Figure 2A549 AT2 cells were infected with Delta SARS - CoV - 2 variant . Proteasome inhibitor Bortezomib ( BZ , 1μM ) was added to cells 18h post - infection . Cells were lysed 24h post - addition of inhibitor and cyclins and viral proteins were detected by western blot . ( N ) nucleocapsid . Densitometry analysis of western blots for D - cyclins ( normalized to actin ) in A549 AT2 cells . Plots are average of 2 ( cyclin D1 ) , 3 ( cyclin D3 ) biological replicates . Bars indicate mean with SD . Statistical analysis was performed using ordinary two way ANOVA ; ns , non - significant ; ∗ p < 0 . 1 . A459 AT2 cells were infected with Delta SARS - CoV - 2 variant . Proteasome inhibitor Bortezomib ( BZ , 1μM ) was added to cells 8h post - infection . Cells were fixed and stained 24h post - addition of inhibitor . ( N ) nucleocapsid . Scale bars : 40μm . Contour lines represent outlines of infected cells . Quantification of D3 cyclin relocalization from nucleus after infection . Uninfected ( - ) and SARS - CoV - 2 infected cells were identified by negative / positive nucleocapsid staining . Ratio between nuclear and cytoplasm ( N / C ratio ) staining intensity of cyclins was measured using ImageJ and Harmony ( PerkinElmer ) . At least 50 cells have been counted . Bars indicate mean with SD . Statistical analysis was performed using ordinary two - way ANOVA ; ∗ ∗ ∗ p < 0 . 001 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A549 AT2 cells were infected with Delta SARS-CoV-2 variant. Proteasome inhibitor Bortezomib (BZ, 1μM) was added to cells 18h post-infection. Cells were lysed 24h post-addition of inhibitor and cyclins and viral proteins were detected by western blot. (N) nucleocapsid. Densitometry analysis of western blots for D-cyclins (normalized to actin) in A549 AT2 cells. Plots are average of 2 (cyclin D1), 3 (cyclin D3) biological replicates. Bars indicate mean with SD. Statistical analysis was performed using ordinary two way ANOVA; ns, non-significant; ∗p < 0.1.A459 AT2 cells were infected with Delta SARS-CoV-2 variant. Proteasome inhibitor Bortezomib (BZ, 1μM) was added to cells 8h post-infection. Cells were fixed and stained 24h post-addition of inhibitor. (N) nucleocapsid.Scale bars: 40μm. Contour lines represent outlines of infected cells.Quantification of D3 cyclin relocalization from nucleus after infection. Uninfected (-) and SARS-CoV-2 infected cells were identified by negative/positive nucleocapsid staining. Ratio between nuclear and cytoplasm (N/C ratio) staining intensity of cyclins was measured using ImageJ and Harmony (PerkinElmer). At least 50 cells have been counted. Bars indicate mean with SD. Statistical analysis was performed using ordinary two-way ANOVA; ∗∗∗p < 0.001.


Figure 3 ( A , D and A - cyclins were depleted using siRNA in A549 AT2 cells . ( A , Representative example of western blot from uninfected cell lysates collected at 72h post - knockdown . B ) D and A - cyclins were depleted using siRNA in A549 AT2 cells . Cells were infected 18h later with Delta ( ∆ ) , Alpha ( α ) or wild type ( WT ) SARS - CoV - 2 variants at MOI 0 . 001 , 0 . 1 , 0 . 1 respectively . Cells were washed 4h post - infection and new media were added . Supernatants and cells were collected 48h later for TCID50 ( B ) . ( B , Virus titres in cell culture supernatants were determined as TCID50 Data information : Graphs represent average of n = 3 ( B , Statistical analysis was performed using one - way ANOVA with Dunnett ' s multiple comparisons test . NT , non - targeting control . ns , non - significant ; ∗ p < 0 . 1 ; ∗ ∗ p < 0 . 01 ; ∗ ∗ ∗ p < 0 . 001 . Bars indicate mean with SD . ( C , D and A - cyclins were depleted using siRNA in VERO AT2 cells . C ) Representative example of western blot from uninfected cell lysates collected at 72h post - knockdown . D ) D and A - cyclins were depleted using siRNA in VERO AT2 cells . Cells were infected 18h later with Alpha ( α ) or wild type ( WT ) SARS - CoV - 2 variants at MOI 0 . 1 . Cells were washed 4h post - infection and new media added . Supernatants and cells were collected at 24h and 48h later . D ) Virus titres in cell culture supernatants were determined as TCID50 in VERO AT2 cells . Data information : Graphs represent average of n = 3 D ) ; Statistical analysis was performed using one - way ANOVA with Dunnett ' s multiple comparisons test . NT , non - targeting control . ns , non - significant ; ∗ p < 0 . 1 ; ∗ ∗ p < 0 . 01 ; ∗ ∗ ∗ p < 0 . 001 . Bars indicate mean with SD . Cyclin D3 was depleted using siRNA in Calu3 cells . Cells were infected 48h later with Delta SARS - CoV - 2 at MOI 0 . 01 . Cells were washed 4h post - infection and new media were added . Supernatants and cells were collected 48h later for western blot and TCID50 . Uninfected cell lysates were used for western blot . Data information : Graphs represent average of n = 4 ( E ) biological replicates . Statistical analysis was performed using one - way ANOVA with Dunnett ' s multiple comparisons test . NT , non - targeting control . ns , non - significant ; ∗ p < 0 . 1 ; ∗ ∗ p < 0 . 01 ; ∗ ∗ ∗ p < 0 . 001 . Bars indicate mean with SD .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A, D and A-cyclins were depleted using siRNA in A549 AT2 cells. (A, Representative example of western blot from uninfected cell lysates collected at 72h post-knockdown.B) D and A-cyclins were depleted using siRNA in A549 AT2 cells. Cells were infected 18h later with Delta (∆), Alpha (α) or wild type (WT) SARS-CoV-2 variants at MOI 0.001, 0.1, 0.1 respectively. Cells were washed 4h post-infection and new media were added. Supernatants and cells were collected 48h later for TCID50 (B). (B, Virus titres in cell culture supernatants were determined as TCID50 Data information: Graphs represent average of n=3 (B, Statistical analysis was performed using one-way ANOVA with Dunnett's multiple comparisons test. NT, non-targeting control. ns, non-significant; ∗p < 0.1; ∗∗p < 0.01; ∗∗∗p < 0.001. Bars indicate mean with SD.(C, D and A-cyclins were depleted using siRNA in VERO AT2 cells. C) Representative example of western blot from uninfected cell lysates collected at 72h post-knockdown.D) D and A-cyclins were depleted using siRNA in VERO AT2 cells. Cells were infected 18h later with Alpha (α) or wild type (WT) SARS-CoV-2 variants at MOI 0.1. Cells were washed 4h post-infection and new media added. Supernatants and cells were collected at 24h and 48h later. D) Virus titres in cell culture supernatants were determined as TCID50 in VERO AT2 cells. Data information: Graphs represent average of n=3 D); Statistical analysis was performed using one-way ANOVA with Dunnett's multiple comparisons test. NT, non-targeting control. ns, non-significant; ∗p < 0.1; ∗∗p < 0.01; ∗∗∗p < 0.001. Bars indicate mean with SD.Cyclin D3 was depleted using siRNA in Calu3 cells. Cells were infected 48h later with Delta SARS-CoV-2 at MOI 0.01. Cells were washed 4h post-infection and new media were added. Supernatants and cells were collected 48h later for western blot and TCID50. Uninfected cell lysates were used for western blot. Data information: Graphs represent average of n=4 (E) biological replicates. Statistical analysis was performed using one-way ANOVA with Dunnett's multiple comparisons test. NT, non-targeting control. ns, non-significant; ∗p < 0.1; ∗∗p < 0.01; ∗∗∗p < 0.001. Bars indicate mean with SD.


Figure 5A549 AT2 cells were depleted for D and A2 cyclins ( A ) A representative western blot from lysates of uninfected knock - down cells . ( E ) A549 AT2 cells were transduced with VSV - G pseudotyped Fucci containing lentiviral particles and VSV - G pseudotyped lentiviral particles containing WT cyclin D3 or T283A mutant cyclin D3 ( mutant not degraded by proteasome ) . Cells were infected 24h later and collected 48h post - infection for flow cytometry analysis of early S and western blot . n = 2 biological replicates ; Ordinary two - way ANOVA with Sidak ' s multiple comparisons test : ns , non - significant ; ∗ ∗ p < 0 . 01 . Bars indicate mean with SD . ( F , G ) VERO AT2 cells were transduced with VSV - G pseudotyped Fucci containing lentiviral particles and 18h later infected with Delta variant SARS - CoV - 2 . Cells were fixed and stained for D - cyclins 24h later . ( F ) Example of acquisition using automated microscopic platform . Cells are identified for infection , expression of cyclin D3 , and cell cycle ( Red / arrow = G1phase ; Green / arrowhead = S / G2 / M ; Red + Green / arrowhead = early S ) . Scale bar : 20μm . ( G ) Quantification of D - cyclins re - localization from nucleus to cytoplasm and correlation with cell cycle phases using ImageJ and Harmony ( PerkinElmer ) . At least 50 - 200 cells were analysed in each condition . Bars indicate mean with SD . Statistical analysis was performed using two - sided unpaired Student ' s t - tests ; ∗ p < 0 . 1 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A549 AT2 cells were depleted for D and A2 cyclins (A) A representative western blot from lysates of uninfected knock-down cells.(E) A549 AT2 cells were transduced with VSV-G pseudotyped Fucci containing lentiviral particles and VSV-G pseudotyped lentiviral particles containing WT cyclin D3 or T283A mutant cyclin D3 (mutant not degraded by proteasome). Cells were infected 24h later and collected 48h post-infection for flow cytometry analysis of early S and western blot. n=2 biological replicates; Ordinary two-way ANOVA with Sidak's multiple comparisons test: ns, non-significant; ∗∗p < 0.01. Bars indicate mean with SD.(F,G) VERO AT2 cells were transduced with VSV-G pseudotyped Fucci containing lentiviral particles and 18h later infected with Delta variant SARS-CoV-2. Cells were fixed and stained for D-cyclins 24h later. (F) Example of acquisition using automated microscopic platform. Cells are identified for infection, expression of cyclin D3, and cell cycle (Red/arrow=G1phase; Green/arrowhead=S/G2/M; Red+Green/arrowhead=early S). Scale bar: 20μm. (G) Quantification of D-cyclins re-localization from nucleus to cytoplasm and correlation with cell cycle phases using ImageJ and Harmony (PerkinElmer). At least 50-200 cells were analysed in each condition. Bars indicate mean with SD. Statistical analysis was performed using two-sided unpaired Student's t-tests; ∗p < 0.1.


Figure 6 ( A ) 293T cells were contransfected with HA - cyclin D3 and Strep - tag - SARS - CoV - 2 E or nsp9 , and control plasmid ( EV ) . Immunoprecipitation was performed using anti - HA antibody . The immunoprecipitates were blotted with anti - Strep , anti - HA , and cyclin D3 antibodies . ( B ) 293T cells were contransfected with HA - cyclin D3 and Strep - tag - SARS - CoV - 2 E , M , both E and M or nsp9 . Immunoprecipitation was performed using anti - HA antibody . The immunoprecipitates were blotted with anti - Strep , anti - HA antibodies . WCL , whole cell lysate . ( C - G ) 293T GFP11 cells were transfected with Spike , and / or with Envelope , Membrane , and cyclin D3 . 24h post - transfection cells were seeded at a 1 : 1 ratio with Vero - GFP10 cells and percentage of GFP + ve area ( syncytia ) were determined 18h later . S , Spike ; E , Envelope ; M , Membrane ; D3 , cyclin D3 . ( C , D ) Representative images of GFP + syncytia . Scale bars : 40μm . ( E - G ) Quantification of cell - to - cell fusion showing percentage of the GFP + ve area to the acquired total cell area . ( E ) n = 4 biological replicates . ( F , G ) n = 4 biological replicates ; technical triplicates shown ; one - way ANOVA with Dunnett ' s multiple comparisons test : ns , non - significant ; ∗ ∗ ∗ ∗ p < 0 . 0001 ; ∗ ∗ ∗ p < 0 . 001 ; ∗ ∗ p < 0 . 01 . Bars indicate mean with SD . ( I ) Representative example of western blot from purified SARS - CoV - 2 Virus Like Particles ( VLPs ) and whole cell lysates ( WCL ) . MCM2 was used as loading control . ( J ) Quantification of protein incorporation ( into VLPs ) or expression ( in WCL ) . n = 4 biological replicates ; one - way ANOVA with Dunnett ' s multiple comparisons test : ns , non - significant ; ∗ ∗ ∗ p < 0 . 001 . Bars indicate mean with SD . N , nucleocapsid ; M , membrane ; E , envelope ; S , spike .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(A) 293T cells were contransfected with HA - cyclin D3 and Strep-tag-SARS-CoV-2 E or nsp9, and control plasmid (EV). Immunoprecipitation was performed using anti-HA antibody. The immunoprecipitates were blotted with anti-Strep, anti-HA, and cyclin D3 antibodies.(B) 293T cells were contransfected with HA - cyclin D3 and Strep-tag-SARS-CoV-2 E, M, both E and M or nsp9. Immunoprecipitation was performed using anti-HA antibody. The immunoprecipitates were blotted with anti-Strep, anti-HA antibodies. WCL, whole cell lysate.(C-G) 293T GFP11 cells were transfected with Spike, and/or with Envelope, Membrane, and cyclin D3. 24h post-transfection cells were seeded at a 1:1 ratio with Vero-GFP10 cells and percentage of GFP+ve area (syncytia) were determined 18h later. S, Spike; E, Envelope; M, Membrane; D3, cyclin D3. (C,D) Representative images of GFP+ syncytia. Scale bars: 40μm. (E-G) Quantification of cell-to-cell fusion showing percentage of the GFP+ve area to the acquired total cell area. (E) n=4 biological replicates. (F,G) n = 4 biological replicates; technical triplicates shown; one-way ANOVA with Dunnett's multiple comparisons test: ns, non-significant; ∗∗∗∗p < 0.0001; ∗∗∗p < 0.001; ∗∗p < 0.01 . Bars indicate mean with SD.(I) Representative example of western blot from purified SARS-CoV-2 Virus Like Particles (VLPs) and whole cell lysates (WCL). MCM2 was used as loading control. (J) Quantification of protein incorporation (into VLPs) or expression (in WCL). n = 4 biological replicates; one-way ANOVA with Dunnett's multiple comparisons test: ns, non-significant; ∗∗∗p < 0.001. Bars indicate mean with SD. N, nucleocapsid; M, membrane; E, envelope; S, spike.


Figure 1 ( A ) Northern blot experiments show the expression of RsaI during growth phase in the HG001 strain in MHB medium with or without the addition of 1 , 5 g / L of D - glucose . Glucose was added either at the beginning of growth ( + glucose , left panel ) or after 3h of growth ( right panel ) . ( B ) Northern blot experiment shows the expression of RsaI during growth phase in the HG001 , ∆ ccpA mutant strain , and ∆ codY mutant strain , in MHB medium with ( + ) or without ( - ) the addition of 1 , 5 g / L of D - glucose . ( C ) Northern blot experiment shows the expression of RsaI in the HG001 and ∆ ccpA mutant strains . Total RNA was prepared after 2 , 3 , 4 , 5 and 6h of culture in BHI medium at 37 ° C . ( D ) Northern blot analysis of RsaI in the HG001 strain grown in MHB medium with or without the addition of 1 g / L of glucose , fructose or xylose . For all the experiments , loading controls were done using the expression of 5S rRNA ( 5S ) as revealed after hybridization of the membranes with a specific probe . However for these controls , we used aliquots of the same RNA preparations but the migration of the samples was performed in parallel to the experiments on a separate agarose gel because RsaI and 5S rRNA have very similar sizes .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(A) Northern blot experiments show the expression of RsaI during growth phase in the HG001 strain in MHB medium with or without the addition of 1,5 g/L of D-glucose. Glucose was added either at the beginning of growth (+ glucose, left panel) or after 3h of growth (right panel).(B) Northern blot experiment shows the expression of RsaI during growth phase in the HG001, ∆ccpA mutant strain, and ∆codY mutant strain, in MHB medium with (+) or without (-) the addition of 1,5 g/L of D-glucose.(C) Northern blot experiment shows the expression of RsaI in the HG001 and ∆ccpA mutant strains. Total RNA was prepared after 2, 3, 4, 5 and 6h of culture in BHI medium at 37°C.(D) Northern blot analysis of RsaI in the HG001 strain grown in MHB medium with or without the addition of 1 g/L of glucose, fructose or xylose. For all the experiments, loading controls were done using the expression of 5S rRNA (5S) as revealed after hybridization of the membranes with a specific probe. However for these controls, we used aliquots of the same RNA preparations but the migration of the samples was performed in parallel to the experiments on a separate agarose gel because RsaI and 5S rRNA have very similar sizes.


Figure 2 ( B ) Gel retardation assays show the formation of the complex between RsaI and icaR , glcU _ 2 and fn3K mRNAs . The 5 ' end - labeled wild - type RsaI ( RsaI ) , RsaI mutant 3 ( RsaI mut3 , deletion of the two G - track motifs ) , and RsaI mutant 4 ( RsaI mut4 , deletion of the C / U rich unpaired interhelical region ) were incubated with increasing concentrations of mRNAs : lane 1 , 0 nM ; lane 2 , 25 nM ; lane 3 , 50 nM ; lane 4 , 100 nM and lane 5 , 200 nM . Below the gels , the predicted interactions between RsaI and its targets are shown . Translation start codons are in green and SD is for Shine Dalgarno sequence . Graphs represented the % of complex formed between either RsaI or its two mutant forms ( RsaI mut3 and RsaI mut4 ) and the target mRNA ( icaR , glcU _ 2 , fn3K ) as the function of mRNA concentrations . ( C ) Gel retardation assays show the formation of the complex between RsaI and RsaG . The 5 ' end - labeled wild - type RsaI ( RsaI ) , RsaI mutant 2 ( RsaI mut2 ) , RsaI mutant 4 ( RsaI mut4 ) , and RsaI mutant 1 ( RsaI mut1 ) were incubated with increasing concentrations of RsaG given in nM on the top of the autoradiographies .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(B) Gel retardation assays show the formation of the complex between RsaI and icaR, glcU_2 and fn3K mRNAs. The 5' end-labeled wild-type RsaI (RsaI), RsaI mutant 3 (RsaI mut3, deletion of the two G-track motifs), and RsaI mutant 4 (RsaI mut4, deletion of the C/U rich unpaired interhelical region) were incubated with increasing concentrations of mRNAs: lane 1, 0 nM; lane 2, 25 nM; lane 3, 50 nM; lane 4, 100 nM and lane 5, 200 nM. Below the gels, the predicted interactions between RsaI and its targets are shown. Translation start codons are in green and SD is for Shine Dalgarno sequence. Graphs represented the % of complex formed between either RsaI or its two mutant forms (RsaI mut3 and RsaI mut4) and the target mRNA (icaR, glcU_2, fn3K) as the function of mRNA concentrations.(C) Gel retardation assays show the formation of the complex between RsaI and RsaG. The 5' end-labeled wild-type RsaI (RsaI), RsaI mutant 2 (RsaI mut2), RsaI mutant 4 (RsaI mut4), and RsaI mutant 1 (RsaI mut1) were incubated with increasing concentrations of RsaG given in nM on the top of the autoradiographies.


Figure 3 ( A ) Toe - print assays showing the effect of RsaI on the formation of the ribosomal initiation complex of glcU _ 2 and fn3K mRNAs , respectively . Lane 1 : incubation control of mRNA alone ; lane 2 : incubation control of mRNA with 30S subunits ; lane 3 : incubation control of mRNA with RsaI ; lane 4 : formation of the ribosomal initiation complex containing mRNA , 30S and the initiator tRNAfMet ( tRNAi ) ; lanes 5 to 9 : formation of the initiation complex in the presence of increasing concentrations of RsaI , respectively : 50 nM ( lane 5 ) , 100 nM ( lane 6 ) , 150 nM ( lane 7 ) , 300 nM ( lane 8 ) , and 400 nM ( lane 9 ) . Lanes T , A , C , G : sequencing ladders . The Shine and Dalgarno ( SD ) sequence , the start site of translation ( START ) and the toe - printing signals ( + 16 ) are indicated . At the bottom of the gels are shown the predicted interactions between RsaI and its targets . Translation start codons are in green , and the Shine and Dalgarno ( SD ) sequence is underlined , the arrowheads depict the toe - printing signals . ( B ) The β - galactosidase activity ( Miller Units ) has been measured from various fusions expressed from a plasmid which also carries rsaI gene under its own promoter : PrpoB : : glcU : : lacZ : : rsaI , PrpoB : : fn3K : : lacZ : : rsaI , PrpoB : : treB : : lacZ : : rsaI , PrpoB : : HG001 _ 1242 : : lacZ : : rsaI , and PrpoB : : HG001 _ 2520 : : lacZ : : rsaI expressed in the mutant strain HG001 - ∆ rsaI . The same constructs were made in the absence of rsaI gene . For the fn3K - lacZ fusion , we also used an additional construct PrpoB : : fn3K : : lacZ : : rsaImut4 expressing both the fusion FN3K - LacZ protein and RsaI mut4 . The β - galactosidase activity was normalized for bacterial density and the results represented the mean of four independent experiments . The error bars are standard deviations and the statistical significance was determined using the Student ' s t - test . * p < 0 . 05 , * * p < 0 . 005 , * * * p < 0 . 0005 , * * * * p < 0 . 0001 , ns is for not significant .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A) Toe-print assays showing the effect of RsaI on the formation of the ribosomal initiation complex of glcU_2 and fn3K mRNAs, respectively. Lane 1 : incubation control of mRNA alone; lane 2 : incubation control of mRNA with 30S subunits; lane 3: incubation control of mRNA with RsaI; lane 4 : formation of the ribosomal initiation complex containing mRNA, 30S and the initiator tRNAfMet (tRNAi); lanes 5 to 9 : formation of the initiation complex in the presence of increasing concentrations of RsaI, respectively : 50 nM (lane 5), 100 nM (lane 6), 150 nM (lane 7), 300 nM (lane 8), and 400 nM (lane 9). Lanes T, A, C, G: sequencing ladders. The Shine and Dalgarno (SD) sequence, the start site of translation (START) and the toe-printing signals (+16) are indicated. At the bottom of the gels are shown the predicted interactions between RsaI and its targets. Translation start codons are in green, and the Shine and Dalgarno (SD) sequence is underlined, the arrowheads depict the toe-printing signals.(B) The β-galactosidase activity (Miller Units) has been measured from various fusions expressed from a plasmid which also carries rsaI gene under its own promoter: PrpoB::glcU::lacZ::rsaI, PrpoB::fn3K::lacZ::rsaI, PrpoB::treB::lacZ::rsaI, PrpoB::HG001_1242::lacZ::rsaI, and PrpoB::HG001_2520::lacZ::rsaI expressed in the mutant strain HG001-∆rsaI. The same constructs were made in the absence of rsaI gene. For the fn3K-lacZ fusion, we also used an additional construct PrpoB::fn3K::lacZ::rsaImut4 expressing both the fusion FN3K-LacZ protein and RsaI mut4. The β-galactosidase activity was normalized for bacterial density and the results represented the mean of four independent experiments. The error bars are standard deviations and the statistical significance was determined using the Student's t-test. * p < 0.05, ** p < 0.005, *** p < 0.0005, **** p < 0.0001, ns is for not significant.


Figure 4 ( B ) Gel retardation assays show the formation of the complex between RsaI and icaR full length , icaR SUBST , icaR - 5 ' UTR , and icaR - 3 ' UTR . The 5 ' end - labeled of RsaI was incubated with increasing concentrations of the various mRNAs . UTR is for untranslated region , SUBST stands for the substitution of UCCCCUG sequence by AGGGGAC . This mutation , which is located in the 3 ' UTR of icaR , significantly destabilizes the long - range interaction , and enhances icaR translation . Lane C represents binding between radiolabelled RsaI and full - length icaR mRNA ( 50 nM ) . Due to the rather short migration of the gel , the full - length mRNA is still observed in the pocket . ( C ) In vivo effect of RsaI on PIA - PNAG synthesis in the S . aureus wild - type ( WT ) 132 strain , the ∆ rsaI mutant , and the strain carrying a deletion of icaR 3 ' UTR ( ∆ 3 ' UTR ) . This last mutant strain has been transformed with the pES plasmids expressing rsaI or rsaI mut5 . The PIA - PNAG exopolysaccharide biosynthesis was quantified using dot - blot assays . Serial dilutions ( 1 / 5 ) of the samples were spotted onto nitrocellulose membranes and PIA - PNAG production was detected with specific anti - PIA - PNAG antibodies . RsaI was detected in the same samples by Northern blot using a probe directed against RsaI . Ethidium bromide staining of rRNA was used as loading controls of the same gel .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(B) Gel retardation assays show the formation of the complex between RsaI and icaR full length, icaR SUBST, icaR-5'UTR, and icaR-3'UTR. The 5' end-labeled of RsaI was incubated with increasing concentrations of the various mRNAs. UTR is for untranslated region, SUBST stands for the substitution of UCCCCUG sequence by AGGGGAC. This mutation, which is located in the 3'UTR of icaR, significantly destabilizes the long-range interaction, and enhances icaR translation. Lane C represents binding between radiolabelled RsaI and full-length icaR mRNA (50 nM). Due to the rather short migration of the gel, the full-length mRNA is still observed in the pocket.(C) In vivo effect of RsaI on PIA-PNAG synthesis in the S. aureus wild-type (WT) 132 strain, the ∆rsaI mutant, and the strain carrying a deletion of icaR 3'UTR (∆3'UTR). This last mutant strain has been transformed with the pES plasmids expressing rsaI or rsaI mut5. The PIA-PNAG exopolysaccharide biosynthesis was quantified using dot-blot assays. Serial dilutions (1/5) of the samples were spotted onto nitrocellulose membranes and PIA-PNAG production was detected with specific anti-PIA-PNAG antibodies. RsaI was detected in the same samples by Northern blot using a probe directed against RsaI. Ethidium bromide staining of rRNA was used as loading controls of the same gel.


Figure 5 ( A ) Ternary complex formation between RsaI , various mRNAs ( glcU _ 2 , HG001 _ 00942 and HG001 _ 1242 ) , and RsaG . The 5 ' - end labeled RsaI was incubated with increasing concentrations of the target mRNA alone or in the presence of 50 nM of RsaG . The various complexes are notified on the sides of the autoradiographies . ( B ) RsaG does not form stable complexes with glcU _ 2 and HG001 _ 01242 . Binding assays were done in the presence of 5 ' end - labeled RsaG and either cold RsaI ( 300 nM ) or increasing concentrations of glcU _ 2 and HG001 _ 01242 . ( C ) Measurements of the half - lives of RsaI and RsaG in HG001 - ∆ rsaG or HG001 - ∆ rsaI mutant strains using Northern blot experiments . The cells were treated with rifampicin at 4h of growth and total RNAs were extracted after 2 , 4 , 8 , 15 , 30 and 60 min at 37 ° C in BHI . 5S rRNA was probed to quantify the yield of RNAs in each lane using the same samples , which were however run on two different gels . Calculated half - lives are shown beneath the autoradiographies and are the average of 2 experiments . The data were normalized to 5S rRNA .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A) Ternary complex formation between RsaI, various mRNAs (glcU_2, HG001_00942 and HG001_1242), and RsaG. The 5'-end labeled RsaI was incubated with increasing concentrations of the target mRNA alone or in the presence of 50 nM of RsaG. The various complexes are notified on the sides of the autoradiographies.(B) RsaG does not form stable complexes with glcU_2 and HG001_01242. Binding assays were done in the presence of 5' end-labeled RsaG and either cold RsaI (300 nM) or increasing concentrations of glcU_2 and HG001_01242.(C) Measurements of the half-lives of RsaI and RsaG in HG001-∆rsaG or HG001-∆rsaI mutant strains using Northern blot experiments. The cells were treated with rifampicin at 4h of growth and total RNAs were extracted after 2, 4, 8, 15, 30 and 60 min at 37°C in BHI. 5S rRNA was probed to quantify the yield of RNAs in each lane using the same samples, which were however run on two different gels. Calculated half-lives are shown beneath the autoradiographies and are the average of 2 experiments. The data were normalized to 5S rRNA.


Figure 1 . factor . a , Duplicate screens depicting HCoV - 229E infection ( 24 hours post - infection ) in Huh7 cells ectopically expressing ISGs . b , HCoV - 229E infection of Huh7 cells expressing LY6E or control vector ( Empty ) . Blue : DAPI , green : HCoV - 229E N protein , red : TagRFP encoded in SCRPSY vector . c - h , Effect of LY6E expression on diverse coronaviruses . Stably transduced cells were infected with HCoV - 229E ( c ) , HCoV - OC43 ( d ) , MERS - CoV ( e ) , SARS - CoV ( f ) , SARS - CoV - 2 ( g ) , MHV - Gluc ( h ) . Viral replication readouts included : titer determination by plaque assay ( c ) , infectivity by nucleoprotein staining and quantitation by flow cytometry ( d ) , limiting dilution assay ( e , f , g ) , luciferase activity shown as relative light units ( RLU ) ( h , j , l ) . i , Western blot of WT A549 or two LY6E KO clones ( # 3 , # 4 ) . j , HCoV - 229E infection of WT or LY6E KO A549 . k , Western blot of WT or LY6E KO A549 reconstituted with CRISPR - resistant LY6E ( CR LY6E ) or control vector ( Empty ) . l , HCoV - 229E infection of CR LY6E - reconstituted WT or LY6E KO A549 .

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Figure 1.factor.a, Duplicate screens depicting HCoV-229E infection (24 hours post-infection) in Huh7 cells ectopically expressing ISGs.b, HCoV-229E infection of Huh7 cells expressing LY6E or control vector (Empty). Blue: DAPI, green: HCoV-229E N protein, red: TagRFP encoded in SCRPSY vector.c-h, Effect of LY6E expression on diverse coronaviruses. Stably transduced cells were infected with HCoV-229E (c), HCoV-OC43 (d), MERS-CoV (e), SARS-CoV (f), SARS-CoV-2 (g), MHV-Gluc (h). Viral replication readouts included: titer determination by plaque assay (c), infectivity by nucleoprotein staining and quantitation by flow cytometry (d), limiting dilution assay (e,f,g), luciferase activity shown as relative light units (RLU) (h,j,l).i, Western blot of WT A549 or two LY6E KO clones (#3, #4).j, HCoV-229E infection of WT or LY6E KO A549.k, Western blot of WT or LY6E KO A549 reconstituted with CRISPR-resistant LY6E (CR LY6E) or control vector (Empty).l, HCoV-229E infection of CR LY6E-reconstituted WT or LY6E KO A549.


Figure 2 . formation . a - d , VSV pseudoparticles ( PP ) harboring spike proteins from HCoV - 229E ( a ) , MERS - CoV ( b ) , SARS - CoV ( c ) , SARS - CoV - 2 ( d ) were inoculated on LY6E - or empty vector - expressing cells . Virus entry efficiency was quantified by measuring virus - encoded luciferase reporter activity . e , VSV pseudoparticles expressing VSV G protein on their surface and encoding CoV S protein and GFP ( VSV * ΔG ( CoV S ) ) were inoculated with LY6E - or empty vector - expressing Huh7 . Syncytia formation was analyzed by immunofluorescence microscopy . Blue : DAPI , green : GFP , red : TagRFP inserted in SCRPSY vector . f , Quantification of VSV * ΔG ( CoV S ) induced syncytia depicted as percentage syncytia area . Three independent areas were analyzed per biological replicate ( circle , square , triangle ) . h , Cell - cell fusion was measured by co - incubating cells transfected with plasmids encoding CoV S proteins and a split - luciferase construct ( DSP1 - 7 ) together with CoV permissive LY6E - expressing or control cells transfected with the second fragment of split - luciferase ( DSP8 - 11 ) .

O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2.formation.a-d, VSV pseudoparticles (PP) harboring spike proteins from HCoV-229E (a), MERS-CoV (b), SARS-CoV (c), SARS-CoV-2 (d) were inoculated on LY6E- or empty vector-expressing cells. Virus entry efficiency was quantified by measuring virus-encoded luciferase reporter activity.e, VSV pseudoparticles expressing VSV G protein on their surface and encoding CoV S protein and GFP (VSV*ΔG(CoV S)) were inoculated with LY6E- or empty vector-expressing Huh7. Syncytia formation was analyzed by immunofluorescence microscopy. Blue: DAPI, green: GFP, red: TagRFP inserted in SCRPSY vector.f, Quantification of VSV*ΔG(CoV S) induced syncytia depicted as percentage syncytia area. Three independent areas were analyzed per biological replicate (circle, square, triangle).h, Cell-cell fusion was measured by co-incubating cells transfected with plasmids encoding CoV S proteins and a split-luciferase construct (DSP1-7) together with CoV permissive LY6E-expressing or control cells transfected with the second fragment of split-luciferase (DSP8-11).


Figure 3 . virus . a - b , Female Ly6efl / fl and Ly6eΔHSC mice were injected with PBS or MHV and monitored for survival ( a ) and weight loss ( b ) . c - n , Mice were assessed at 3 ( c - h ) or 5 ( i - n ) days post - infection for serum ALT ( c , i ) , viral titers in liver ( d , j ) and spleen ( e , k ) , and liver necrosis and inflammation ( f - h , l - n ) .

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Figure 3.virus.a-b, Female Ly6efl/fl and Ly6eΔHSC mice were injected with PBS or MHV and monitored for survival (a) and weight loss (b).c-n, Mice were assessed at 3 (c-h) or 5 (i-n) days post-infection for serum ALT (c,i), viral titers in liver (d,j) and spleen (e,k), and liver necrosis and inflammation (f-h, l-n).


Figure 4 . mice . a - e , Transcriptomic analysis from Ly6efl / fl ( WT ) and Ly6eΔHSC ( KO ) mice . a - b , Heat maps displaying significant changes ( mean RPKM > 0 . 5 ; fold change > 2 ; FDR ≤ 0 . 05 ) in liver ( a ) and spleen ( b ) . Dendrograms are normalized read data ( row z - score ) clustered with complete linkage method employing Spearman Rank correlation distance measurement . d - e , Expression of select genes in liver ( d ) and spleen ( e ) . f - g , Immune cell counts from liver ( f ) and spleen ( g ) . h , Infection of cultured splenocytes . For a - e , n = 3 . For f - g , n = 8 MHV - injected , n = 4 PBS - injected from two pooled experiments .

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Figure 4.mice.a-e, Transcriptomic analysis from Ly6efl/fl (WT) and Ly6eΔHSC (KO) mice. a-b, Heat maps displaying significant changes (mean RPKM > 0.5; fold change > 2; FDR ≤ 0.05) in liver (a) and spleen (b). Dendrograms are normalized read data (row z-score) clustered with complete linkage method employing Spearman Rank correlation distance measurement.d-e, Expression of select genes in liver (d) and spleen (e).f-g, Immune cell counts from liver (f) and spleen (g).h, Infection of cultured splenocytes. For a-e, n=3. For f-g, n=8 MHV-injected, n=4 PBS-injected from two pooled experiments.


Figure 1 ( A ) Differential intron retention ( IR ) analysis of RNA - seq data from samples under PABPN1 knockdown ( siPABPN1 ) and control ( NC ) conditions . Significantly repressed and induced intron retention events ( retained introns , RIs ) are indicated by blue and red dots , respectively . The threshold of IR difference ( ΔIR , siPABPN1 - siNC ) is 0 . 15 , and the FDR cutoff is 0 . 01 . ( C ) Cumulative frequency distributions of retained introns ( RIs ) , up - regulated ( red ) , and down - regulated ( blue ) upon PABPN1 knockdown , and of all retained introns in control ( NC , green ) samples by their relative intronic ranks in genes . Validation of last intron retention of six representative genes in response to PABPN1 knockdown by semi - quantitative PCR in HeLa cells treated with three different NC and PABPN1 siRNAs ( D ) . ( G ) Western blot validating the degradation effect of PABPN1 under the treatment of dTAG13 for 0 , 3 , and 6 hours .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(A) Differential intron retention (IR) analysis of RNA-seq data from samples under PABPN1 knockdown (siPABPN1) and control (NC) conditions. Significantly repressed and induced intron retention events (retained introns, RIs) are indicated by blue and red dots, respectively. The threshold of IR difference (ΔIR, siPABPN1-siNC) is 0.15, and the FDR cutoff is 0.01.(C) Cumulative frequency distributions of retained introns (RIs), up-regulated (red), and down-regulated (blue) upon PABPN1 knockdown, and of all retained introns in control (NC, green) samples by their relative intronic ranks in genes.Validation of last intron retention of six representative genes in response to PABPN1 knockdown by semi-quantitative PCR in HeLa cells treated with three different NC and PABPN1 siRNAs (D).(G) Western blot validating the degradation effect of PABPN1 under the treatment of dTAG13 for 0, 3, and 6 hours.


Figure 2 ( B ) Western blots of the C1 - C4 reporters for two endogenous regulated genes ( LARP7 and COPS5 ) and three endogenous non - regulated control genes ( TDRD9 , CENPV , and SMTN ) . ( C ) RT - PCR analysis of the spliced and unspliced isoforms from the C3 and C4 reporters for two endogenous regulated genes LARP7 and COPS5 . ( E ) RT - PCR quantification of MS2 reporters of histone 3 ´ end ( left ) and Malat1 3 ´ end ( right ) for two PABPN1 - regulated genes LARP7 and COPS5 .

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Figure 2(B) Western blots of the C1-C4 reporters for two endogenous regulated genes (LARP7 and COPS5) and three endogenous non-regulated control genes (TDRD9, CENPV, and SMTN).(C) RT-PCR analysis of the spliced and unspliced isoforms from the C3 and C4 reporters for two endogenous regulated genes LARP7 and COPS5.(E) RT-PCR quantification of MS2 reporters of histone 3´end (left) and Malat1 3´end (right) for two PABPN1-regulated genes LARP7 and COPS5.


Figure 3 ( D ) RNA quantification by RT - PCR for the spliced and unspliced isoforms ( top ) and calculated IR scores ( bottom ) of the reporters Data information : Data are from n = 3 biologically independent experiments . The error bars represent SD . The P - values are calculated by unpaired t test ( * * * * P < 0 . 0001 ; * * * P < 0 . 001 ; * * P < 0 . 01 ; * P < 0 . 05 ; ns , non - significant ) . ( E ) Quantification of the spliced ( EEJ ) over unspliced isoforms ( EIJ ) of the reporters in Figure 3F . Data information : Data are from n = 3 biologically independent experiments . The error bars represent SD . The P - values are calculated by unpaired t test ( * * * * P < 0 . 0001 ; * * * P < 0 . 001 ; * * P < 0 . 01 ; * P < 0 . 05 ; ns , non - significant ) . ( G ) Splicing analysis of the C3 , C4 , and polyA reporters in Figure 3F for endogenous genes LARP7 , COPS5 , and NSUN5 by RT - PCR upon PABPN1 knockdown ( top ) and the quantified intron retention levels ( IR score ) of these reporters ( bottom ) . Data information : Data are from n = 3 biologically independent experiments . The error bars represent SD . The P - values are calculated by unpaired t test ( * * * * P < 0 . 0001 ; * * * P < 0 . 001 ; * * P < 0 . 01 ; * P < 0 . 05 ; ns , non - significant ) . ( J ) Relative ratios of the unspliced isoforms ( EIJ ) to the spliced isoforms ( EEJ ) of the last introns for LARP7 , DUSP12 , and BRCA1 genes in HeLa cells treated with si - PABPN1 and then rescuing PABPN1 mutants constructed in G . Data information : Data are from n = 3 biologically independent experiments . The error bars represent SD . The P - values are calculated by unpaired t test ( * * * * P < 0 . 0001 ; * * * P < 0 . 001 ; * * P < 0 . 01 ; * P < 0 . 05 ; ns , non - significant ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(D) RNA quantification by RT-PCR for the spliced and unspliced isoforms (top) and calculated IR scores (bottom) of the reporters Data information: Data are from n=3 biologically independent experiments. The error bars represent SD. The P-values are calculated by unpaired t test (****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05; ns, non-significant).(E) Quantification of the spliced (EEJ) over unspliced isoforms (EIJ) of the reporters in Figure 3F. Data information: Data are from n=3 biologically independent experiments. The error bars represent SD. The P-values are calculated by unpaired t test (****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05; ns, non-significant).(G) Splicing analysis of the C3, C4, and polyA reporters in Figure 3F for endogenous genes LARP7, COPS5, and NSUN5 by RT-PCR upon PABPN1 knockdown (top) and the quantified intron retention levels (IR score) of these reporters (bottom). Data information: Data are from n=3 biologically independent experiments. The error bars represent SD. The P-values are calculated by unpaired t test (****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05; ns, non-significant).(J) Relative ratios of the unspliced isoforms (EIJ) to the spliced isoforms (EEJ) of the last introns for LARP7, DUSP12, and BRCA1 genes in HeLa cells treated with si-PABPN1 and then rescuing PABPN1 mutants constructed in G. Data information: Data are from n=3 biologically independent experiments. The error bars represent SD. The P-values are calculated by unpaired t test (****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05; ns, non-significant).


Figure 4Splicing analysis of NSUN5 and LARP7 reporter mutants by RT - PCR ( E ) Splicing analysis of NSUN5 and LARP7 reporter mutants by Western blot ( F ) .

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Figure 4Splicing analysis of NSUN5 and LARP7 reporter mutants by RT-PCR (E)Splicing analysis of NSUN5 and LARP7 reporter mutants by Western blot (F).


Figure 5 ( B ) Volcano plots of the enriched proteins identified by PABPN1 TurboID - MS . Significantly enriched proteins are colored in orange , and a few representative proteins are highlighted with labels . ( C ) RT - PCR analysis of the last intron splicing of five representative PABPN1 - dependent endogenous genes upon RBM26 / 27 knockdown with quantified IR values . Data information Data are from n = 3 biologically independent experiments . The error bars represent SD . The P - values are calculated by unpaired t test ( * * * * P < 0 . 0001 ; * * * P < 0 . 001 ; * * P < 0 . 01 ; * P < 0 . 05 ; ns , non - significant ) . ( G ) Cumulative frequency distributions of up - regulated RIs upon PABPN1 ( red ) , RBM26 & 27 ( blue ) , ZC3H3 ( green ) , and ZFC3H1 ( yellow ) knockdown by their relative intronic rank in genes . ( H ) Western blot validation of endogenous PABPN1 interacting with the myc - tagged RBM26 and RBM27 . ( I ) Representative immunofluorescence images showing the localization of endogenous PABPN1 relative to SC35 , and the mCherry - tagged RBM26 & 27 relative to SRRM2 in vivo . ( K - L ) Western blot of endogenous PABPN1 immunoprecipitated myc - tagged RBM27 mutated proteins .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O

Figure 5(B) Volcano plots of the enriched proteins identified by PABPN1 TurboID-MS. Significantly enriched proteins are colored in orange, and a few representative proteins are highlighted with labels.(C) RT-PCR analysis of the last intron splicing of five representative PABPN1-dependent endogenous genes upon RBM26/27 knockdown with quantified IR values. Data information Data are from n=3 biologically independent experiments. The error bars represent SD. The P-values are calculated by unpaired t test (****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05; ns, non-significant).(G) Cumulative frequency distributions of up-regulated RIs upon PABPN1 (red), RBM26&27 (blue), ZC3H3 (green), and ZFC3H1 (yellow) knockdown by their relative intronic rank in genes.(H) Western blot validation of endogenous PABPN1 interacting with the myc-tagged RBM26 and RBM27.(I) Representative immunofluorescence images showing the localization of endogenous PABPN1 relative to SC35, and the mCherry-tagged RBM26&27 relative to SRRM2 in vivo.(K-L) Western blot of endogenous PABPN1 immunoprecipitated myc-tagged RBM27 mutated proteins.


Figure 1The expression of SAMHD1 is negatively correlated with EV71 viral replication ability . HEK293T and RD cells were infected with EV71 at a MOI of 0 . 1 , then the cells and supernatants were harvested at the indicated time points . Immunoblotting ( IB ) analysis of EV71 VP1 in cells , with tubulin as a loading control . EV71 - VP1 protein in the supernatants was detected after ultracentrifugation . The expression of SAMHD1 is negatively correlated with EV71 viral replication ability . HEK293T and RD cells were infected with EV71 at a MOI of 0 . 1 , then the cells and supernatants were harvested at the indicated time points . EV71 viral RNA levels in cell lysates and supernatants were detected by RT - qPCR with GAPDH as a control . ( n = 3 , mean ± SD , ns stands for no significance , paired t - test ) The expression of SAMHD1 is negatively correlated with EV71 viral replication ability . HEK293T and RD cells were infected with EV71 at a MOI of 0 . 1 , then the cells and supernatants were harvested at the indicated time points . The mRNA levels of the host restriction factors were detected by RT - qPCR in infected or uninfected HEK293T or RD cells , and the expression levels of the target genes were normalized to GAPDH . ( n = 3 , mean ± SD , ns stands for no significance , paired t - test ) . The expression of SAMHD1 is negatively correlated with EV71 viral replication ability . HEK293T and RD cells were infected with EV71 at a MOI of 0 . 1 , then the cells and supernatants were harvested at the indicated time points . IB analysis of SAMHD1 and BST - 2 protein levels with tubulin as a loading control . The densities of bands were analyzed with ImageJ software to calculate the values relative to that of tubulin . SAMHD1 knockdown enhances EV71 replication . The stable cell lines pLKO . 1 and sh - SAMHD1 constructed in HEK293T cells were infected with EV71 at a MOI of 0 . 1 and 0 . 05 respectively , and cells and supernatants were harvested at the indicated time points . IB analysis of EV71 VP1 and SAMHD1 in cells was performed with tubulin as a loading control . EV71 - VP1 protein in the supernatants was detected after ultracentrifugation . SAMHD1 knockdown enhances EV71 replication . EV71 viral RNA in cell lysates was detected by RT - qPCR with GAPDH as a control ( n = 3 , mean ± SD , * P < 0 . 05 , * * P < 0 . 01 , paired t - test ) . SAMHD1 knockdown enhances EV71 replication . Viral titers in the supernatants were measured by the cytopathic effect method . SAMHD1 knockdown enhances EV71 replication . The stable cell lines pLKO . 1 and sh - SAMHD1 constructed in RD ( H ) cells were infected with EV71 at a MOI of 0 . 1 and 0 . 05 respectively , and cells and supernatants were harvested at the indicated time points . IB analysis of EV71 VP1 and SAMHD1 in cells was performed with tubulin as a loading control . EV71 - VP1 protein in the supernatants was detected after ultracentrifugation . SAMHD1 knockdown enhances EV71 replication . EV71 viral RNA in cell lysates was detected by RT - qPCR with GAPDH as a control ( n = 3 , mean ± SD , * P < 0 . 05 , * * P < 0 . 01 , paired t - test ) . SAMHD1 knockdown enhances EV71 replication . Viral titers in the supernatants were measured by the cytopathic effect method . HEK293T cells transfected with VR1012 , which is a eukaryotic expression vector , or Vpx - HA were infected with EV71 at a MOI of 0 . 1 , and then cells and supernatants were harvested at the indicated time points . IB analysis of EV71 - VP1 , SAMHD1 and Vpx - HA in cell lysates was performed with tubulin as a loading control . EV71 - VP1 was detected in the supernatants after ultracentrifugation . EV71 viral RNA was detected in cell lysates by RT - qPCR with GAPDH as a control ( n = 3 , mean ± SD , * * P < 0 . 01 , paired t - test ) . Viral titres in the supernatants were measured by the cytopathic effect method . The results represent the means ± SD from three independent experiments . Statistical significance was analyzed using Student ' s t test ( * * P < 0 . 01 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1The expression of SAMHD1 is negatively correlated with EV71 viral replication ability. HEK293T and RD cells were infected with EV71 at a MOI of 0.1, then the cells and supernatants were harvested at the indicated time points. Immunoblotting (IB) analysis of EV71 VP1 in cells, with tubulin as a loading control. EV71-VP1 protein in the supernatants was detected after ultracentrifugation.The expression of SAMHD1 is negatively correlated with EV71 viral replication ability. HEK293T and RD cells were infected with EV71 at a MOI of 0.1, then the cells and supernatants were harvested at the indicated time points. EV71 viral RNA levels in cell lysates and supernatants were detected by RT-qPCR with GAPDH as a control. (n=3, mean±SD, ns stands for no significance, paired t-test)The expression of SAMHD1 is negatively correlated with EV71 viral replication ability. HEK293T and RD cells were infected with EV71 at a MOI of 0.1, then the cells and supernatants were harvested at the indicated time points. The mRNA levels of the host restriction factors were detected by RT-qPCR in infected or uninfected HEK293T or RD cells, and the expression levels of the target genes were normalized to GAPDH. (n=3, mean±SD, ns stands for no significance, paired t-test).The expression of SAMHD1 is negatively correlated with EV71 viral replication ability. HEK293T and RD cells were infected with EV71 at a MOI of 0.1, then the cells and supernatants were harvested at the indicated time points. IB analysis of SAMHD1 and BST-2 protein levels with tubulin as a loading control. The densities of bands were analyzed with ImageJ software to calculate the values relative to that of tubulin.SAMHD1 knockdown enhances EV71 replication. The stable cell lines pLKO.1 and sh-SAMHD1 constructed in HEK293T cells were infected with EV71 at a MOI of 0.1 and 0.05 respectively, and cells and supernatants were harvested at the indicated time points. IB analysis of EV71 VP1 and SAMHD1 in cells was performed with tubulin as a loading control. EV71-VP1 protein in the supernatants was detected after ultracentrifugation.SAMHD1 knockdown enhances EV71 replication. EV71 viral RNA in cell lysates was detected by RT-qPCR with GAPDH as a control (n=3, mean±SD, * P < 0.05, ** P < 0.01, paired t-test).SAMHD1 knockdown enhances EV71 replication. Viral titers in the supernatants were measured by the cytopathic effect method.SAMHD1 knockdown enhances EV71 replication. The stable cell lines pLKO.1 and sh-SAMHD1 constructed in RD (H) cells were infected with EV71 at a MOI of 0.1 and 0.05 respectively, and cells and supernatants were harvested at the indicated time points. IB analysis of EV71 VP1 and SAMHD1 in cells was performed with tubulin as a loading control. EV71-VP1 protein in the supernatants was detected after ultracentrifugation.SAMHD1 knockdown enhances EV71 replication. EV71 viral RNA in cell lysates was detected by RT-qPCR with GAPDH as a control (n=3, mean±SD, * P < 0.05, ** P < 0.01, paired t-test).SAMHD1 knockdown enhances EV71 replication. Viral titers in the supernatants were measured by the cytopathic effect method.HEK293T cells transfected with VR1012, which is a eukaryotic expression vector, or Vpx-HA were infected with EV71 at a MOI of 0.1, and then cells and supernatants were harvested at the indicated time points. IB analysis of EV71-VP1, SAMHD1 and Vpx-HA in cell lysates was performed with tubulin as a loading control. EV71-VP1 was detected in the supernatants after ultracentrifugation.EV71 viral RNA was detected in cell lysates by RT-qPCR with GAPDH as a control (n=3, mean±SD, ** P <0.01, paired t-test).Viral titres in the supernatants were measured by the cytopathic effect method. The results represent the means ± SD from three independent experiments. Statistical significance was analyzed using Student's t test (**P < 0.01).


Figure 2Stable SAMHD1 - HA - overexpressing cell lines were constructed in HEK293T and RD cells and detected by IB with pLVX as a negative control . RD SAMHD1 - HA cell lines were treated with dimethyl sulfoxide ( DMSO ) or 10 μM MG132 for 12 h prior to harvest and subjected to IB with tubulin as a loading control . RD SAMHD1 - HA cell lines were treated as in ( B ) , and then HA immunoprecipitation ( IP ) and IB were performed . The IP elution was analyzed by mass spectrometry . TRIM21 induced the degradation of SAMHD1 , and MG132 rescued the TRIM21 - mediated degradation of SAMHD1 . HEK293T cells were cotransfected with SAMHD1 and VR1012 or TRIM21 expression plasmids with or without MG132 treatment for 12 h prior to harvest , and then subjected to IB with tubulin as a loading control . TRIM25 ( E ) induced the degradation of SAMHD1 HEK293T cells were cotransfected with SAMHD1 and VR1012 or TRIM25 expression plasmids and then subjected to IB with tubulin as a loading control . Reverse co - IP confirmed the interaction between SAMHD1 and TRIM21 . SAMHD1 - flag was transfected with TRIM21 - HA into HEK293T cells , and then the cells were treated as in ( B ) and subjected to HA IP and IB . Endogenous TRIM21 but not TRIM25 still interacted with SAMHD1 . RD - SAMHD1 - HA cells treated as in ( B ) were subjected to TRIM21 or TRIM25 IP and IB . mRNA levels of TRIM21 in RD and HEK293T cells . protein levels of TRIM21 in RD and HEK293T cells . TRIM21 ( J ) but not TRIM25 ( K ) colocalized with SAMHD1 . Images were taken under a Zeiss LZM710 confocal microscope , Bars , 10μm .

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Figure 2Stable SAMHD1-HA-overexpressing cell lines were constructed in HEK293T and RD cells and detected by IB with pLVX as a negative control.RD SAMHD1-HA cell lines were treated with dimethyl sulfoxide (DMSO) or 10 μM MG132 for 12 h prior to harvest and subjected to IB with tubulin as a loading control.RD SAMHD1-HA cell lines were treated as in (B), and then HA immunoprecipitation (IP) and IB were performed. The IP elution was analyzed by mass spectrometry.TRIM21 induced the degradation of SAMHD1, and MG132 rescued the TRIM21-mediated degradation of SAMHD1. HEK293T cells were cotransfected with SAMHD1 and VR1012 or TRIM21 expression plasmids with or without MG132 treatment for 12 h prior to harvest, and then subjected to IB with tubulin as a loading control.TRIM25 (E) induced the degradation of SAMHD1 HEK293T cells were cotransfected with SAMHD1 and VR1012 or TRIM25 expression plasmids and then subjected to IB with tubulin as a loading control.Reverse co-IP confirmed the interaction between SAMHD1 and TRIM21. SAMHD1-flag was transfected with TRIM21-HA into HEK293T cells, and then the cells were treated as in (B) and subjected to HA IP and IB.Endogenous TRIM21 but not TRIM25 still interacted with SAMHD1. RD-SAMHD1-HA cells treated as in (B) were subjected to TRIM21 or TRIM25 IP and IB.mRNA levels of TRIM21 in RD and HEK293T cells.protein levels of TRIM21 in RD and HEK293T cells.TRIM21 (J) but not TRIM25 (K) colocalized with SAMHD1. Images were taken under a Zeiss LZM710 confocal microscope, Bars, 10μm.


Figure 3TRIM21 Knockout in RD cells released SAMHD1 inhibition on EV71 replication . Negative control or RD - CRISPR - Cas9 - TRIM21 RD cells were infected with EV71 at a MOI of 0 . 05 for the indicated time and harvested for SAMHD1 , TRIM21 and EV71 - VP1 detection by IB . Tubulin served as a loading control . TRIM21 Knockout in RD cells released SAMHD1 inhibition on EV71 replication . EV71 RNA levels in ( B ) were detected by RT - qPCR with GAPDH as a control ( n = 3 , mean ± SD , * * P < 0 . 01 , paired t - test ) . TRIM21 Knockout in RD cells released SAMHD1 inhibition on EV71 replication . Viral titres in the supernatant were measured by the cytopathic effect method . The results represent the means ± SD from three independent experiments . Statistical significance was analyzed using Student ' s t test ( * * P < 0 . 01 ) . Overexpression of TRIM21 in HEK293T cells increased EV71 replication by degrading SAMHD1 . HEK293T cells were transfected with VR1012 or TRIM21 - HA for 24 h and then infected with EV71 at a MOI of 0 . 05 . Cells were harvested at the indicated time points and subjected to IB with tubulin as a loading control . Overexpression of TRIM21 in HEK293T cells increased EV71 replication by degrading SAMHD1 . HEK293T cells were transfected with VR1012 or TRIM21 - HA for 24 h and then infected with EV71 at a MOI of 0 . 05 . EV71 viral RNA in cell lysates was detected by RT - qPCR with GAPDH as a control ( n = 3 , mean ± SD , * * P < 0 . 01 , paired t - test ) ) . TRIM21 itself has no effect on EV71 replication . VR1012 or TRIM21 was transfected into pLKO . 1 or sh - SAMHD1 HEK293T cells for 24 h and infected with EV71 . Cells were harvested and subjected to IB analysis . TRIM21 itself has no effect on EV71 replication . VR1012 or TRIM21 was transfected into pLKO . 1 or sh - SAMHD1 HEK293T cells for 24 h and infected with EV71 . Viral titers in the supernatants were measured by the cytopathic effect method . The results represent the means ± SD from three independent experiments . Statistical significance was analyzed using Student ' s t test ( * P < 0 . 05 ) .

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Figure 3TRIM21 Knockout in RD cells released SAMHD1 inhibition on EV71 replication. Negative control or RD-CRISPR-Cas9-TRIM21 RD cells were infected with EV71 at a MOI of 0.05 for the indicated time and harvested for SAMHD1, TRIM21 and EV71-VP1 detection by IB. Tubulin served as a loading control.TRIM21 Knockout in RD cells released SAMHD1 inhibition on EV71 replication. EV71 RNA levels in (B) were detected by RT-qPCR with GAPDH as a control (n=3, mean±SD, ** P < 0.01, paired t-test).TRIM21 Knockout in RD cells released SAMHD1 inhibition on EV71 replication. Viral titres in the supernatant were measured by the cytopathic effect method. The results represent the means ± SD from three independent experiments. Statistical significance was analyzed using Student's t test (**P < 0.01).Overexpression of TRIM21 in HEK293T cells increased EV71 replication by degrading SAMHD1. HEK293T cells were transfected with VR1012 or TRIM21-HA for 24 h and then infected with EV71 at a MOI of 0.05. Cells were harvested at the indicated time points and subjected to IB with tubulin as a loading control.Overexpression of TRIM21 in HEK293T cells increased EV71 replication by degrading SAMHD1. HEK293T cells were transfected with VR1012 or TRIM21-HA for 24 h and then infected with EV71 at a MOI of 0.05. EV71 viral RNA in cell lysates was detected by RT-qPCR with GAPDH as a control (n=3, mean±SD, ** P < 0.01, paired t-test)).TRIM21 itself has no effect on EV71 replication. VR1012 or TRIM21 was transfected into pLKO.1 or sh-SAMHD1 HEK293T cells for 24 h and infected with EV71. Cells were harvested and subjected to IB analysis.TRIM21 itself has no effect on EV71 replication. VR1012 or TRIM21 was transfected into pLKO.1 or sh-SAMHD1 HEK293T cells for 24 h and infected with EV71. Viral titers in the supernatants were measured by the cytopathic effect method. The results represent the means ± SD from three independent experiments. Statistical significance was analyzed using Student's t test (*P < 0.05).


Figure 4The mRNA levels of SAMHD1 and TRIM21 in different tissues of neonatal mice were detected by RT - qPCR with GAPDH as a control , and the expression of SAMHD1 and TRIM21 in the heart was set as 100 % ( n = 3 , mean ± SD , * P < 0 . 05 , paired t - test ) . The protein levels of SAMHD1 and TRIM21 in different tissues of mice were examined by Near Infrared Spectrum ( NIR ) western blot with tubulin as a control . Immunohistochemical ( IHC ) staining results of SAMHD1 in various tissues of mice . Bars , 100μm . The mRNA levels of EV71 virus and host factors in mock or EV71 infected mice were detected by RT - qPCR with GAPDH as a control . The expression of EV71 ( D ) were presented , and the levels of corresponding gene in mock infected mice were set as 100 % The mRNA levels of EV71 virus and host factors in mock or EV71 infected mice were detected by RT - qPCR with GAPDH as a control . The expression of , IFNα and IFNβ ( E ) were presented , and the levels of corresponding gene in mock infected mice were set as 100 % The mRNA levels of EV71 virus and host factors in mock or EV71 infected mice were detected by RT - qPCR with GAPDH as a control . The expression of SAMHD1 and TRIM21 ( F ) were presented , and the levels of corresponding gene in mock infected mice were set as 100 %

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Figure 4The mRNA levels of SAMHD1 and TRIM21 in different tissues of neonatal mice were detected by RT-qPCR with GAPDH as a control, and the expression of SAMHD1 and TRIM21 in the heart was set as 100% (n=3, mean±SD, * P < 0.05, paired t-test).The protein levels of SAMHD1 and TRIM21 in different tissues of mice were examined by Near Infrared Spectrum (NIR) western blot with tubulin as a control.Immunohistochemical (IHC) staining results of SAMHD1 in various tissues of mice. Bars, 100μm.The mRNA levels of EV71 virus and host factors in mock or EV71 infected mice were detected by RT-qPCR with GAPDH as a control. The expression of EV71 (D) were presented, and the levels of corresponding gene in mock infected mice were set as 100%The mRNA levels of EV71 virus and host factors in mock or EV71 infected mice were detected by RT-qPCR with GAPDH as a control. The expression of , IFNα and IFNβ (E) were presented, and the levels of corresponding gene in mock infected mice were set as 100%The mRNA levels of EV71 virus and host factors in mock or EV71 infected mice were detected by RT-qPCR with GAPDH as a control. The expression of SAMHD1 and TRIM21 (F) were presented, and the levels of corresponding gene in mock infected mice were set as 100%


Figure 5IB analysis of THP - 1 cells treated with scrambled shRNA , TRIM21 - specific shRNA or SAMHD1 - specific shRNA . Tubulin served as loading control . Quantification of SAMHD1 and TRIM21 levels in whole cells in which the indicated genes were knocked down by shRNA . Data are normalized to tubulin and expressed as the fold change over cells treated with pLKO . 1 without HIV infection ( n = 3 , mean ± SD ) . sh - SAMHD1 and sh - TRIM21 THP - 1 cells were treated with PMA for 24 h and infected with the VSV - G pseudotyped reporter HIV - 1 , then detected 48 h later by flow cytometry analysis ( n = 3 , mean ± SD , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , paired t - test ) . Quantification of SAMHD1 and TRIM21 levels in whole cells in which the indicated genes were knocked down by shRNA . Data are normalized to tubulin and expressed as the fold change over cells treated with pLKO . 1 without HIV infection ( n = 3 , mean ± SD ) . IB analysis of MDM cells from 3 donors transfected with TRIM21 siRNA twice at days 1 and 3 , respectively , after MDM differentiation . MDM cells treated with TRIM21 siRNA were infected with VSV - G - pseudotyped HIV - 1 reporter and then detected 48 h later by flow cytometry analysis ( n = 3 , mean ± SD , * * P < 0 . 01 , paired t - test ) . Quantification of SAMHD1 and TRIM21 levels in whole cells in which the indicated genes were knocked down by shRNA . Data are normalized to tubulin and expressed as the fold change over cells treated with pLKO . 1 without HIV infection ( n = 3 , mean ± SD ) . IB analysis of Jurkat and KG - 1 cells treated with scrambled shRNA or TRIM21 - specific shRNA . Jurkat and KG - 1 cells treated with TRIM21 shRNA were infected with HIV - 1 , then detected 48 h later by flow cytometry analysis ( n = 3 , mean ± SD , ns stands for no significance , paired t - test ) .

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Figure 5IB analysis of THP-1 cells treated with scrambled shRNA, TRIM21-specific shRNA or SAMHD1-specific shRNA. Tubulin served as loading control. Quantification of SAMHD1 and TRIM21 levels in whole cells in which the indicated genes were knocked down by shRNA. Data are normalized to tubulin and expressed as the fold change over cells treated with pLKO.1 without HIV infection (n=3, mean±SD).sh-SAMHD1 and sh-TRIM21 THP-1 cells were treated with PMA for 24 h and infected with the VSV-G pseudotyped reporter HIV-1, then detected 48 h later by flow cytometry analysis (n=3, mean±SD, * P < 0.05, ** P < 0.01, *** P < 0.001, paired t-test).Quantification of SAMHD1 and TRIM21 levels in whole cells in which the indicated genes were knocked down by shRNA. Data are normalized to tubulin and expressed as the fold change over cells treated with pLKO.1 without HIV infection (n=3, mean±SD). IB analysis of MDM cells from 3 donors transfected with TRIM21 siRNA twice at days 1 and 3, respectively, after MDM differentiation.MDM cells treated with TRIM21 siRNA were infected with VSV-G-pseudotyped HIV-1 reporter and then detected 48 h later by flow cytometry analysis (n=3, mean±SD, ** P < 0.01, paired t-test).Quantification of SAMHD1 and TRIM21 levels in whole cells in which the indicated genes were knocked down by shRNA. Data are normalized to tubulin and expressed as the fold change over cells treated with pLKO.1 without HIV infection (n=3, mean±SD). IB analysis of Jurkat and KG-1 cells treated with scrambled shRNA or TRIM21-specific shRNA.Jurkat and KG-1 cells treated with TRIM21 shRNA were infected with HIV-1, then detected 48 h later by flow cytometry analysis (n=3, mean±SD, ns stands for no significance, paired t-test).


Figure 6TRIM21 interacts with SAMHD1 via PRY and SPRY domains . The effect of TRIM21 on the degradation of SAMHD1 . SAMHD1 - flag was cotransfected with VR1012 , TRIM21 WT or the indicated mutant into HEK293T cells for 48 h , and the cells were subjected to IB with tubulin as loading control . TRIM21 interacts with SAMHD1 via PRY and SPRY domains . SAMHD1 - flag was cotransfected with VR1012 or TRIM21 WT or the indicated mutant for 24 h , and the cells were then treated with 10 μM MG132 for 12 h before harvest and subjected to HA IP and IB . The effect of TRIM21 on SAMHD1 WT or its mutants . SAMHD1 - HA or the indicated mutant were cotransfected with VR1012 or TRIM21 - flag into HEK293T cells for 48 h , and the cells were subjected to IB with tubulin as a loading control . SAMHD1 - flag 1 - 547 was cotransfected with VR1012 or TRIM21 - HA for 24 h , and the cells were then treated with 10 μM MG132 for 12 h before harvest and subjected to HA IP and IB . SAMHD1 109 - 626 followed with a His tag or TRIM21 - PRYSPRY followed with a GST tag were expressed in Rosetta ( DE3 ) , and pull - down assay were performed with Ni - Sepharose ( up ) and GST Sepharose ( down ) , respectively . FRET analysis indicates interaction between YFP - SAMHD1 and CFP - TRIM21 . A representative image of SAMHD1 - YFP ( yellow ) and ECFP - TRIM21 ( cyan ) expressing cells before and after photobleaching the acceptor fluorophore , YFP . The region chosen for photobleaching is marked ( white open box ) , Bars , 10μm . The quantization of fluorescence brightness was analyzed by Image J ( n = 3 , mean ± SD , ns stands for no significance , * P < 0 . 05 , paired t - test ) The microscale thermophoresis curve for the interaction between SAMHD1 and TRIM21 . Alexa Fluor ® 647 labelled SAMHD1 at a concentration of 20 nM were incubated with two - fold dilution series of unlabeled TRIM21 ( 3 μM to 9 . 16E - 05 μM ) . The curve represents the signal recorded from three measurements . The normalized fluorescence thermophoretic signals were plotted against the concentration of TRIM21 with mean values ± standard deviation as well as a fitting to a 1 : 1 binding model ( NanoTemper ® analysis software , F Norm = F Hot / F Cold ) . The Kd of this interaction was determined to be 270 ± 92nM . The data was a representive of three independent experiments using different dilution .

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Figure 6TRIM21 interacts with SAMHD1 via PRY and SPRY domains. The effect of TRIM21 on the degradation of SAMHD1. SAMHD1-flag was cotransfected with VR1012, TRIM21 WT or the indicated mutant into HEK293T cells for 48 h, and the cells were subjected to IB with tubulin as loading control.TRIM21 interacts with SAMHD1 via PRY and SPRY domains. SAMHD1-flag was cotransfected with VR1012 or TRIM21 WT or the indicated mutant for 24 h, and the cells were then treated with 10 μM MG132 for 12 h before harvest and subjected to HA IP and IB.The effect of TRIM21 on SAMHD1 WT or its mutants. SAMHD1-HA or the indicated mutant were cotransfected with VR1012 or TRIM21-flag into HEK293T cells for 48 h, and the cells were subjected to IB with tubulin as a loading control.SAMHD1-flag 1-547 was cotransfected with VR1012 or TRIM21-HA for 24 h, and the cells were then treated with 10 μM MG132 for 12 h before harvest and subjected to HA IP and IB.SAMHD1 109-626 followed with a His tag or TRIM21-PRYSPRY followed with a GST tag were expressed in Rosetta (DE3), and pull-down assay were performed with Ni-Sepharose (up) and GST Sepharose (down), respectively.FRET analysis indicates interaction between YFP-SAMHD1 and CFP-TRIM21. A representative image of SAMHD1-YFP (yellow) and ECFP-TRIM21 (cyan) expressing cells before and after photobleaching the acceptor fluorophore, YFP. The region chosen for photobleaching is marked (white open box), Bars, 10μm. The quantization of fluorescence brightness was analyzed by Image J (n=3, mean±SD, ns stands for no significance, * P < 0.05, paired t-test)The microscale thermophoresis curve for the interaction between SAMHD1 and TRIM21. Alexa Fluor®647 labelled SAMHD1 at a concentration of 20 nM were incubated with two-fold dilution series of unlabeled TRIM21 (3 μM to 9.16E-05 μM). The curve represents the signal recorded from three measurements. The normalized fluorescence thermophoretic signals were plotted against the concentration of TRIM21 with mean values ± standard deviation as well as a fitting to a 1:1 binding model (NanoTemper® analysis software, F Norm = F Hot / F Cold). The Kd of this interaction was determined to be 270±92nM. The data was a representive of three independent experiments using different dilution.


Figure 7The effect of TRIM21 on SAMHD1 proteins from various species . HEK293T cells were transfected with VR1012 or TRIM21 and the indicated SAMHD1 expression vector , then subjected to IB analysis . Tubulin served as a loading control . The effect of TRIM21 on hSAMHD1 mutants . HEK293T cells were transfected with VR1012 or TRIM21 and SAMHD1 mutants for 48 h and then subjected to IB analysis . Tubulin served as a loading control . SAMHD1 - flag WT or the G153S or G183R mutant was cotransfected with VR1012 or TRIM21 - HA for 24 h , and the cells were then treated with 10 μM MG132 for 12 h before harvest and subjected to HA IP and IB . Tubulin served as a loading control . SAMHD1 - HA WT or the indicated mutants were cotransfected with VR1012 or TRIM21 - flag into HEK293T cells for 48 h , and the cells were subjected to IB with tubulin as a loading control . SAMHD1 - HA WT and VR1012 or TRIM21 were cotransfected with K48 - only and K63 - only ubiquitin - flag into HEK293T cells for 24 h , and the cells were then treated with 10 μM MG132 for 12 h before harvest and subjected to HA IP and IB with tubulin as a loading control . K48 - only ubiquitin - flag and SAMHD1 - HA WT or mutant K622R were cotransfected with VR1012 or TRIM21 into HEK293T cells as indicated . The cells were then treated with 10 μM MG132 for 12 h before harvest and subjected to HA IP and IB with tubulin as a loading control . VR1012 , SAMHD1 - HA WT or the K622R mutant was transfected into HEK293T - shSAMHD1 cells for 24 h . The cells were then infected with EV71 at a MOI of 0 . 1 and harvested at the indicated time points . SAMHD1 - HA , TRIM21 and EV71 - VP1 were detected by IB with tubulin as a loading control .

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Figure 7The effect of TRIM21 on SAMHD1 proteins from various species. HEK293T cells were transfected with VR1012 or TRIM21 and the indicated SAMHD1 expression vector, then subjected to IB analysis. Tubulin served as a loading control.The effect of TRIM21 on hSAMHD1 mutants. HEK293T cells were transfected with VR1012 or TRIM21 and SAMHD1 mutants for 48 h and then subjected to IB analysis. Tubulin served as a loading control.SAMHD1-flag WT or the G153S or G183R mutant was cotransfected with VR1012 or TRIM21-HA for 24 h, and the cells were then treated with 10 μM MG132 for 12 h before harvest and subjected to HA IP and IB. Tubulin served as a loading control.SAMHD1-HA WT or the indicated mutants were cotransfected with VR1012 or TRIM21-flag into HEK293T cells for 48 h, and the cells were subjected to IB with tubulin as a loading control.SAMHD1-HA WT and VR1012 or TRIM21 were cotransfected with K48-only and K63-only ubiquitin-flag into HEK293T cells for 24 h, and the cells were then treated with 10 μM MG132 for 12 h before harvest and subjected to HA IP and IB with tubulin as a loading control.K48-only ubiquitin-flag and SAMHD1-HA WT or mutant K622R were cotransfected with VR1012 or TRIM21 into HEK293T cells as indicated. The cells were then treated with 10 μM MG132 for 12 h before harvest and subjected to HA IP and IB with tubulin as a loading control.VR1012, SAMHD1-HA WT or the K622R mutant was transfected into HEK293T-shSAMHD1 cells for 24 h. The cells were then infected with EV71 at a MOI of 0.1 and harvested at the indicated time points. SAMHD1-HA, TRIM21 and EV71-VP1 were detected by IB with tubulin as a loading control.


Figure 1 ( D ) Absolute numbers of lymphocytes subpopulations in the bone marrow , thymus , and spleen . B cell development in the bone marrow was assessed by FACS analysis for surface expression of : B220 + CD43 + CD19 − IgM − IgD − ( pre - pro B ) ; B220 + CD43 + CD19 + IgM − IgD − ( pro B ) ; B220 + CD43 − CD19 + IgM − IgD − ( pre B ) ; CD19 + IgM + IgD − ( immature ) ; CD19 + IgM + IgD + ( mature ) . T cell development in the thymus was assessed by FACS analysis for surface expression of : CD4 − CD8 − ( double negative , DN ) ; CD4 + CD8 + ( double positive , DP ) ; CD4 + CD8 − ( CD4 single positive , SP ) ; CD4 − CD8 + ( CD8 SP ) . Splenic B cell populations were assessed by FACS analysis for surface expression of : B220 + CD21 + CD23 + ( follicular B cells , FOB ) ; B220 + CD21 + CD23low ( marginal zone B cells , MZB ) . Each symbol represents an individual mouse . Horizontal bars indicate mean values . Two - tailed Mann - Whitney test , ns = not significant , * P < 0 . 05 , and * * P < 0 . 01 .

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Figure 1(D) Absolute numbers of lymphocytes subpopulations in the bone marrow, thymus, and spleen. B cell development in the bone marrow was assessed by FACS analysis for surface expression of: B220+CD43+CD19−IgM−IgD− (pre-pro B); B220+CD43+CD19+IgM−IgD− (pro B); B220+CD43−CD19+IgM−IgD− (pre B); CD19+IgM+IgD− (immature); CD19+IgM+IgD+ (mature). T cell development in the thymus was assessed by FACS analysis for surface expression of: CD4−CD8− (double negative, DN); CD4+CD8+ (double positive, DP); CD4+CD8− (CD4 single positive, SP); CD4−CD8+ (CD8 SP). Splenic B cell populations were assessed by FACS analysis for surface expression of: B220+CD21+CD23+ (follicular B cells, FOB); B220+CD21+CD23low (marginal zone B cells, MZB). Each symbol represents an individual mouse. Horizontal bars indicate mean values. Two-tailed Mann-Whitney test, ns = not significant, *P < 0.05, and **P < 0.01.


Figure 2 ( B ) Maximum Ca2 + flux ( peak height ) at each anti - IgM concentration for FOB and MZB cells . Paired t test , ns = not significant , * P < 0 . 05 , and * * * P < 0 . 001 .

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Figure 2(B) Maximum Ca2+ flux (peak height) at each anti-IgM concentration for FOB and MZB cells. Paired t test, ns = not significant, *P < 0.05, and ***P < 0.001.


Figure 3 ( B ) Co - immunoprecipitation of HA - tagged Pacs1 by FLAG - Wdr37 and HA - Wdr37 by FLAG - Pacs1 in co - transfected 293T cells . ( E ) Wdr37 + / + and Wdr37 − / − splenocytes were labeled with Indo - 1 , stained for cell surface markers to identify FOB cells , and stimulated with the indicated amounts of anti - IgM ( black arrowhead ) . Normalized traces from three ( 2 . 5 mcg / ml anti - IgM ) or four independent experiments ( 10 mcg / ml and 5 mcg / ml anti - IgM ) are shown ( Wdr37 + / + gray , Wdr37 − / − pink ) . Mean Ca2 + flux for each genotype is overlaid in bold ( Wdr37 + / + black , Wdr37 − / − red ) .

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Figure 3(B) Co-immunoprecipitation of HA-tagged Pacs1 by FLAG-Wdr37 and HA-Wdr37 by FLAG-Pacs1 in co-transfected 293T cells.(E) Wdr37+/+ and Wdr37−/− splenocytes were labeled with Indo-1, stained for cell surface markers to identify FOB cells, and stimulated with the indicated amounts of anti-IgM (black arrowhead). Normalized traces from three (2.5 mcg/ml anti-IgM) or four independent experiments (10 mcg/ml and 5 mcg/ml anti-IgM) are shown (Wdr37+/+ gray, Wdr37−/− pink). Mean Ca2+ flux for each genotype is overlaid in bold (Wdr37+/+ black, Wdr37−/− red).


Figure 4 ( A ) Immunoblot of ER mass , ER stress , and autophagy markers in Pacs1 + / + and Pacs1 - / - splenic B cells that were left unstimulated or stimulated overnight with 5 mcg / ml IgM .

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Figure 4(A) Immunoblot of ER mass, ER stress, and autophagy markers in Pacs1+/+ and Pacs1-/- splenic B cells that were left unstimulated or stimulated overnight with 5 mcg/ml IgM.


Figure 5 ( B ) Real - time quantitative PCR of IP3R and SERCA2 transcripts in B cells . Data are presented as mean ± SD from three independent Pacs1 + / + and Pacs1 - / - pairs of mice .

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Figure 5(B) Real-time quantitative PCR of IP3R and SERCA2 transcripts in B cells. Data are presented as mean ± SD from three independent Pacs1+/+ and Pacs1-/- pairs of mice.


Figure 6 ( D ) Peak cytosolic Ca2 + concentration based on aequorin measurements as performed in ( C ) . Symbols represent individual wells containing ~ 5 x 105 transfected 3T3 or Pacs1 - / - cells from one experiment . Horizontal bars indicate mean . Two - tailed unpaired t test , * * P < 0 . 01 . ( E ) Pacs1 + / + and Pacs1 - / - NIH - 3T3 cells ( C1 and C2 from ( A ) ) were transfected with ER - GCamP6 - 210 . ER Ca2 + was measured before and after treatment with 10 µM ATP . Kinetic traces show the mean 488 / 405 nm excitation ratio of each cell line with error bars indicating SEM . N = 30 cells ( 3T3 ) , 16 cells ( C1 ) , 25 cells ( C2 ) . Data are representative of 2 independent experiments .

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Figure 6(D) Peak cytosolic Ca2+ concentration based on aequorin measurements as performed in (C). Symbols represent individual wells containing ~5 x 105 transfected 3T3 or Pacs1-/- cells from one experiment. Horizontal bars indicate mean. Two-tailed unpaired t test, **P < 0.01.(E) Pacs1+/+ and Pacs1-/- NIH-3T3 cells (C1 and C2 from (A)) were transfected with ER-GCamP6-210. ER Ca2+ was measured before and after treatment with 10 µM ATP. Kinetic traces show the mean 488/405 nm excitation ratio of each cell line with error bars indicating SEM. N=30 cells (3T3), 16 cells (C1), 25 cells (C2). Data are representative of 2 independent experiments.


Figure 7 ( B , C ) Pacs1 + / + and Pacs1 − / − mice were immunized with alum - ova and one week later with NP - Ficoll . Anti - ova IgG and anti - NP IgM titers were measured at 14 days and 7 days after immunization , respectively . Each symbol represents an individual mouse . ( H , I ) Pacs1 + / + and Pacs1 - / - mice were injected with EdU and the fraction of EdU + FOB and MZB cells were measured in the spleen at 1 , 4 , and 7 days post - injection . Data are from one experiment .

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Figure 7(B, C) Pacs1+/+ and Pacs1−/− mice were immunized with alum-ova and one week later with NP-Ficoll. Anti-ova IgG and anti-NP IgM titers were measured at 14 days and 7 days after immunization, respectively. Each symbol represents an individual mouse.(H, I) Pacs1+/+ and Pacs1-/- mice were injected with EdU and the fraction of EdU+ FOB and MZB cells were measured in the spleen at 1, 4, and 7 days post-injection. Data are from one experiment.


Figure 8 ( D , E ) The fraction of proliferating ( D ) and recovered ( E ) donor cells from the experiment in ( C ) . Symbols represent individual recipient mice and data are from two independent adoptive transfer experiments . Two - tailed unpaired t test , * * P < 0 . 01 , * * * P < 0 . 001 . ( I ) Enumeration of CD3 + B220 + cells in the peripheral blood and lymph nodes of Faslpr / lpr dependent on Pacs1 expression . Horizontal bars indicate median value . Two - tailed Mann - Whitney test , * * P < 0 . 01 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8(D, E) The fraction of proliferating (D) and recovered (E) donor cells from the experiment in (C). Symbols represent individual recipient mice and data are from two independent adoptive transfer experiments. Two-tailed unpaired t test, **P < 0.01, ***P < 0.001.(I) Enumeration of CD3+B220+ cells in the peripheral blood and lymph nodes of Faslpr/lpr dependent on Pacs1 expression. Horizontal bars indicate median value. Two-tailed Mann-Whitney test, **P < 0.01.