Interferon-stimulated gene TDRD7 interacts with AMPK and inhibits its activation to suppress viral replication and pathogenesis

ABSTRACT Many viruses activate cellular autophagy in infected cells to facilitate their replication. Recently, we identified an interferon (IFN)-stimulated gene (ISG) Tudor domain containing 7 (TDRD7), which inhibits viral replication by blocking autophagy pathway. Here, we present a molecular mechanism for TDRD7 action and its relative contribution to protection against viral pathogenesis. TDRD7 inhibited the activation of adenosine monophosphate (AMP)-activated protein kinase (AMPK), a kinase required for initiating autophagy. Mechanistically, TDRD7 interacted directly with AMPK in the cytosolic compartment. Domain-mapping analyses revealed C-terminal Tudor domain of TDRD7 interacted with auto-inhibitory domain of AMPK. Deletion of Tudor domains abolished anti-AMPK and antiviral activities of TDRD7. We evaluated physiological relevance of TDRD7 function against viral replication using newly engineered TDRD7 knockout mice and the derived primary cells. TDRD7 knockout primary cells displayed increased AMPK activation, which led to a higher viral load. Subsequently, TDRD7 knockout mice showed enhanced susceptibility upon intranasal Sendai virus infection. Therefore, our study revealed a new antiviral function of IFN, mediated by TDRD7-AMPK, inhibiting viral replication and pathogenesis. IMPORTANCE Virus infection triggers induction of interferon (IFN)-stimulated genes (ISGs), which ironically inhibit viruses themselves. We identified Tudor domain-containing 7 (TDRD7) as a novel antiviral ISG, which inhibits viral replication by interfering with autophagy pathway. Here, we present a molecular basis for autophagy inhibitory function of TDRD7. TDRD7 interacted with adenosine monophosphate (AMP)-activated protein kinase (AMPK), the kinase that initiates autophagy, to inhibit its activation. We identified domains required for the interaction; deleting AMPK-interacting domain blocked antiAMPK and antiviral activities of TDRD7. We used primary cells and mice to evaluate the TDRD7-AMPK antiviral pathway. TDRD7-deficient primary mouse cells exhibited enhanced AMPK activation and viral replication. Finally, TDRD7 knockout mice showed increased susceptibility to respiratory virus infection. Therefore, our study revealed a new antiviral pathway of IFN and its contribution to host response. Our results have therapeutic potential; a TDRD7-derived peptide may be an effective AMPK inhibitor with application as antiviral agent.


IFN-stimulated gene TDRD7 interacts with AMPK and inhibits its activation
In recent studies, we reported TDRD7 as a new viral restriction factor, which inhibits cellular autophagy pathway to suppress viral replication (9,10).Tdrd7 knockdown mouse cells, as expected, displayed increased LC3-II, a marker of autophagy, upon SeV infection compared to control cells (Fig. 1A, lanes 2 and 4).We noted increased LC3-II levels in Tdrd7-knockdown uninfected cells (Fig. 1A, lanes 1 and 3), indicating Tdrd7 also inhibited basal autophagy.We previously showed TDRD7 blocks autophagy by interfering with the autophagy-initiating kinase AMPK; however, the mechanism is unknown.We used serum starvation (SS) and AICAR, two known activators of AMPK, for investigating TDRD7-mediated AMPK inhibition.SS-induced activation of AMPK, analyzed by its phosphorylation on Thr 172 (pAMPK) in TDRD7 knockout cells, was inhibited by restora tion of TDRD7 expression (Fig. 1B).TDRD7 expression also inhibited pAMPK, induced by AICAR, a pharmaceutical activator of AMPK (Fig. 1C).To investigate the molecular basis of antiAMPK activity of TDRD7, we inquired whether TDRD7 and AMPK formed a complex.We used ectopic co-expression of epitope-tagged TDRD7 and AMPK in HEK293T cells, followed by co-immunoprecipitation (co-IP).Our results indicate AMPK co-immunoprecipitated with TDRD7 (Fig. 1D).We validated co-IP results using confocal microscopy, which showed robust co-localization of AMPK and TDRD7 proteins (Fig. 1E).We confirmed these results by proximity ligation assay, which detects closely localized proteins (Fig. 1F).Similar to ectopically expressed proteins, endogenous TDRD7 and AMPK proteins also co-localized, as analyzed by confocal microscopy (Fig. 1G).Together, TDRD7-mediated inhibition of AMPK was accompanied by their interaction.

TDRD7 interacts with regulatory domain of AMPK via its C-terminal Tudor domain
Since TDRD7 and AMPK co-expression led them to interact, we mapped the interacting domains between the two proteins.We designed N-terminally epitope-tagged TDRD7 mutants by deleting functional [OST (∆OST) and Tudor (∆Tud)] domains (Fig. 2A).We validated, using co-IP assay, both human and mouse TDRD7 proteins interacted with AMPK (Fig. 2B).Deletion of all three Tudor domains (ΔTud) abolished AMPK binding, indicating that Tudor domains were required for AMPK interaction (Fig. 2B).To determine the subcellular localization of TDRD7:AMPK complex, we isolated cytosolic, nuclear, and mitochondrial fractions from the co-expressed cells.Co-IP results indicate that TDRD7:AMPK complex was localized predominantly in cytosolic but not nuclear or mitochondrial fractions (Fig. 2C).We confirmed the requirement of Tudor domain for interaction by co-IP analysis in cytosolic fractions, in which only the full-length TDRD7, but not its ΔTud mutant, interacted with AMPK (Fig. 2D).To further narrow down the interacting domain, we tested additional deletion mutants of TDRD7 (ΔOST, ΔTud 2+3 , and ΔTud 3 ) using co-IP, which revealed that ΔOST mutant interacted with AMPK (Fig. 2E).Deletion of either Tud-3 alone (ΔTud 3 ) or Tud-2 and Tud-3 together (ΔTud 2+3 ), however, blocked AMPK interaction (Fig. 2E), indicating that Tud-3 was critical for TDRD7:AMPK interaction.To confirm these results, we performed reverse co-IP by pulling down His-AMPK, which showed strong interaction with Wt and ΔOST but not with Tud-deleted mutants (Fig. 2F).Therefore, Tudor domains of TDRD7 were sufficient for AMPK binding.To confirm ΔOST fragment was sufficient for directly binding AMPK, we expressed them bacterially and purified the recombinant proteins (Fig. S1).Recombinant ΔOST and AMPK were used for in vitro interaction, which confirmed that ΔOST interacted directly with AMPK (Fig. 2G).These results, together, demonstrated that Tud-3 domain of TDRD7 was critical for direct interaction with AMPK.
We next mapped TDRD7-interacting domain of AMPK by deleting its critical func tional domains, as shown in Fig. 3A.Using co-IP, we revealed Wt and catalytic domaindeleted (ΔCat) AMPK interacted with TDRD7 (Fig. 3B and C).Deletion of auto-inhibitory domain (AID) in ΔCat + AID mutant, however, inhibited TDRD7:AMPK interaction (Fig. 3C).As a negative control, we used ΔTud fragment, which, as expected, did not interact with Wt AMPK (Fig. 3C).These results indicated that AMPK AID was required for interac tion with TDRD7.Next, we used C-terminal Tud domain, the Tud-3 fragment (Fig. 3B), and examined its interaction with AMPK and its mutants.Co-IP analyses revealed that Tud-3 interacted with Wt and ΔCat but not with ΔCat + AID, as observed for Wt TDRD7 (Fig. 3D).These results demonstrated that Tud-3 was sufficient for interacting with AID of AMPK.Domain mapping results, together, enabled us to model a putative TDRD7:AMPK complex by molecular docking of the Tud-3 and AID fragments, as we have done previously with other proteins (25,26).Modeled complex indicated putative contact sites between the two proteins (Fig. 3E).Together, TDRD7 and AMPK interacted directly using specific domains.

TDRD7:AMPK interaction is required for antiAMPK and antiviral activities of TDRD7
To determine whether TDRD7:AMPK interaction is responsible for TDRD7-mediated inhibition of AMPK, we expressed Wt TDRD7 (AMPK interacting) and ΔTud mutant (AMPK noninteracting) in TDRD7 KO human cells at similar protein levels (Fig. 4A).In control (empty vector [EV]) cells, AICAR induced robust pAMPK, which was inhibited by Wt TDRD7 but not ΔTud (Fig. 4B and C).SS-induced pAMPK, similarly, was also inhibited by Wt TDRD7 but not ΔTud (Fig. 4D and E).These results indicated that Tudor domains were critical for inhibiting AMPK.Next, we examined whether ΔOST, which interacted directly with AMPK, was sufficient for AMPK inhibition.SS-induced pAMPK, indeed, was strongly inhibited by both Wt and ΔOST mutant of TDRD7, compared to control (EV) cells (Fig. 4F).Similarly, SeV-mediated pAMPK was also inhibited in ΔOST-expressing cells (Fig. 4G).These results demonstrated that ΔOST was sufficient for antiAMPK activity of TDRD7.
In previous studies, we demonstrated that antiviral function of TDRD7 depends on its antiAMPK activity; AMPK inhibitor alleviates the antiviral activity of TDRD7 (10).To investigate this genetically, we used Wt TDRD7 (AMPK inhibitory) and ΔTud (AMPK noninhibitory) against HSV-1 and SeV, which require AMPK activity for viral replication (9,10).Ectopic expression of Wt TDRD7 but not ΔTud mutant in HEK293T cells inhibited ICP0 expression in HSV-1-infected cells (Fig. 5A).Encouraged by these results, we and His-AMPK plasmids; AMPK was pulled down by Ni-NTA-agarose from the cell lysates and analyzed for TDRD7 by immunoblot.(G) TDRD7 ΔOST mutant and AMPK were expressed bacterially; the recombinant proteins were used for in vitro interaction followed by co-immunoprecipitation, as indicated.FL, full length.
The results are representative of at least three experiments.examined viral replication in stably expressed cells.Stable expression of Wt TDRD7 but not ΔTud mutant inhibited ICP0 and ICP8 expression in TDRD7 KO cells (Fig. 5B).As expected, ΔOST, which was sufficient for interacting with and inhibiting AMPK, sup pressed HSV-1 replication, similar to Wt TDRD7 (Fig. 5C).Infectious virion release, as expected, was inhibited by Wt TDRD7 and ΔOST but not ΔTud (Fig. 5D).We further examined the antiviral function of TDRD7 and its mutants against SeV; ΔOST mutant inhibited SeV viral protein (SeV C) expression (Fig. 5E).Antiviral activities of TDRD7 mutants were further quantified using a flow-cytometry-based assay for viral infectivity, which we used previously (9).Wt TDRD7 significantly inhibited SeV replication compared to EV control (Fig. 5F).As expected, ΔOST but not ΔTud mutant also inhibited SeV replication (Fig. 5F).These results demonstrated TDRD7 interacted with AMPK via its Tudor domain to inhibit AMPK activation and virus replication.

Tdrd7 knockout primary cells and mice exhibit increased AMPK activation, viral replication, and pathogenesis
To evaluate the relative contribution of Tdrd7 to prevent viral infection and pathogenesis, we generated Tdrd7 conditional knockout mice by crossing floxed (fl)-Tdrd7 (Tdrd7 fl/fl ) mice with Cre CMV deleter mice (Fig. 6A and B).We confirmed Tdrd7 deletion in tissues (Fig. 6C) and primary mouse embryonic fibroblasts (MEFs) (Fig. 6D), isolated from Tdrd7 fl/fl and Tdrd7 Δ/Δ mice.In primary MEFs, Tdrd7 was expressed in naïve cells, and treatment with IFNβ or transfection of polyI:C strongly upregulated its expression in Tdrd7 fl/fl but not in Tdrd7 Δ/Δ cells (Fig. 6D).We tested whether endogenous TDRD7 and AMPK proteins interact in mouse tissues; co-IP results from liver homogenates indicated interaction between these proteins (Fig. S2).We examined whether Tdrd7 deficiency led to increased AMPK activation.In primary MEFs, SS-induced pAMPK was enhanced in Tdrd7 Δ/Δ MEFs, compared to Tdrd7 fl/fl cells (Fig. 6E).In Tdrd7 Δ/Δ primary splenocytes, SS-induced pAMPK was increased compared to Tdrd7 fl/fl cells (Fig. 6F).Tdrd7 knockout primary cells displayed increased AMPK activation compared to the control cells.
We next examined viral load in Tdrd7 Δ/Δ primary cells; in Tdrd7 Δ/Δ primary splenocytes and BMDMs, levels of SeV transcript and genomic RNA were enhanced, compared to Tdrd7 fl/fl cells (Fig. 7A through D).To investigate the role of Tdrd7 against viral pathogene sis, we infected Tdrd7 fl/fl and Tdrd7 Δ/Δ mice intranasally with SeV and analyzed for pAMPK and viral antigen levels in infected lungs and monitored body weight changes.Tdrd7 Δ/Δ mouse lungs showed increased pAMPK compared to control mice (Fig. 7E).Tdrd7 Δ/Δ mouse lungs showed increased SeV antigen levels, compared to control mice, as analyzed by immunohistochemistry (Fig. 7F; S3A).Infectious viral load also significantly enhanced in Tdrd7 Δ/Δ mouse lungs compared to the control mice (Fig. 7G).Elevated levels of viral antigen expression and infectious viral load in Tdrd7 Δ/Δ mice led to increased lung pathology.hematoxylin and eosin (H&E) staining revealed increased immune cell infiltration in Tdrd7 Δ/Δ lungs, compared to Tdrd7 fl/fl control (Fig. 7H; S3B).As a result, Tdrd7 Δ/Δ mice were more susceptible to infection, as analyzed by loss in body weight, compared to the Tdrd7 fl/fl mice (Fig. 7I).In summary, Tdrd7 deficiency led to increased AMPK activation and viral infection in primary cells and mice.

DISCUSSION
In previous studies, we showed TDRD7 is a novel ISG and inhibits virus-induced autoph agy by interfering with AMPK activation to suppress virus replication.Here, we demonstrated a molecular mechanism for TDRD7-mediated inhibition of AMPK (Fig. 8).TDRD7 interacted directly with AMPK via specific domains, and the interaction was required for its antiAMPK functions.TDRD7 consists of RNA-binding domains (OST), and arginine-methylated protein binding domains (Tudor) (27).Our results suggest that Cterminal Tudor domain (Tud-3) of TDRD7 interacted directly with AID of AMPK.Molecular modeling of TDRD7:AMPK complex revealed a putative interface between Tud-3 and AID, and future studies will further narrow down the specific amino acids involved in this interaction.Since AMPK gets activated by LKB1:AMPK complex formation on lysosome membrane, we speculate that TDRD7 likely targets activation step by disrupting the complex.AMPK activators are used clinically to treat diabetes (28,29); however, unregu lated AMPK activities may be harmful.A small peptide of TDRD7 blocking AMPK may inhibit these effects.The TDRD7:AMPK interacting domains may also be therapeutically targeted for potential anti-AMPK and antiviral compounds.Viruses, however, can evade cellular antiviral response by inhibiting this complex.Autophagy-regulatory activity of TDRD7 has also been demonstrated in lens development and spermatogenesis by preventing autophagosome from fusion with lysosome (30).Therefore, TDRD7 likely regulates autophagy by targeting multiple steps of the pathway.Using Tdrd7 knockout mice and derived primary cells, we evaluated the physiological relevance of Tdrd7 functions.Tdrd7 knockout primary cells exhibited enhanced AMPK activation and elevated viral load.Tdrd7 knockout mice displayed increased susceptibil ity to SeV pathogenesis compared to the Wt control.A recent study using transcriptomic screens of humans, ferrets, and mice revealed TDRD7 as an antiviral ISG against influenza A virus (31).TDRD7 is known to localize in cytoplasmic RNA granules to regulate lens development (32).TDRD7 regulates the mRNAs critical for lens development, and as a result, Tdrd7 mutant mice develop cataracts.A recent study showed that TDRD3 is an antiviral protein and functions via recruitment to stress granules (SGs) and modulating innate immune signaling (33).TDRD3 containing SGs recruit effector innate immune signaling proteins, TRAF3 and IRF7.Whether TDRD7 also functions via SGs for its antiviral response will require future investigation.The autophagy-regulating functions of TDRD7 may also be related to its SG localization and the ability to bind RNA.Our results have implications for interplay between IFN response and metabolic pathway.IFN has earlier been shown to trigger intracellular PI3K signaling to interfere with AMPK activation (34).Therefore, TDRD7 may be an effector ISG for IFN-mediated inhibition of AMPK, suggesting that an IFN-inducible protein can further amplify IFN's anti-AMPK effects.Because viruses utilize AMPK differentially, it will be interesting to investigate whether IFN-mediated direct AMPK inhibition has any role in viral infection, particularly where IFN is deleterious to the host.On the other hand, AMPK has been shown to regulate IFN synthesis and signaling (22,23).AMPK regulation by an ISG may provide a regulatory circuit for cellular IFN response.Because AMPK has largely been studied in the context of autophagy, its role in autophagy-independent response is unclear.Our recent study in autophagy-deficient cells revealed that HSV-1 utilizes an autophagy-independent function of AMPK (10,35).Future studies will reveal the nature of such a function and whether IFN and its induced proteins may also influence those.Since IFN treatment or virus infection triggers induction of many ISGs, some may also share anti-AMPK function.Screening of an AMPK-interacting motif may help identify additional anti-AMPK ISGs.
How viruses activate and utilize AMPK is unclear; the answer depends on the virus (16).Our studies indicate that SeV takes advantage of the AMPK-autophagy pathway for effective replication, whereas HSV-1 utilizes an autophagy-independent function of AMPK (9).AMPK is a well-studied metabolic kinase, and its activation is regulated by cellular energy levels (12,13).Low cellular AMP causes activation of LKB1, which directly phosphorylates Thr 172 of AMPK.Active AMPK is a protein complex of α, β, and γ subunits, which form a productive assembly for subsequent downstream functions.AMPK α (E) Tdrd7 fl/fl and Tdrd7 Δ/Δ mice were infected intranasally with SeV (125,000 pfu/mouse).Lung lysates from two mice from each genotype were analyzed for pAMPK 7 days post-infection (dpi) by immunoblot (E).(F-I) Tdrd7 fl/fl and Tdrd7 Δ/Δ mice were infected intranasally with SeV (as in panel E), and the following analyses were performed: lung sections from the SeV-infected mice (7 dpi) were immuno-stained with antiSeV antibody and analyzed by confocal microscopy (F; scale bar, 500 µm); SeV infectious load in lung homogenates was analyzed by plaque assay (G); lung sections were H&E stained for histological analyses (H; scale bar, 100 µm), and the infected mice were monitored for body weight post-infection (I).The results are representative of three experiments; the data represent mean ± SEM, *P < 0.05. subunit has α1 and α2 isoforms, which expressed cell type specifically and activated simultaneously via phosphorylation of consensus Thr 172 residue (13,36).In addition to LKB1, AMPK can be directly phosphorylated by CaMKK, which gets activated by increased Ca 2+ levels.Many viruses cause increased Ca 2+ levels during infection, activat ing AMPK via CaMKK.In the current study, we focused on the phosphorylation of Thr 172 , which leads to downstream functions of AMPK.Moreover, AMPK activation is regulated by other posttranslational modifications, e.g., AKT-mediated phosphorylation of Ser 485 inhibits phosphorylation of Thr 172 (21,(36)(37)(38).Hepatitis C virus regulates AMPK activity using this mechanism (21).Alternatively, TDRD7 may interact with AMPK to recruit AKT to trigger phosphorylation of Ser 485 .Future studies will test a possibility that TDRD7 triggers dephosphorylation of pThr 172 by activating and recruiting the phosphatase PP2A (39).However, TDRD7 may rely on these strategies to efficiently inhibit the proviral AMPK activity.
In summary, our results provide a molecular mechanism for IFN-mediated inhibi tion of AMPK.The mechanism may apply to other protein inhibitors of AMPK and other ISGs that exhibit antiviral activity by inhibiting AMPK.TDRD7 mutations lead to cataract formation, and studies are required using the cells from these individuals to determine whether AMPK pathways are altered and whether AMPK activity regulates lens development.Primary cells from individuals with TDRD7 mutations would further provide a translational potential of our results.Various inducers, e.g., virus infection, serum starvation (SS), or AICAR, activate LKB1, the kinase that directly phosphorylates AMPK.Inhibition of AMPK activation by TDRD7 is a molecular basis for its viral restriction activity.The TDRD7-AMPK branch is a novel antiviral pathway of the IFN system.

Knockdown and ectopic expression
For generating stable Tdrd7 knockdown mouse cells, the shRNA CAGGATTTGCCTCA GATTA was lentivirally expressed, and transduced cells were selected in the puromycincontaining medium, as described before (9).The stable knockdown cells were evaluated for levels of Tdrd7 by qRT-PCR.Human cell lines, stably expressing epitope (V5) tagged Wt or mutants of TDRD7, were generated in TDRD7 KO HT1080 cells.These cells were generated by lentiviral transduction followed by selection in the puromycin-containing medium.The stable cells were used for viral infection and other biochemical analyses.

Virus infections
SeV Cantell (VR-907) and SeV 52 (VR-105) strains (Charles River) and HSV-1 KOS strain (40), and the infection procedures have been previously described (10).Briefly, the cells were infected by the viruses (at an multiplicity of infection [MOI] specified in the figure legends) in serum-free DMEM for 2 h, after which the cells were washed and replaced with the normal growth medium.The virus-infected cells were analyzed at indicated time for viral protein expression or as described in figure legends.For quantification of infectious HSV-1 particles in the culture medium, the TCID 50 assay was performed as described previously (10,41).

Treatment of cells for analysis of AMPK activation
For analyses of AMPK activation through serum starvation, the cells were washed with and incubated in serum-free DMEM for 4 h or the time period indicated in figure legends or treated with 500 µM of AICAR for 2 h.The cells were harvested, and pAMPK was analyzed by immunoblot.

Cell lysis and immunoblot
Immunoblot analyses were performed using previously described procedures (9,42).Briefly, cells were lysed in 50 mM Tris buffer, pH 7.4 containing 150 mM of NaCl, 0.1% Triton X-100, 1 mM sodium orthovanadate, 10 mM of sodium fluoride, 10 mM of β-glycerophosphate, 5 mM sodium pyrophosphate, protease, and phosphatase inhibitors (Roche).Total protein extracts were analyzed by SDS-PAGE followed by immunoblot.The immunoblots were developed using a Syngene imaging system and processed using Adobe Photoshop for further analyses.The density of protein bands on the immunoblots was quantified, wherever indicated, using the Image J program.

Generation and expression of TDRD7 and AMPK mutants
Expression vectors of human and mouse, TDRD7/Tdrd7 gene, were obtained from Origene and subcloned with V5-tag into lentiviral vector pLVX-IRES-puro.AMPK expression plasmid was obtained from Addgene and was subcloned into pLVX-IRESpuro as HA or His-tagged AMPK.The TDRD7 and AMPK mutants were generated either by megaprimer PCR or NEB site-directed mutagenesis kit, and the mutations were confirmed by sequencing and immunoblot analysis.Recombinant TDRD7 (ΔOST, doubly-tagged with His and V5) and AMPK (doubly-tagged with His and HA) proteins were purified from Escherichia coli BL-21 strain using the previously described procedure (43).

Co-immunoprecipitation and in vitro protein-protein interaction assay
Co-immunoprecipitation assays were performed by co-transfection of epitope (HA or His)-tagged AMPK and V5-tagged TDRD7 constructs for 24 h followed by lysis in 3-[4-(2-hydroxyethyl)piperazin-1-yl]propane-1-sulfonic acid (EPPS) buffer containing protease inhibitors by repetitive freeze-thaw cycles, and lysates were immunoprecipita ted with anti-V5-agarose beads or pulled down with Ni-NTA-agarose beads overnight at 4°C.Beads were washed twice with EPPS buffer and once with RIPA buffer, and bound proteins and eluates were analyzed by immunoblot.For in vitro interaction, bacterially purified TDRD7 and AMPK proteins were incubated at 37°C for 2 h, and co-IP assays were performed.For co-IP of endogenous TDRD7 and AMPK proteins, mouse liver homoge nates were prepared in lysis buffer and immunoprecipitated by anti-AMPK antibody followed by immunoblot with anti-TDRD7 antibody.

Molecular docking analyses
The AMPK three-dimensional structure was modeled on template 6C9J, and the TDRD7 structure was built on template 5m9n.2 based on sequence homology using SWISS-MODEL because there was no structure available for full-length TDRD7.The templates were selected based on the close sequence similarity and were used for docking analysis.The 3D model structure of the AMPK:TDRD7 complex was predicted using Z-DOCK.The auto-inhibitory domain of AMPK and Tudor domain of TDRD7 were used for binding, and non-interacting residues were blocked in the input for predictions from Z-DOCK.The top 10 predictions were analyzed, and the most likely conformation was selected based on the binding affinity and optimal binding arrangement as a representative.

Isolation of subcellular fractions
Cytosolic, nuclear, and mitochondrial fractions from transfected cells were isolated as described previously using the Mitochondria Isolation Kit (Pierce Biotechnology), following the manufacturer's instructions (44).Isolated mitochondrial and nuclear fractions were washed with phosphate-buffered saline (PBS) and extracted in lysis buffer for co-immunoprecipitation and immunoblot analysis.

Flow cytometry and analysis
HEK293T cells were transfected with TDRD7 Wt or mutants; 24 h later, the cells were infected with SeV (Cantell) at an MOI of 10.After 16 h, cells were harvested, fixed with 1% paraformaldehyde in PBS for 10 min, permeabilized with 0.1% (wt/vol) Triton X-100, and incubated with an anti-SeV polyclonal antibody and an Alexa Fluor conjugated secondary antibody.Cells were analyzed on BD Canto (flow cytometer) using Flow Jo.The relative infectivity in each condition was normalized to EV to obtain percent infection.

Statistical analyses
Statistical analyses were performed using GraphPad Prism 9 software or Microsoft Excel for Windows 10.The P values were calculated using two-tailed, unpaired Student's t-tests (for comparing two groups) or one-way analysis of variance (ANOVA) (for comparing more than two groups), based on the number of sets for comparison.P < 0.05 was considered statistically significant.The results presented here are representatives of three or more independent experiments.

FIG 1
FIG 1 TDRD7 interacts with AMPK and inhibits its activation by various stimuli.(A) L929 cells expressing Tdrd7-specific shRNA were infected with SeV, and LC3-II was analyzed by immunoblot at 8 hours post-infection (hpi).(B) TDRD7 knockout (KO) cells, ectopically expressing V5-TDRD7, were serum starved and analyzed for pAMPK by immunoblot.(C) TDRD7 KO cells, ectopically expressing V5-TDRD7, were treated with AICAR and analyzed for pAMPK by immunoblot 2 h post-treatment.(D) HEK293T cells were co-transfected with V5-TDRD7 and HA-AMPKα1 plasmids.TDRD7 was immunoprecipitated from the cell lysates 24 h post-transfection, and the immunoprecipitates were analyzed for AMPK by immunoblot.(E) HEK293T cells were co-transfected with V5-TDRD7 and HA-AMPKα1 plasmids.TDRD7 and AMPK were immuno-stained and analyzed by confocal microscopy.(F) HEK293T cells were co-transfected with V5-TDRD7 and HA-AMPKα1 plasmids, and TDRD7:AMPK interaction was analyzed by duolink assay; the red dots indicate the duolink signals.(G) HT1080 cells were immuno-stained using anti-TDRD7 and antiAMPK antibodies and analyzed by confocal microscopy.NT, nontargeting, EV, empty vector; scale bar, 5 µm.The results are representative of at least three experiments.

FIG 2
FIG 2 The Tudor-3 domain of TDRD7 is required for interaction with AMPK.(A) Wild-type (Wt) TDRD7 with its functional domains (OST and Tud) and the deletion mutants used in the study.(B) HEK293T cells were co-transfected with V5-TDRD7 (Hu, human; Mu, mouse), V5-Tdrd7 (mouse), or Tud-deleted mutant (ΔTud) and HA-AMPKα1 plasmids.TDRD7 was immunoprecipitated from the cell lysates, and the immunoprecipitates were analyzed for AMPK by immunoblot.(C) HEK293T cells were co-transfected with V5-TDRD7 and HA-AMPKα1 plasmids.TDRD7 was immunoprecipitated from cytosolic, mitochondrial, and nuclear fractions, and the immunoprecipitates were analyzed for AMPK by immunoblot.(D) HEK293T cells were co-transfected with Wt TDRD7 or the Tudor-deleted mutant (ΔTud) and HA-AMPKα1 plasmids; TDRD7 was immunoprecipitated from cytosolic fractions.The immunoprecipitates were analyzed for AMPK by immunoblot.(E) HEK293T cells were co-transfected with V5-TDRD7 deletion mutants and HA-AMPKα1 plasmids, as indicated.TDRD7 was immunoprecipitated from cell lysates, and the immunoprecipitates were analyzed for AMPK by immunoblot.(F) HEK293T cells were co-transfected with Wt V5-TDRD7 or the deletion mutants (as indicated)

FIG 3
FIG 3 The auto-inhibitory domain (AID) of AMPK is required for interaction with TDRD7.(A, B) As shown, wild-type (Wt) and deletion mutants of AMPKα1 and TDRD7 were used for following experiments.(C) HEK293T cells were co-transfected with Wt or deletion mutants of HA-AMPK and Wt or Tudor domain-deleted (ΔTud) V5-TDRD7 plasmids, as indicated.TDRD7 was immunoprecipitated from cell lysates, and the immunoprecipitates were analyzed for AMPK by immunoblot.(D) HEK293T cells were co-transfected with Wt or deletion mutants of HA-AMPK and the Tud-3 mutant of V5-TDRD7 plasmids, as indicated.TDRD7 was immunoprecipitated from the cell lysates, and the immunoprecipitates were analyzed for AMPK by immunoblot.(E) Tudor-3 (Tud-3) and AID domains, as indicated, were used for homology modeling and molecular docking analyses.A modeled structure of the TDRD7:AMPK complex is shown, and arrows indicate the putative contact sites.EV, empty vector.The results are representative of three experiments.

FIG 4 7 FIG 5
FIG 4 TDRD7:AMPK interaction is required for AMPK inhibition by TDRD7.(A) Expression levels of Wt of ΔTud mutant of TDRD7 in TDRD7 KO HT1080 cells were analyzed by immunoblot.(B, C) The cells (as in panel A) were treated with AICAR (for 2 h), and pAMPK was analyzed by immunoblot (B), which was quantified by Image J (C). (D, E) The cells (as in panel A) were serum starved (SS, 2 h), and pAMPK was analyzed by immunoblot (D), which was quantified by Image J (E). (F) TDRD7 KO HT1080 cells, stably expressing Wt or ΔOST mutant of TDRD7, were left untreated (NT) or serum starved (SS, 2 h), and pAMPK was analyzed by immunoblot.(G) TDRD7 KO HT1080 cells, stably expressing ΔOST mutant of TDRD7, were left untreated (NT) or infected with SeV, and pAMPK was analyzed by immunoblot.The results are representative of three experiments; the data represent mean ± SEM, *P < 0.05.

FIG 6
FIG 6 Increased AMPK activation in Tdrd7 knockout primary mouse cells.(A, B) A diagram showing the newly generated Tdrd7 fl/fl mice and the derived Tdrd7 Δ/Δ strain (A) and the genotypes of relevant strains (B).The location of primers (P) used for genotyping is shown in A. (C) Liver homogenates from Tdrd7 fl/fl and Tdrd7 Δ/Δ mice were analyzed for TDRD7 by immunoblot.(D) Tdrd7 fl/fl and Tdrd7 Δ/Δ primary MEFs were treated with IFNβ or polyI:C for 16 h, and the Tdrd7 mRNA levels were analyzed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR).(E) Tdrd7 fl/fl and Tdrd7 Δ/Δ primary MEFs were serum starved (SS) for the indicated times, and pAMPK was analyzed by immunoblot.(F) Tdrd7 fl/fl and Tdrd7 Δ/Δ primary splenocytes were serum starved (SS) and analyzed for pAMPK by immunoblot.The results are representative of three experiments using two to four mice from each group; the data represent mean ± SEM; *P < 0.05.

FIG 8 A
FIG 8 A model representing IFN-inducible protein TDRD7 interacts with pro-viral AMPK and inhibits its activation to suppress viral replication and pathogenesis.Virus infection, via IRF3/IFN pathway, transcriptionally induces TDRD7, which interacts with AMPK via the C-terminal Tudor domain, to inhibit AMPK activation.