerol for FluB PB2_cap-GDP were used. After soaking, the crystals were flash cooled in liquid nitrogen and maintained at 100 K in cooled nitrogen gas for data collection. All of the data were processed with HKL 2000. The FluB Q325F PB2_cap-m⁷GDP crystal structure was solved by molecular replacement using Molrep in the CCP4 suite (23) with the H3N2 PB2_cap-m⁷GTP structure (Protein Data Bank code 4EQK) used as a search model. The 424-loop was rebuilt manually in Coot (24). The FluB Q325F PB2_cap-GDP and FluB PB2_cap-GDP were solved with the FluB Q325F PB2_cap-m⁷GDP structure used as a search model. All the initial structures were refined by REFMAC5 (25) and built in Coot (24). The structure model of FluB Q325F PB2_cap-GDP was further refined with the geometry restraints by the refinement program in PHENIX (26). The final models were assessed with the program PROCHECK (27). The final statistics for data collection and structure refinement are presented in Table 1.

TABLE 1.

Data collection and refinement statistics

Mol/Asym, molecules per asymmetric unit; r.m.s., root mean square.

Protein Data Bank code	4OR4	4OR6	4Q46
Protein	FluB Q325F PB2cap	FluB Q325F PB2cap	FluB PB2cap
Ligand	m7GDP	GDP	GDP
Data collection
Space group	P 212121	P 212121	P 1211
Cell dimensions
a, b, c (Å)	42.94, 90.70, 95.77	42.97, 90.15, 95.71	43.07, 88.54 52.15
α, β, γ (°)	90, 90, 90	90, 90, 90	90, 105.18, 90
Mol/Asym	2	2	2
Wavelength (Å)	0.9793	1.0000	0.9792
Resolution (Å)	50-2.20 (2.24-2.20)	50-2.30 (2.30-2.36)	50-1.80 (1.83-1.80)a
Rmerge (%)b	16.0 (76.9)	21.2 (83.0)	7.7 (21.7)
I/σ	22.6 (4.6)	10.9 (3.8)	19.9 (8.5)
Completeness (%)	99.9 (100)	99.9 (99.9)	94.6 (98.2)
Redundancy	7.8 (7.9)	9.3 (9.3)	5.5 (5.3)
Refinement
Resolution (Å)	42.35-2.21	30.13-2.29	24.57-1.80
No. reflections	19,227	17,022	31,459
Rwork/Rfree (%)c	17.6/24.2	18.5/25.0	17.0/23.0
Average B-factors (Å2)	26.84	25.22	18.0
Protein (chain A, B)	27.02, 25.88	25.54, 24.37	16.51,17.77
Ligand (chain A, B)	21.87, 24.53	20.59, 22.85	13.79, 14.01
Water	34.91	30.94	30.91
r.m.s. deviations
Bond lengths (Å)	0.022	0.002	0.025
Bond angles (°)	2.197	0.614	2.170
Ramachandran plot (%)
Favored region	97.5	96.9	98.5
Allowed region	2.5	3.1	1.5
Outlier region	0	0	0

^a Numbers in parentheses are for highest resolution shell.

^bR_merge = Σ|I_i − I|/Σ|I_i|.

^c R = Σh|F_o(h) − F_c(h)|/Σ|hF_o(h)|. R_free is calculated using 5% of the data excluded from the refinement.

Isothermal Titration Calorimetry

ITC was carried out at 20 °C with a MicroCal iTC₂₀₀ (MicroCal Inc.). All proteins were desalted into a buffer containing 10 mm HEPES, pH 7.4, 150 mm NaCl with either m⁷GDP or GDP dissolved in the same buffer. For FluA PB2_cap and FluB PB2_cap, 240–280 μm protein (in the cell) and 2.5 mm m⁷GDP or GDP (in the syringe) were used for titration. For FluB Q325F PB2_cap, 85 μm protein (in the cell) and 1.2 mm m⁷GDP or GDP (in the syringe) were used. The first injection of 0.5 μl was followed by 34 injections of 1 μl with a 110-s spacer. All of the data were fitted to a single binding site model using Origin 7.0 program analysis.

RESULTS

The Crystal Structures of FluB Q325F PB2_cap with Bound m⁷GDP and GDP

Unlike FluA PB2_cap, FluB PB2_cap readily precipitated out of solution at room temperature. During protein purification efforts, we noticed that the addition of m⁷GTP improved the stability of FluB PB2_cap, which is similar to FluA PB2_cap. We obtained crystals of m⁷GTP with FluA PB2_cap (17) but not m⁷GTP with FluB PB2_cap. Comparison of the residues of the cap-binding pocket between FluA and FluB revealed that the key difference(s) in the m⁷GTP-binding site (cap-binding pocket) of PB2_cap might result in the weak stability of FluB PB2_cap. Based on the sequence alignment (Fig. 1), Gln³²⁵, Trp³⁵⁹, and Tyr⁴³⁴ are the three key residues in the cap-binding pocket of FluB PB2_cap that are different from FluA, and we thus mutated these residues to the corresponding residues of FluA PB2 (Q325F, W359H, and Y434H) and screened the three single mutations. FluB W359H PB2_cap was insoluble, and FluB Y434H PB2_cap exhibited the same instability as the wild type. However, FluB Q325F PB2_cap showed greater stability than the wild type and was used for further crystallographic studies.

FIGURE 1.

Structure-based amino acid sequence alignments of FluB and FluA PB2_caps. Amino acid sequence alignments of the PB2_caps, including FluA (influenza A/Puerto Rico/8/34 (H1N1)) and FluB (influenza B/Jiangxi/BV/2006), were analyzed by ESPript (37). The secondary structure and amino acid number of FluB PB2_cap-GDP are shown at the top. There are 14 residues associated with substrate binding in FluB or FluA PB2_caps that are marked with blue triangles, and the respective amino acid number is noted for FluB and FluA PB2_caps.

We first solved the crystal structure of FluB Q325F PB2_cap-m⁷GTP at 2.21-Å resolution, and it was refined with an R-factor of 0.176 (R_free = 0.242) (Fig. 2A and Table 1). In the crystal structure, there was clear electron density for the substrate without the γ-phosphate observed (Fig. 3B), and we therefore named this structure as FluB Q325F PB2_cap-m⁷GDP.

FIGURE 2.

Crystal structure of the FluB Q325F PB2_cap with m⁷GDP. A, ribbon diagram of the structure of FluB Q325F PB2_cap-m⁷GDP. The overall structure is in green with m⁷GDP colored purple. B, structural superposition of FluB Q325F PB2_cap-m⁷GDP (colored as in A) with FluA PB2_cap-m⁷GTP (pale cyan; Protein Data Bank code 4EQK). C, close-up view of the cap-binding pocket of FluB Q325F PB2_cap-m⁷GDP. Side chains are shown together with a translucent surface, and hydrogen bonds are indicated by dotted lines. Water molecules W1–W6 are presented as red spheres. D, comparison of the cap-binding pocket between FluB Q325F PB2_cap-m⁷GDP and FluA PB2_cap-m⁷GTP (colored as in B). The structure models in all figures were prepared with PyMOL (21).

FIGURE 3.

Comparison of the cap-binding pockets of FluB Q325F PB2_cap with m⁷GDP and GDP, and FluA PB2_cap-m⁷GTP. A and B, electron density of the cap-binding pocket of molecule A of FluB Q325F PB2-m⁷GDP (purple) with the vertical F_o − F_c map (green) of the N7-methyl group in A and the 2F_o − F_c map (blue) of single m⁷GDP in B. C, the cap-binding pocket of FluA PB2_cap-m⁷GTP. Phe³²³, His³⁵⁷, and Phe⁴⁰⁴ are shown to bind m⁷GTP. D and E, electron density of the cap-binding pocket of molecule A of FluB Q325F PB2-GDP (white) in D with the electron density of single GDP in E. The dotted lines in A, C, and D point to the shortest distance between Phe³²⁵ and N7 of m⁷GDP/GDP/m⁷GTP. The 2F_o − F_c (blue) and F_o − F_c (green; in A) maps contoured at 1.4 and 2.5 σ, respectively, were generated by the FFT program in CCP4 (23) and drawn by PyMOL (21).

The structure of FluB Q325F PB2_cap-m⁷GDP shares a similar conformation with FluA PB2_cap-m⁷GTP (Protein Data Bank code 4EQK) with a 1.31-Å root mean square deviation mainly in the 424-loop and C-terminal loop (Fig. 2B). Surprisingly, FluB shows a distinct binding pattern to the substrate in the cap-binding pocket (Fig. 2, C and D). Compared with FluA PB2_cap-m⁷GTP, the guanine moiety of m⁷GDP in FluB Q325F PB2_cap flips 180° around the long axis of the base, resulting in the N7-methyl group and hydroxyl group facing outward of the binding pocket and leaving room for a water molecule (W3) inside of the pocket. The broadened F_o − F_c map (Fig. 3A, green map) suggests oscillation of the N7-methyl group, which may result because it points out into the solvent. In this case, Lys³⁷⁸ and Gln⁴⁰⁸ lose their interaction to the guanine moiety, whereas Arg³³⁴ forms a new 2.59-Å hydrogen bond with the hydroxyl group. Glu³⁶³ keeps conserved hydrogen bonds with the N1 and N2 of the guanine moiety. Trp³⁵⁹ sandwiches the methylated guanine moiety instead of His³⁵⁷ in FluA PB2_cap. Phe⁴⁰⁶ and Phe³²⁵ (the Q325F mutation) form the same upper cover as FluA PB2_cap for stacking interactions to the guanine moiety (Fig. 3, A and C). Phe³²⁵ has a 4.52-Å distance to the N7 of the guanine moiety (Fig. 3A) compared with the 3.4-Å distance between Phe⁴⁰⁴ and the N7-methyl group in FluA PB2_cap (Fig. 3C). The ribose moiety is also inverted in the structure, and the ribose hydroxyl groups face inward toward the pocket and are close to Phe³²⁷ and Met⁴³³. The O4 of the ribose moiety forms a 2.85-Å hydrogen bond with Trp³⁵⁹. Although the guanine and ribose moieties invert, the diphosphate moiety of m⁷GDP shows a similar trend as that of m⁷GTP in FluA PB2_cap. The diphosphate moiety of m⁷GDP forms hydrogen bonds with Ser⁴³¹ (α-phosphate, 2.78 Å) and Lys³⁴¹ (β-phosphate, 2.69 and 3.18 Å) with three well ordered water molecules internally placed (W4, W5, and W6). Tyr⁴³⁴ loses its interaction with the substrate, whereas in FluA PB2_cap, His⁴³² binds to the α-phosphate of the m⁷GTP at the corresponding position. As found in the FluA PB2_caps structure (9, 17), there are also two water molecules (W1 and W2) that are buried among residues Glu³⁶³, Lys³⁷⁸, and Gln⁴⁰⁸.

The FluB polymerase recognizes both the m⁷G-capped RNAs and the unmethylated GpppG-RNAs efficiently (15). The structure of FluB Q325F PB2_cap-m⁷GDP shows the N7-methyl group pointing out toward the solvent. To test whether the unmethylated GpppG-RNA binds in the same orientation, we determined the crystal structure of GDP-bound FluB Q325F PB2_cap. The crystal structure of FluB Q325F PB2_cap-GDP was obtained within 2–4 days after co-crystallization with 5 mm GDP. The 2.29-Å FluB Q325F PB2_cap-GDP structure was refined with an R-factor of 0.185 (R_free = 0.250) (Table 1). In the cap-binding pocket here, GDP shows the same conformation as m⁷GDP in FluB Q325F PB2_cap-m⁷GDP (Fig. 3, D and E).

Gln³²⁵ Is Flexible in the Cap-binding Pocket of Wild-type FluB PB2_cap

The structure of the wild-type FluB PB2_cap with its substrate is essential for understanding how the wild-type residue Gln³²⁵ interacts with substrate. As mentioned above, we failed to crystallize FluB PB2_cap with m⁷GTP even though the addition of m⁷GTP improved the stability of FluB PB2_cap. Based on the successful crystallization of FluB Q325F PB2_cap with m⁷GDP and GDP, we further screened the crystals of wild-type FluB PB2_cap with m⁷GDP and GDP. We obtained crystals and solved the structure of FluB PB2_cap (amino acids 318–484) bound with GDP but not with the methylated substrate m⁷GDP.

The structure of FluB PB2_cap-GDP was solved to 1.8-Å resolution and refined with an R-factor of 0.170 (R_free = 0.230) (Fig. 4, A and B, and Table 1). The GDP in the structure (Fig. 4, C and D) shows the same orientation as the GDP and m⁷GDP in FluB Q325F PB2_cap (Fig. 3, D and A). There are two molecules in the crystallographic asymmetric unit. In molecule B, the side chain of Gln³²⁵ shows a 3.96-Å distance to GDP (Fig. 4D), and in molecule A, the side chain of Gln³²⁵ is disordered with weak electron density (Fig. 4C). These observations suggest that Gln³²⁵ is flexible and may contribute weakly to cap binding of FluB PB2_cap.

FIGURE 4.

Crystal structure of the cap-binding pocket of FluB PB2_cap-GDP. A, close-up view of the cap-binding pocket of FluB PB2_cap-GDP (FluB PB2_cap is colored cyan, and GDP is colored white). B, electron density of GDP in molecule A of FluB PB2_cap-GDP. C and D, electron density of the cap-binding pockets of molecule A (C) and molecule B (D) of FluB PB2_cap-GDP. E, proposed structural model of FluB PB2 binding m⁷GDP based on the structures shown in Figs. 3, A and C, and 4D. The dotted lines in D and E point to the shortest distance between Gln³²⁵ and N7 of GDP/m⁷GDP. The 2F_o − F_c (blue) map contoured at 1.4 σ was generated by the FFT program in CCP4 (23) and drawn by PyMOL (21).

Structural analysis revealed that the N7-methyl group is oscillating in FluB Q325F PB2_cap-m⁷GDP (Fig. 3A) and that Gln³²⁵ in the wild-type FluB PB2_cap is more flexible than Phe³²⁵ of the mutant (Fig. 4C). These data may explain why we could not obtain the crystal of FluB PB2_cap-m⁷GDP. Based on the crystal structures solved in this investigation, we propose a model for wild-type FluB PB2_cap binding to m⁷GDP (Fig. 4E). In the model, m⁷GDP keeps the same orientation as m⁷GDP in the crystal structure of FluB Q325F PB2_cap-m⁷GDP (Fig. 3A) and is consistent with GDP in FluB Q325F PB2_cap-GDP (Fig. 3D) and FluB PB2_cap-GDP (Fig. 4, C and D). The orientation of Gln³²⁵ is the same as that of Gln³²⁵ in FluB PB2_cap-GDP (Fig. 4D) and is similar to that of Phe³²⁵ in FluB Q325F PB2_cap-m⁷GDP (Fig. 3A) and FluB Q325F PB2_cap-GDP (Fig. 3D). All three available crystal structures and the model presented here show that the guanine and ribose moieties of substrates invert in FluB PB2_caps compared with those of FluA PB2_cap.

FluB and FluA PB2_caps Show Different Substrate Specificity and Affinity

To investigate the cap binding affinity resulting from the structural differences between FluB and FluA PB2_caps, we examined the affinity of m⁷GDP and GDP to FluA, FluB, and FluB Q325F PB2_caps using ITC, respectively (Fig. 5). ITC was carried out at 20 °C in a buffer containing 10 mm HEPES, pH 7.4, 150 mm NaCl. Consistent with previous reports (9), FluA PB2_cap was favored a methylated substrate with a dissociation equilibrium constant (K_d) of 17 μm for m⁷GDP and 118 μm for GDP. In contrast, FluB PB2_cap showed an approximated binding to m⁷GDP (167 μm) and GDP (169 μm) with about one-tenth of the affinity of FluA PB2_cap to m⁷GDP and slightly weaker affinity to GDP than FluA PB2_cap. Notably, the Q325F mutation significantly improves the affinity of FluB PB2_cap to both m⁷GDP (26.8 μm; 6.2-fold) and GDP (4.9 μm; 34.5-fold) and shows great specificity for GDP versus m⁷GDP. Compared with FluA PB2_cap, FluB Q325F PB2_cap shows a similar affinity to m⁷GDP and tighter binding characteristics to GDP. These results indicate that FluB PB2_cap possesses different substrate specificity and affinity compared with those of FluA PB2_cap.

FIGURE 5.

Comparison of the substrate specificity and affinity between FluA, FluB and FluB Q325F PB2_caps by ITC. The binding of FluA PB2_cap, FluB PB2_cap, and FluB Q325F PB2_cap to m⁷GDP and GDP was analyzed by ITC. The raw data are depicted in the top panels with the integrated data in the bottom panels. All data are shown with continuous lines fitting to a one-site binding model. The dissociation equilibrium constants (K_d) are listed in the table. The standard errors of affinity represent data fitted to binding isotherms in the lower panel.

DISCUSSION

Influenza B virus has a devastating mortality rate in humans, especially in children (3, 4, 6), which is an essential characteristic of seasonal influenza. Structural information for FluB PB2_cap, a core executor for the transcription initiation of influenza polymerase, had previously been unknown. The structure of the whole FluB polymerase was reported recently; however, it still lacks substrate binding information for cap snatching (12). Whether FluB PB2_cap functions with the same cap binding mechanism as FluA is unclear. Conversely, the neuraminidase structures of FluA and FluB viruses show a common drug-binding pocket (28); therefore, FluB can be treated with the same clinical drugs, such as oseltamivir (Tamiflu®) and zanamivir (Relenza®), as FluA. As a potential drug target, whether FluB PB2_cap and FluA PB2_cap can bind to the same inhibitors is currently unknown. Herein, we have determined the structures of substrate-binding FluB PB2_cap and FluB Q325F PB2_cap and characterized their substrate specificity and affinity.

Our results suggest that, unlike FluA PB2_cap, FluB PB2_cap has a novel cap recognition feature. In both structures of FluB wild-type and Q325F PB2_caps, the guanine and ribose moieties of m⁷GDP (GDP) invert around the long axis of the base compared with FluA PB2_cap, leaving the N7-methyl group and hydroxyl group of the guanine moiety facing outward and the ribose hydroxyl group facing toward the cap-binding pocket (Fig. 2). According to the structure and sequence alignments between FluA and FluB Q325F PB2_caps (Figs. 1 and 2D), this inversion may be caused by the orientation of Trp³⁵⁹ in FluB PB2_caps, whereas a His³⁵⁷ occupies the same position of FluA PB2_cap.

Lys³⁷⁸ and Gln⁴⁰⁸ of FluB PB2_cap lose their interactions with m⁷GDP (GDP) due to the inversion of the guanine moiety, consistent with the report that these two residues contribute less to cap binding (15). Conversely, Arg³³⁴ of FluB PB2_cap is a newly identified residue utilized for the recognition of the outward facing hydroxyl group of guanine moiety that functions similarly to Lys³⁷⁶ in FluA PB2_cap (at the corresponding position of Lys³⁷⁸ of FluB PB2_cap; Figs. 1 and 2D). Conservatively, Glu³⁶³ of FluB PB2_cap retains recognition of N1 and N2 of guanine moiety as in FluA (Figs. 1 and 2D). In conclusion, the available structural information for PB2_caps indicates that both FluA and FluB need an acidic glutamate and an alternative basic residue for the recognition of the guanine moiety in the cap-snatching mechanism (Fig. 2D).

The combination of Trp³⁵⁹, Phe⁴⁰⁶, and Gln³²⁵ is used to clamp the guanine moiety and is associated with the weak cap binding of FluB PB2_cap to m⁷GDP. In contrast, the mutation Q325F can recover the affinity of FluB PB2_cap to m⁷GDP (Fig. 5). The weak cap binding affinity of methylated substrate to FluB PB2_cap could further explain why the RNA elongation and cap binding activities of the FluB polymerase are lower than those of the FluA polymerase (15). However, it is unclear whether some other factors contribute to the unexpectedly low affinity of the FluB PB2_cap to substrates under physiological conditions.

The inversion of the guanine moiety also allows FluB PB2_cap to accept GDP well. The ITC results revealed that FluB PB2_cap has an equal affinity for GDP and m⁷GDP (Fig. 5). The side chain of Gln³²⁵ shows conformational flexibility in molecule A of FluB PB2_cap-GDP (Fig. 4C). These observations suggest that the flexibility of Gln³²⁵ may contribute to the weak recognition of the N7-methyl and unmethylated substrates.

In contrast, both FluB Q325F PB2_cap and FluA PB2_cap could distinguish m⁷GDP from GDP (Fig. 5) because Phe³²⁵ in FluB Q325F PB2_cap and Phe⁴⁰⁴ in FluA PB2_cap are used for rigid packing to the N7-methyl group (Fig. 3, A and C). In a comparison of FluA PB2 with FluB PB2, FluB PB2 could recognize GpppG for cap primer generation, although it fails in elongation (15). It has been reported that the transcript from the non-methylated cap primer could not be translated by host ribosomes (29–31). Whether the recognition of non-methylated cap primer by FluB has a physiological role is unclear; therefore further investigation is needed.

Cap-binding proteins in humans, such as the heterodimeric nuclear cap-binding complex (32) and eIF4E (33), show a higher affinity to methylated substrates and significant discrimination against non-methylation (34–36). Therefore, the cap recognition of FluB PB2_cap for both m⁷GDP and GDP with a low affinity provides a good opportunity for rational inhibitor design. First, the binding cavity allows FluB PB2_cap to accommodate both GDP and m⁷GDP derivatives. Second, a modification of N3 of the guanine moiety, such as the addition of a hydroxyl to N3 (Protein Data Bank code 3G9L), may recover its interaction with Lys³⁷⁸ and Gln⁴⁰⁸. FluA PB2_cap may also utilize this modification, which has the potential to interact with Arg³³² of FluA PB2_cap (Fig. 2D). Third, N7 of the guanine moiety could be modified to interact with Gln³²⁵. Finally, modification of the ribose hydroxyl groups facing inward toward the pocket is likely to build a stronger interaction to Tyr⁴³⁴ or Phe³²⁷. In summary, our data have provided the structural and biochemical basis for cap recognition of FluB PB2_cap, which in turn helps in understanding how FluB PB2_cap executes the cap binding process and provides further insight into rational inhibitor design targeting FluB PB2_cap.