INTRODUCTION
Influenza A virus (IAV) is a major causative pathogen for severe respiratory infections and causes 250,000–500,000 annual fatalities worldwide (
1–3). The recent emergence of IAV strains resistant to commonly used therapeutic agents, such as neuraminidase (NA) inhibitors and viral endonuclease inhibitors, which target essential viral proteins, has accelerated the development of new therapies that target cellular proteins used for viral propagation (
4,
5). Previously, genome-wide RNA interference screening identified 295 host-cell factors required for early-stage IAV replication in A549 human lung epithelial cells (
6). Among them, 10 proteins, including nuclear trafficking protein CSE1L and Ca
2+/calmodulin-dependent protein kinase IIβ(CaMKIIβ), are needed for the post-entry processes of IAV replication. Furthermore, the CaMKII inhibitor KN-93 suppressed IAV replication in cells, indicating that CaMKII is a promising target to protect against IAV infection. However, the efficacy of CaMKII inhibitors has not been shown in an animal infection model, and, importantly, the molecular function of CaMKII in IAV replication is unknown.
CaMKII plays an important role in a wide range of intracellular signaling events that are associated with diverse neuronal functions (
7–13), inflammation, and cardiovascular pathology (
14–18). CaMKII holoenzyme is comprised of a large symmetrical complex of 12 monomers assembled through the interaction of multiple COOH-terminal association domains (
19–22) that results in the cooperative activation of each CaMKII by Ca
2+/CaM (
23). Recently, among the inhibitors of CaMKII (
24,
25), the most widely used inhibitor, KN-93, has been shown to bind directly to Ca
2+/CaM to inhibit its interaction with CaMKII (
26); thus, it may have off-target effects on other Ca
2+/CaM-dependent molecules.
Retinoic acid-inducible gene I (RIG-I), a member of the RIG-I-like receptor (RLR) family, is a key sensor of viral RNAs for the transduction of antiviral signals (
27–29). Activated RIG-I interacts with mitochondrial antiviral signaling protein (MAVS) to induce its activation and oligomerization, stimulating the formation of a signal complex. The complex activates IκB kinase (IKK) and Tank binding kinase-1 (TBK1), resulting in the activation of nuclear factor-κB (NF-κB) and interferon regulatory factor (IRF) 3/7, respectively. This activation induces the expression of type I interferon (IFN) and proinflammatory cytokines, which inhibit IAV propagation through enhanced expression of a series of IFN-stimulated genes (ISGs) (
30). In contrast, the induction of mRNAs for type I IFN and proinflammatory cytokines can enhance IAV propagation. This is due to cap-snatching (
31), in which viral RNA-dependent RNA polymerase cleaves 5′ 7-methyl guanosine (m7G) caps from the predominant host-cell pre-mRNAs, using the caps as primers to initiate viral mRNA transcription (
32,
33). However, the mechanism by which IAV propagates efficiently by cap-snatching in the presence of large amounts of antiviral host protein mRNAs induced by infection is unknown.
In this study, we identified CaMKII inhibitory peptide M3 by targeting the kinase domain using affinity-based screening of a tailored random peptide library, which is highly effective for identifying high-affinity binding motifs against multi-subunit target proteins (
34–38). M3, but not KN-93, efficiently inhibited IAV propagation
in vitro and
in vivo. M3 specifically inhibited the CaMKII-dependent acute-phase activation of RIG-I, which provides small but sufficient amounts of the capped 5’-ends to promote viral mRNA synthesis. The discovery of this novel pathway provides a promising target for the treatment of influenza.
DISCUSSION
In this study, we identified M3 as a novel CaMKII inhibitory peptide by targeting the catalytic domain using affinity-based screening of a random peptide library customized for CaMKII. Our studies with M3 revealed that early in IAV infection, CaMKII activates a novel non-canonical RIG-I pathway that, in turn, activates TBK1 and IRF3, which induce transcription of the genes for IFN α/β and proinflammatory cytokines. The capped 5′-ends of these transcripts are used preferentially to enhance virus propagation via the cap-snatching mechanism. Thus, this pathway acts in an opposite manner compared with the canonical RIG-I pathway, which functions later in infection to induce high levels of mRNA for IFN α/β and then for several antiviral proteins (
Fig. 7). Inhibition of CaMKII by M3 during only the first hour after infection strongly inhibited the cytopathicity of infection (
Fig. 3c), indicating that this pathway provides a promising target for the treatment of influenza.
Here, we identified six high-affinity binding motifs to the catalytic domain of CaMKII that shared the sequence Arg-Arg/Ile-Arg/Ile-Leu-Leu-Leu-Leu-Leu-
Ala-Arg/Leu-His/Leu-His. Notably, the sequences are substantially different from the predicted optimal substrate motif for CaMKII, as determined by a peptide library screening (
53), a representative CaMKII substrate peptide GluN2B (NMDA receptor peptide substrate 1289–1310) (
42), or previously identified CaMKII inhibitory peptides (
24), such as AIP (
54) and CaMKIINtide (
24,
55)—all sequences that are very similar to each other (Fig. S11). Focusing on M3, we characterized the interaction of M3 with the CaMKII catalytic region using molecular dynamics simulations (Fig. S12). In five independent trials, we observed strong interactions between the catalytic cleft of CaMKII-KD and the entire functional region of M3. The binding free energy of the interaction between the catalytic cleft and M3 (−166.94 kcal/mol) was even lower than with CaMKIINtide (−146.78 kcal/mol). In particular, the (−3)Leu of M3 interacts stably with Phe98 of CaMKII-KD through hydrophobic interaction (Fig. S12b), whereas the (−3)Arg of CaMKIINitde interacts electrostatically with Glu96 of CaMKII-KD (
42). The (−5)Ile, which corresponds to the (−4)Leu of M3, and (−2)Ser of CaMKIINitde form hydrophobic interactions with P210 and Trp213 and hydrogen bonds with Glu139 and Lys137, respectively. In contrast, the (−4)Leu and (−2)Leu of M3 fully occupy a hydrophobic pocket comprised of Tyr179, P211, Phe213, Trp214, and Leu221, further indicating a unique and effective inhibitory interaction of M3 with CaMKII-KD.
Here, we found a non-canonical RIG-I pathway that is activated by CaMKII early in infection to enhance virus propagation through cap-snatching. Cells with a
RIG-I KO showed decreased expression of IFN mRNAs and subsequent NP mRNA early in infection, which then protected cells from cytopathicity 24 h after infection. On the other hand, later in infection, a
RIG-I KO abolishes the antiviral activity of the canonical RIG-I pathway, which induces high levels of IFN mRNA expression and then causes the expression of various antiviral proteins, resulting in the induction of delayed but sufficient NP mRNA (
Fig. 5e) to support virus propagation (Fig. S13). Previous studies using
RIG-I KO mice also demonstrated that RIG-I is critical for protection against IAV infection, including the clearance of IAV from the lung through the enhanced expressions of IFNβ (
56,
57). In contrast to the
RIG-I KO cells, M3 treatment of cells specifically inhibited the CaMKII-dependent RIG-I pathway early in infection without affecting the canonical RIG-I pathway. This resulted in highly efficient inhibition of not only the cytopathicity induced by the infection but also virus propagation. The importance of this novel RIG-I pathway in supporting virus propagation was confirmed when the loss of this pathway by
RIG-I KO almost completely inhibited NP mRNA expression up to 6 h after infection (
Fig. 5e), which has not been reported previously.
Previously, it has been shown that the TLR3/4 pathway causes CaMKII activation to induce the mRNA expression of inflammatory cytokines, such as IL-6, TNFα, and IFNα/β in macrophages (
43). However, in this study, the enhanced IFNβ mRNA expression observed early in infection, which was markedly suppressed by M3 and overexpression of kinase-inactive CaMKII mutant (
Fig. 4a and d), was also significantly inhibited by RIG-I knockout (
Fig. 5d). Furthermore, IFNβ mRNA expression induced by 3p-hpRNA, which can specifically activate RIG-I, was efficiently inhibited by M3 and CaMKII knockdown (Fig. S10). These observations clearly indicate that the RIG-I pathway (non-canonical RIG-I pathway), but not the TLR3/4 pathway, is involved in the enhanced IFNβ mRNA expression early in infection.
At present, it is not clear how CaMKII activates RIG-I early in infection. Activation of RIG-I by a series of post-translational modifications (
58) begins with the recognition of viral dsRNA through the C-terminal RNA interaction domain (CTD) that induces a conformational change in RIG-I. Subsequently, dephosphorylation of Ser8 and Thr170 in the two N-terminal caspase activating and recruiting domains (CARD), and Ser854, Ser855, and Thr770 in the CTD by protein phosphatases (PPase), such as PP1-α/γ (
59), stimulate the ubiquitination of each domain by their respective E3 ubiquitin ligases, enhancing their interaction with MAVS that transduce the downstream signaling (
58). The most important difference between the novel CaMKII-dependent RIG-I pathway and the canonical RIG-I pathway is the amount of viral RNAs sensed by RIG-I. The amount of NP RNA at 3 h after infection, when IFNβ mRNA provided a sufficient source of mRNA for cap-snatching (
Fig. 4c), was less than 1% of the amount at 6 or 9 h after infection (
Fig. 3b). LGP2, another RLR family member, which lacks any CARDs and was originally identified as a negative regulator of RLR signaling, facilitates vRNA recognition by RIG-I through its ATPase domain (
60). Thus, it is possible that CaMKII is involved in the LGP2-dependent process by enhancing the sensing of low amounts of viral RNAs by RIG-I. Another possibility is that activated CaMKII enhances F-actin remodeling, as reported previously (
61), and then activates RIG-I by inducing its dephosphorylation. This idea is based on the recent observation that several RNA viruses, including IAV, result in the relocalization of PPP1R12C, a regulatory subunit of PP1-α/γ, from filamentous actin to the cytosol through actin remodeling to allow activation of PP1-α/γ followed by dephosphorylation-mediated priming of RIG-I (
62). However, a better understanding of the precise mechanisms by which CaMKII activates the RIG-I pathway remains to be elucidated.
Coadministration of M3, but not KN-93, with IAV infection rescued mice from the lethality of IAV PR8 infection, indicating that M3 is highly effective against IAV
in vivo. In addition, M3 also efficiently inhibited the cytopathicity induced by other IAV strains, Tokyo/UTHP013/2016 (H1N1 pdm) and Aichi/2/1968 (H3N2), or IBV Wisconsin/01/2010 (Yamagata lineage). RIG-I signaling is also essential for IBV (B/Shangdong/7/97)-induced IRF3 activation and subsequent IFN mRNA expressions in mouse embryonic fibroblasts, occurring earlier and faster than for IAV infection (
63). Thus, M3 may act similarly against IBV and IAV. Our finding that M3 is a novel CaMKII inhibitory peptide and our discovery of the non-canonical CaMKII-dependent RIG-I pathway could provide a new strategy to treat infections with various types of influenza viruses.
MATERIALS AND METHODS
Antibodies
Antibodies were obtained from the vendors and used at the indicated dilution as shown in Table S1 in the supplemental material.
Cell culture experiments
All cell lines were cultured in accordance with the supplier’s recommendation (see Table S2).
Preparation of recombinant CaMKII-KD
Recombinant histidine-tagged CaMKII-KD was expressed in Sf21 cells using baculovirus as follows. A DNA fragment encoding CaMKII-KD was obtained from cDNA of a full length of rat CaMKII by PCR using specific primers (Table S3) and then cloned into a pBacPAK8 transfer vector (Clontech). Recombinant baculovirus was generated using the BacPAK Baculovirus Expression System (Clontech). Sf21 cells were infected with the recombinant baculovirus, and CaMKII-KD was prepared as described previously (
64).
Peptides and library screening
Tetravalent peptide libraries, peptide monomers, and tetravalent peptides were synthesized using N-α-Fmoc-protected amino acids and standard BOP/HOB coupling chemistry as described previously (
34,
65). Recombinant CaMKII kinase domain (0.2 mg) bound to Ni
2+-sepharose beads were incubated with 120 µg of a given library peptide, and the bound peptides were eluted with 30% acetic acid and sequenced on an Applied Biosystems model 477A protein sequencer. The molar ratio of each amino acid recovered from each degenerate position was calculated. The sum of each ratio was normalized to 19 (the number of total amino acids) to evaluate the relative amino acid preference at each degenerate position. Each amino acid would have a value of 1 in the absence of selectivity.
Kinase assay
Autocamtide-2 peptide (Lys-Lys-Ala-Leu-Arg-Arg-Gln-Glu-Thr-Val-Asp-Ala-Leu), which was synthesized as described above, was phosphorylated by CaMKII-KD in the presence or absence of the indicated amount of inhibitory peptides in the kinase buffer (10 mM HEPES-NaOH (pH 7.5), 2 mM MgCl
2, 0.2 mM EGTA, 4 mM β-glycero phosphate, 0.004% NP40, 0.2 mM DTT, 20 µM ATP, 2 µCi [γ-
32P]ATP) for 5 min at 37°C. The amount of radioactivity incorporated into the substrate peptide was determined using the phosphocellulose assay as described previously (
66).
Kinetics analysis of the binding between peptides and CaMKII-KD
The binding of inhibitory peptides to immobilized recombinant CaMKII-KD was quantitated using a BIAcore T100 system instrument (GE Healthcare Sciences, USA) as described previously (
36). Purified His-tagged CaMKII-KD (20 µg/mL) was injected into the system and fixed on the Ni
2+-chelate sensor chip. The resonance unit (RU) is an arbitrary unit (AU) used by the BIAcore system. Binding kinetics were analyzed using BIAevaluation software, v1.1.1 (GE Healthcare Sciences).
ELISA to measure cytokine production
RAW264.7 cells were incubated with each peptide for 30 min and then treated with LPS (10 ng/mL) for 24 h. The culture medium was applied onto each well of a 96-well enzyme-linked immunosorbent assay (ELISA) plate and incubated for 24 h. After blocking, TNFα production was measured using BD OptEIA™ Mouse TNF ELISA Set II (BD Biosciences, Heidelberg, Germany, Cat#558534).
Virus preparation
IAV strain A/Puerto Rico/8/1934 (PR8), mouse-adapted IAV strain A/Tokyo/UTHP013/2016 (H1N1 pdm), mouse-adapted A/Aichi/2/1968 (H3N2), and mouse-adapted type B influenza virus (IBV) B/Wisconsin/01/2010 (Yamagata lineage) were prepared as described previously (
67). These viruses (H1N1 pdm, H3N2, and IBV) were kindly provided by Drs. Yoshihiro Kawaoka and Mutsumi Ito (Division of Virology, Institute of Medical Science, University of Tokyo, Japan).
Cytopathicity assay
Confluent MDCK cell monolayers cultured on a 96-well plate were incubated with various concentrations of compounds at 37°C for 30 min and then infected with IAV strain PR8 at 20 or 0.001 MOI. At the indicated time periods, the relative numbers of living cells were determined using cell count reagent SF (Nacalai tesque) according to the manufacturer’s instructions. KN-93, Baloxavir acid, and GSK8612 were purchased from Wako, Shionogi Inc, Sigma-Aldrich, respectively.
Measurement of virus propagation
Confluent MDCK cell monolayers cultured on a 24-well plate were incubated with various concentrations of M3 at 37°C for 30 min. The cells were infected with IAV at 0.2 MOI in Trypsin(-)-MEM for 16 h. The culture medium was used to determine of the virus titer using a regular plaque-forming assay using the MDCK cell monolayers (
67).
Transfection
A construct for the Flag-tagged human CaMKIIβ was obtained from VectorBuilder Inc. (IL, USA). A kinase-inactive mutant construct with an amino-acid substitution of Lys to Arg (Flag-hCaMKII-K43R) was prepared by PCR. Transfection of the plasmid into MDCK cells was performed using Cell Line Nucleofector™ Kit L (Lonza, Basel, Switzerland) according to the manufacturer’s instructions.
Coprecipitation assay using biotinylated M3
For coprecipitation from cell lysates, MDCK cells cultured on a 10 cm dish were incubated for 30 min at 37°C in the presence or absence of biotinylated M3 (3 µM). The cell lysates were treated with avidin-agarose beads (Sigma-Aldrich) for 1 h at 4°C. After extensive washing, the beads were analyzed by western blot.
Western blot
Western blot analysis was performed as described previously (
36). See Table S1 for antibodies.
Quantitative polymerase chain reaction (qPCR)
After harvesting MDCK cells, total RNA was extracted using the FastGene RNA Premium Kit (NIPPON Genetics Co., Ltd, Tokyo, Japan), and transcribed into cDNA using the ReverTra Ace® qPCR RT Master Mix (TOYOBO, Osaka, Japan) according to the manufacturer’s protocols. For the reverse transcription of NP vRNA, NP cRNA, or NP mRNA, a tag sequence specific for each RNA (Table S3) was used as described previously (
68). Polymerase chain reaction (PCR) was performed for 40 cycles using the obtained cDNA as a template and specific primers (Table S3). The mRNA levels of each gene were quantified by RT-qPCR using THUNDERBIRD® Next SYBR® qPCR Mix (TOYOBO) and the same primers. Data were analyzed by relative quantification based on the ddCt methods using
GAPDH as the reference gene, and expressed as the fold increase over the average mRNA levels of non-treated control cells.
Establishment of MDCK cell clones
MDCK-derived knockout clones of
RIG-I were prepared using CRISPR-Cas9 genome editing (
69). For the construction of sgRNA-Cas9 co-expression vector, DNA fragments (Table S3) were inserted into pSpCas9(BB)−2A-GFP (px458) vector according to the original protocols. px458 vector (Addgene, MA, USA, plasmid #48138) was a gift from Feng Zhang (
69). MDCK cells were transfected with sgRNA-Cas9 co-expression vector. After 72 h, EGFP-positive cells were sorted into 96-well plates using an FACSAria II cell sorter (BD Biosciences). Each clone was screened based on protein expression analyzed by western blot using specific antibodies against RIG-I.
Infection of mice with IAV
Female 6- to 8-week-old, specific pathogen-free BALB/c mice (Shimizu Laboratory Supplies, Japan) were intranasally infected with IAV strains PR8 (2,000 pfu, equivalent to 10 LD50 values) in the presence or absence of the indicated amount of M3 or KN-93. The survival rate was analyzed by Kaplan-Meier survival analysis. Under the same condition, the lung was harvested 3 days after infection and homogenized. After centrifugation, the obtained supernatant was used for the measurement of the expression levels of viral and host mRNAs by qPCR.
Statistical analysis and general methods
Significant differences between the two groups were analyzed using unpaired two-sided Student’s t-test or Welch’s t-test. Multiple comparisons of differences among every group were analyzed using a one-way analysis of variance (ANOVA) followed by Tukey’s test. Significant differences between each group and the control group were analyzed using one-way ANOVA followed by Dunnett’s test. The non-parametric Mann–Whitney U test was also used to analyze significant differences in data distribution between the two groups. Significant differences in survival rates were analyzed using the log-rank test. All statistical analysis was performed using IBM SPSS Statistics software (ver. 28.0.0.0).
ACKNOWLEDGMENTS
We thank Drs. Yoshihiro Kawaoka and Mutsumi Ito (Division of Virology, Institute of Medical Science, University of Tokyo, Japan) for providing virus strain resources.
This work was supported by grants from the Japan Society for the Promotion of Science (JSPS) KAKENHI grant number 21H02629, 21K19344, 22H00432, and JP20H05873, the Japan Science and Technology (JST) SPRING grant number JPMJSP2129, the Research Support Project for Life Science and Drug Discovery (Basis for Supporting Innovative Drug Discovery and Life Science Research; BINDS) from the Japan Agency for Medical Research and Development (AMED) grant number JP22ama121029j0001, JP21am0101093, and JP223fa627005, and the Takeda Science Foundation.
S.H., M.W.-T., H.N., O.J., M.T., T.S., K.M., E.S., T.N., M.F., Y.O., A.I., A. K., K. F., K. I., T.K., N.T.-S., and T.H. performed the experiments, analyzed and interpreted the data. M.W.-T., H.N., and T.N. prepared the recombinant protein and performed peptide library screening experiments. S.H., M.W.-T. and E.S. synthesized the peptides and the peptide libraries. M.T., T.S., and K.M. performed the binding analysis of peptides. T.M. assisted with the qPCR analysis. T.S. and T.H. performed structure-based analysis, interpreted the data, and wrote the manuscript. M.Y. provided virus strain resources. H.T., Ke.N., H.B., and K.N. supervised the project. S.H., T.H., S.T., and K.N. analyzed and interpreted the data, and wrote the manuscript.