Journal: EMBO Reports

Article Title: Inhibition of IRGM establishes a robust antiviral immune state to restrict pathogenic viruses

doi: 10.15252/embr.202152948

Figure Lengend Snippet: RNA isolated from control and IRGM knockdown HT‐29 cells and subjected to qRT–PCR with indicated viral restriction factor genes ( n = 3, mean ± SD, ** P < 0.005, *** P < 0.0005, Student’s unpaired t ‐test). The graph depicts the knockdown efficiency of control and IRGM shRNA stable HT‐29 cells ( n = 3, mean ± SD, **** P < 0.00005, Student’s unpaired t ‐test). The graph depicts the IFN‐β levels in serum of Irgm1 wild‐type and KO mice ( n = 3 mice each group, mean ± SD, **** P < 0.00005, Student’s unpaired t ‐test). The qRT–PCR analysis of APOBEC3G, OAS1, ISG15, MX1 and IFITM3 with RNA isolated from control or Flag IRGM overexpressing HT‐29 cells ( n = 3, Mean ± SD, * P < 0.05, ** P < 0.005, *** P < 0.0005, Student's unpaired t ‐test). The qRT–PCR analysis of OAS1, ISG15 and MX1 with RNA isolated from control or IRGM shRNA or Flag IRGM complemented IRGM shRNA HT‐29 cells. n = 3, Mean ± SD, * P < 0.05, ** P < 0.005, *** P < 0.0005, Student's unpaired t ‐test. Pictorial representation of sequential events of antigen uptake, processing, and presentation via Class I and Class II MHC Pathways. Created using Biorender.com. The graph depicts the knockdown efficiency upon transfection of control and si‐IRGM in THP‐1 cells ( n = 3, mean ± SD, *** P < 0.0005, Student’s unpaired t ‐test). Transferrin uptake assay shown by representative confocal images and flow cytometry analysis of control and si‐IRGM transfected THP‐1 cells treated with AF488 Transferrin (green) (10 μg/ml, 30 min). Graph depicts the mean fluorescence intensity of transferrin uptake in control and si‐IRGM transfected THP‐1 cells treated with AF488 transferrin. Scale, 5 μm (upper panel); Scale, 3 μm, (lower panel). Representative confocal images of H‐2Kb‐SIINFEKL (red) in Irgm1 +/+ and Irgm1 −/− BMDMs treated with OVA (2 mg/ml, 3 h). Scale, 25 μm.

Article Snippet: Primary antibodies used in Western blotting with dilutions: Actin (Abcam #ab6276; 1:5,000), IRGM antibody rodent specific (CST #14979; 1:1,000), IRGM (Abcam #ab69494; 1:500), MX1 (CST #37849; 1:1,000), OAS1 (CST #14498; 1:1,000), ISG15 (CST #2743; Santacruz sc‐166755; 1:1,000; 1:1,000), SAMHD1 (CST #12361; 1:1,000), BST2 (CST #19277; 1:1,000), viperin (CST #13996; 1:1,000), APOBEC3G (CST #43584; 1:1,000), IFITM3 (CST #59212; 1:1,000), PKR B10 (sc‐6282; 1:1,000), p‐PKR (#ab32036; 1:1,000), EIF2 Alpha (CST #9722; 1:1,000), p‐EIF2 Alpha (CST #3398S; 1:1,000), G3BP (BD Bioscience #611126; 1:1,000), GAPDH (CST #2118; 1:1,000), ACE2 (R&D systems #AF933; 1:1,000), HRP‐conjugated secondary antibodies were purchased from Santa Cruz (1:2,000) or Promega (1:5,000) or Abcam (1:10,000) or Novus (1:5,000).

Techniques: Isolation, Quantitative RT-PCR, shRNA, Transfection, Flow Cytometry, Fluorescence

Journal: EMBO Reports

Article Title: Inhibition of IRGM establishes a robust antiviral immune state to restrict pathogenic viruses

doi: 10.15252/embr.202152948

Figure Lengend Snippet: A–D Control and si‐IRGM transfected THP‐1 IFN reporter cells were kept uninfected (Mock) or infected with (A) JEV (MOI 5) or (B) CHIKV (MOI 5) or (C) HSV‐1 (MOI 2.5) or (D) VSV‐eGFP (MOI 1) and the supernatant collected 8 hpi were subjected to luciferase assay. The graphs depict fold change in interferon response. ( n = 3, mean ± SD, * P < 0.05, ** P < 0.005, *** P < 0.0005, Student’s unpaired t ‐test). E–J Control and si‐IRGM transfected HT‐29 cells uninfected or infected with CHIKV and qRT–PCR analysis were performed with several ISG’s (E) SAMHD1 (F) HERC5 (G) ISG15 (H) viperin/RSAD2 (I) MX1 (J) APOBEC3G. ( n = 3, mean ± SE, ** P < 0.005, *** P < 0.0005, **** P < 0.00005, Student’s unpaired t ‐test). K Western blot analysis with cell lysates of mock and CHIKV (MOI 5, 24 h) infected control and si‐IRGM transfected THP‐1 cells and probed with the indicated antibodies. S.E, short exposure; L.E, long exposure. L Western blot analysis with cell lysates of mock and CHIKV (MOI 5, 24 h) infected control and IRGM knockdown HT‐29 cells and probed with the indicated antibodies. M Western blot analysis with cell lysates of mock and JEV (MOI 5, 24 h) infected control and si‐IRGM transfected HT‐29 cells and probed with the indicated antibodies. N Western blot analysis with cell lysates of THP‐1 control or IRGM knockdown cells, untransfected or transfected with, heat‐killed whole CHIKV or CHIKV viral RNA and probed with the indicated antibodies. O qRT–PCR analysis to determine the knockdown efficiencies of PRR’s and adaptor proteins as indicated ( n = 3, mean ± SE, *** P < 0.0005, Student’s unpaired t ‐test). P qRT–PCR analysis with total RNA isolated from control and IRGM knockdown HT‐29 cells transfected with siRNA combinations as indicated that were infected with CHIKV (MOI 5, 24 h). ( n = 3, mean ± SE, *** P < 0.0005, **** P < 0.00005, Student’s unpaired t ‐test). Q Western blot analysis with cell lysates of mock and CHIKV (MOI 5, 24 h) infected control and IRGM knockdown HT‐29 cells transfected with indicated siRNA and probed with the indicated antibodies. Source data are available online for this figure.

Article Snippet: Primary antibodies used in Western blotting with dilutions: Actin (Abcam #ab6276; 1:5,000), IRGM antibody rodent specific (CST #14979; 1:1,000), IRGM (Abcam #ab69494; 1:500), MX1 (CST #37849; 1:1,000), OAS1 (CST #14498; 1:1,000), ISG15 (CST #2743; Santacruz sc‐166755; 1:1,000; 1:1,000), SAMHD1 (CST #12361; 1:1,000), BST2 (CST #19277; 1:1,000), viperin (CST #13996; 1:1,000), APOBEC3G (CST #43584; 1:1,000), IFITM3 (CST #59212; 1:1,000), PKR B10 (sc‐6282; 1:1,000), p‐PKR (#ab32036; 1:1,000), EIF2 Alpha (CST #9722; 1:1,000), p‐EIF2 Alpha (CST #3398S; 1:1,000), G3BP (BD Bioscience #611126; 1:1,000), GAPDH (CST #2118; 1:1,000), ACE2 (R&D systems #AF933; 1:1,000), HRP‐conjugated secondary antibodies were purchased from Santa Cruz (1:2,000) or Promega (1:5,000) or Abcam (1:10,000) or Novus (1:5,000).

Techniques: Transfection, Infection, Luciferase, Quantitative RT-PCR, Western Blot, Isolation

Journal: Oncology Letters

Article Title: Correlation of APOBEC3G expression with liver function indexes of patients with chronic hepatitis B and comparison in chronic hepatitis B, liver cirrhosis and liver cancer

doi: 10.3892/ol.2020.11257

Figure Lengend Snippet: Comparison of APOBEC3G expression in liver tissues in patients with chronic hepatitis B, liver cirrhosis or liver cancer. (A) Detection of APOBEC3G expression in liver tissues via western blotting. (B) APOBEC3G protein expression in patients with chronic hepatitis B, liver cirrhosis and liver cancer. (C) APOBEC3G mRNA expression in patients with chronic hepatitis B, liver cirrhosis and liver cancer (*P<0.05, &P<0.01). mRNA, messenger ribonucleic acid.

Article Snippet: Anti-APOBEC3G monoclonal primary antibody (Cell Signaling Technology) was used in this study, and β-actin was the internal control.

Techniques: Expressing, Western Blot

Journal: Oncology Letters

Article Title: Correlation of APOBEC3G expression with liver function indexes of patients with chronic hepatitis B and comparison in chronic hepatitis B, liver cirrhosis and liver cancer

doi: 10.3892/ol.2020.11257

Figure Lengend Snippet: Immunohistochemical detection of APOBEC3G expression in liver tissues.

Article Snippet: Anti-APOBEC3G monoclonal primary antibody (Cell Signaling Technology) was used in this study, and β-actin was the internal control.

Techniques: Immunohistochemical staining, Expressing

Journal: Oncology Letters

Article Title: Correlation of APOBEC3G expression with liver function indexes of patients with chronic hepatitis B and comparison in chronic hepatitis B, liver cirrhosis and liver cancer

doi: 10.3892/ol.2020.11257

Figure Lengend Snippet: Immunohistochemical detection of APOBEC3G expression in liver tissues. (A) hepatitis, (B) liver cirrhosis, (C) liver cancer.

Article Snippet: Anti-APOBEC3G monoclonal primary antibody (Cell Signaling Technology) was used in this study, and β-actin was the internal control.

Techniques: Immunohistochemical staining, Expressing

Journal: Oncology Letters

Article Title: Correlation of APOBEC3G expression with liver function indexes of patients with chronic hepatitis B and comparison in chronic hepatitis B, liver cirrhosis and liver cancer

doi: 10.3892/ol.2020.11257

Figure Lengend Snippet: Correlation between APOBEC3G and ALT in patients with chronic hepatitis B. (A) Correlation between APOBEC3G mRNA and ALT in liver tissues. (B) Correlation between APOBEC3G protein and ALT in liver tissues. ALT, alanine aminotransferase; mRNA, messenger ribonucleic acid.

Article Snippet: Anti-APOBEC3G monoclonal primary antibody (Cell Signaling Technology) was used in this study, and β-actin was the internal control.

Techniques:

Journal: Retrovirology

Article Title: Natural APOBEC3C variants can elicit differential HIV-1 restriction activity

doi: 10.1186/s12977-018-0459-5

Figure Lengend Snippet: A3C-Ile188 exhibits enhanced HIV-1 restriction activity in 293T cells. a Single cycle infectivity data for Vif-deficient HIV-1 viruses produced in the presence of untagged A3C-S188, A3C-I188, or catalytic mutant derivatives (E68Q). Immunoblots are shown below for viral particles (anti-A3G, anti-A3C, and anti-p24) and producer cells (anti-A3G, anti-A3C, and anti-tubulin). b Single cycle infectivity data for Vif-deficient HIV-1 viruses produced in the presence of N-terminally HA-tagged A3C-S188 or A3C-I188. Immunoblots are shown below for viral particles (anti-HA and anti-p24) and producer cells (anti-HA and anti-tubulin). All single cycle experiments were repeated at least 3 times, with representative infectivity data (mean ± SD) and immunoblots shown for one experiment

Article Snippet: Primary antibodies used in these studies were rabbit anti-A3C (Proteintech 10591-1-AP), rabbit anti-A3G (NIH AIDS Reagent Program 10201 courtesy of J. Lingappa), rabbit anti-HA (Cell signaling C29F4), mouse anti-Tubulin (Sigma T5168), mouse anti-HIV-1 p24/CA (NIH AIDS Reagent Program 3537 courtesy of B. Chesebro and K. Wehrly), and mouse anti-HIV-1 Vif (NIH AIDS Reagent Program 6459 courtesy of M. Malim).

Techniques: Activity Assay, Infection, Produced, Mutagenesis, Western Blot

Journal: Nucleic Acids Research

Article Title: Enterovirus 71 antagonizes the inhibition of the host intrinsic antiviral factor A3G

doi: 10.1093/nar/gky840

Figure Lengend Snippet: A3G and the A3G C291S mutant both inhibit EV71 replication in HEK293T cells infected with EV71 virus. HEK293T cells were transfected with pcDNA3.1, A3G-myc or A3G C291S-myc and then infected with EV71 virus at an MOI of 1.0 at 24 h post-transfection. The cells and supernatants were harvested at 24 h, 48 h and 72 h post-infection. ( A ) A3G and viral VP1 levels in the cells and supernatants were detected by immunoblotting analyses using anti-VP1, anti-myc and anti-tubulin antibodies. The supernatants from transfected HEK293T cells were concentrated using 25% sucrose prior to immunoblotting analysis. ( B ) EV71 RNA levels in cells were detected by RT-qPCR. GAPDH was used as a control. EV71 RNA levels of cells transfected with pcDNA3.1 for 24 h were set as 100%. ( C ) Viral titres in the supernatants were determined by the cytopathic effect method. The results are the means with SD from three independent experiments. The asterisks indicate statistically significant differences between groups as assessed by Student's t -test (** P < 0.01). The endogenous expression levels of PPIA or ElonginC in HEK293T cells ( D ) and A3C in Jurkat cells ( E ) were detected by immunoblotting analyses at 72 h post-infection.

Article Snippet: The following antibodies were used in this study: polyclonal antibody (pAb) against EV71 and CA16 was obtained from rabbits immunized with EV71 and CA16 whole viruses in our laboratory respectively, anti-hemagglutinin (anti-HA) monoclonal antibody (mAb, Covance, Princeton, NJ, USA, MMS-101R-10000), anti-tubulin mAb (Abcam, Cambridge, MA, USA, ab11323,), anti-V5 mAb (Invitrogen, R960-25), anti-myc mAb (Millipore, Billerica, MA, USA), anti-GFP pAb (Invitrogen, A-21311), anti-A3G (Cell signaling technology, 43584), anti-flag mAb (Sigma, F1804), anti-APOBEC3C rabbit pAb (Proteintech, 10591-1-AP), anti-PPIA rabbit pAb (Sangon Biotec, Shanghai, CHN, D122908), anti-ElonginC rabbit pAb (Sangon Biotec, D123299), goat anti-Mouse IgG (H+L) Highly Cross Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Invitrogen, A32723).

Techniques: Mutagenesis, Infection, Transfection, Western Blot, Quantitative RT-PCR, Expressing

Journal: Nucleic Acids Research

Article Title: Enterovirus 71 antagonizes the inhibition of the host intrinsic antiviral factor A3G

doi: 10.1093/nar/gky840

Figure Lengend Snippet: H9 cells expressing A3G have lower EV71 replication capacity than Jurkat cells without A3G expression. ( A and B ) EV71 replication was lower in H9 cells than in Jurkat cells. H9 and Jurkat cells were infected with DMEM or EV71 virus at an MOI of 1.0. The infected cells were harvested at the indicated time points post-infection. (A) A3G and viral VP1 levels in cells were detected by immunoblotting analyses using anti-VP1, anti-A3G and anti-tubulin antibodies. (B) EV71 RNA levels were lower in H9 cells than in Jurkat cells according to RT-qPCR detection. EV71 RNA levels of Jurkat cells infected with EV71 for 24 h were set as 100%. (C–F) Silencing A3G in H9 cells enhanced EV7 replication. H9 cells stably expressing A3G shRNA were established. A3G protein ( C ) and mRNA ( D ) levels at different time points are shown. ( E ) EV71 RNA levels were higher in A3G knockdown H9 cells than in negative control pLKO.1 cells at all time points. EV71 RNA levels of negative control pLKO.1 cells infected with EV71 for 48 h were set as 100%. ( F ) Cytotoxicity induced by EV71 in A3G knockdown H9 cells and control pLKO.1 cells was detected by CCK8 assays. Uninfected H9 cells at 0 h were set as 100%. (B, D–F) The results are the means with SD from three independent experiments. The asterisks indicate statistically significant differences between groups as assessed by Student's t -test (* P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: The following antibodies were used in this study: polyclonal antibody (pAb) against EV71 and CA16 was obtained from rabbits immunized with EV71 and CA16 whole viruses in our laboratory respectively, anti-hemagglutinin (anti-HA) monoclonal antibody (mAb, Covance, Princeton, NJ, USA, MMS-101R-10000), anti-tubulin mAb (Abcam, Cambridge, MA, USA, ab11323,), anti-V5 mAb (Invitrogen, R960-25), anti-myc mAb (Millipore, Billerica, MA, USA), anti-GFP pAb (Invitrogen, A-21311), anti-A3G (Cell signaling technology, 43584), anti-flag mAb (Sigma, F1804), anti-APOBEC3C rabbit pAb (Proteintech, 10591-1-AP), anti-PPIA rabbit pAb (Sangon Biotec, Shanghai, CHN, D122908), anti-ElonginC rabbit pAb (Sangon Biotec, D123299), goat anti-Mouse IgG (H+L) Highly Cross Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Invitrogen, A32723).

Techniques: Expressing, Infection, Western Blot, Quantitative RT-PCR, Stable Transfection, shRNA, Negative Control

Journal: Nucleic Acids Research

Article Title: Enterovirus 71 antagonizes the inhibition of the host intrinsic antiviral factor A3G

doi: 10.1093/nar/gky840

Figure Lengend Snippet: A3G inhibits EV71 5′UTR activity. ( A ) Bicistronic plasmid construction. (B–D) pcDNA3.1, A3G or A3G C291S plus the pIRIGF negative vector or bicistronic pIRIGF-5′UTR expression plasmid were co-transfected into HEK293T cells, which were harvested at 48 h post-transfection. ( B ) A3G and A3G C291S expression was detected by immunoblotting analysis. ( C ) Effects of A3G and A3G C291S on luciferase mRNA levels according to RT-qPCR analysis. GAPDH was used as a control. mRNA levels of luciferase downstream of CMV in the absence of A3G were set as 100%. ( D ) Effects of A3G and A3G C291S on luciferase activity. Luciferase activity downstream of CMV in the absence of A3G was set as 100%. (C and D) The results are the means with SD from at least three independent experiments. The asterisks indicate statistically significant differences between groups as assessed by Student's t -test (*** P < 0.001).

Article Snippet: The following antibodies were used in this study: polyclonal antibody (pAb) against EV71 and CA16 was obtained from rabbits immunized with EV71 and CA16 whole viruses in our laboratory respectively, anti-hemagglutinin (anti-HA) monoclonal antibody (mAb, Covance, Princeton, NJ, USA, MMS-101R-10000), anti-tubulin mAb (Abcam, Cambridge, MA, USA, ab11323,), anti-V5 mAb (Invitrogen, R960-25), anti-myc mAb (Millipore, Billerica, MA, USA), anti-GFP pAb (Invitrogen, A-21311), anti-A3G (Cell signaling technology, 43584), anti-flag mAb (Sigma, F1804), anti-APOBEC3C rabbit pAb (Proteintech, 10591-1-AP), anti-PPIA rabbit pAb (Sangon Biotec, Shanghai, CHN, D122908), anti-ElonginC rabbit pAb (Sangon Biotec, D123299), goat anti-Mouse IgG (H+L) Highly Cross Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Invitrogen, A32723).

Techniques: Activity Assay, Plasmid Preparation, Expressing, Transfection, Western Blot, Luciferase, Quantitative RT-PCR

Journal: Nucleic Acids Research

Article Title: Enterovirus 71 antagonizes the inhibition of the host intrinsic antiviral factor A3G

doi: 10.1093/nar/gky840

Figure Lengend Snippet: A3G competitively binds to the EV71 5′UTR with PCBP1. ( A and B ) A3G and PCBP1 expression was detected by immunoblotting analysis. pcDNA3.1, A3G-HA, A3G C291S-HA or PCBP1-HA was co-transfected with the 5′UTR expression vector into HEK293T cells. Cell lysates were prepared at 48 h post-transfection. Part of the cell lysates were immunoprecipitated with anti-HA agarose beads. (B) Binding capacity of A3G or PCBP1 to the EV71 5′UTR. The results are the means with SD from at least three independent experiments. ( C ) The interaction between A3G or PCBP1 with the 5′UTR of EV71 according to RNA pull-down assay. (D–F) Competitive binding assay using immunoprecipitation. HEK293T cells were transfected with increasing doses of A3G-myc and PCBP1-HA plus the 5′UTR. At 48 h post-transfection, half of the cells were harvested and immunoprecipitated with anti-HA agarose beads, and the other cells were precipitated with anti-myc agarose beads. The cell lysates and immunoprecipitated products were analysed by immunoblotting ( D ) and RT-qPCR analyses. ( E ) 5′UTR RNA input in cell lysates. GAPDH was used as a control. ( F ) Increasing amounts of A3G disrupted the interaction of PCBP1 with the EV71 5′UTR. The binding between the 5′UTR and PCBP1 in the absence of A3G was set as 100%. The results are the means with SD from at least three independent experiments. The asterisks indicate statistically significant differences between groups as assessed by Student's t -test (* P < 0.05, ** P < 0.01, *** P < 0.001). ( G ) Increasing amounts of A3G decreased the interaction of PCBP1 with the 5′UTR of EV71 according to RNA pull-down assay.

Article Snippet: The following antibodies were used in this study: polyclonal antibody (pAb) against EV71 and CA16 was obtained from rabbits immunized with EV71 and CA16 whole viruses in our laboratory respectively, anti-hemagglutinin (anti-HA) monoclonal antibody (mAb, Covance, Princeton, NJ, USA, MMS-101R-10000), anti-tubulin mAb (Abcam, Cambridge, MA, USA, ab11323,), anti-V5 mAb (Invitrogen, R960-25), anti-myc mAb (Millipore, Billerica, MA, USA), anti-GFP pAb (Invitrogen, A-21311), anti-A3G (Cell signaling technology, 43584), anti-flag mAb (Sigma, F1804), anti-APOBEC3C rabbit pAb (Proteintech, 10591-1-AP), anti-PPIA rabbit pAb (Sangon Biotec, Shanghai, CHN, D122908), anti-ElonginC rabbit pAb (Sangon Biotec, D123299), goat anti-Mouse IgG (H+L) Highly Cross Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Invitrogen, A32723).

Techniques: Expressing, Western Blot, Transfection, Plasmid Preparation, Immunoprecipitation, Binding Assay, Pull Down Assay, Competitive Binding Assay, Quantitative RT-PCR

Journal: Nucleic Acids Research

Article Title: Enterovirus 71 antagonizes the inhibition of the host intrinsic antiviral factor A3G

doi: 10.1093/nar/gky840

Figure Lengend Snippet: The RNA-binding property of A3G is required for its binding to the EV71 5′UTR and EV71 inhibition. ( A ) The amino-terminal domain of A3G alone could inhibit EV71 replication, but the carboxy-terminal could not. HEK293T cells were transfected with pcDNA3.1(–), A3G or the indicated mutants. At 24 h post-transfection, HEK293T cells were infected with EV71 at an MOI of 0.1. At 72 h post-infection, the cells were harvested and loaded for immunoblotting analyses using anti-VP1, anti-HA and anti-tubulin antibodies. The amino-terminal of A3G alone could inhibit EV71 replication. ( B ) Immunoprecipitation assay. HEK293T cells were transfected with A3G-HA or the indicated mutants plus the 5′UTR expression vector. At 48 h post-transfection, the cells were harvested and immunoprecipitated with anti-HA agarose beads. The cell lysates and immunoprecipitated products were analysed by immunoblotting. ( C ) The amino-terminal domain of A3G lost the ability to interact with the 5′UTR according to RT-qPCR analysis. GAPDH was used as a control. ( D ) EMSA of the EV71 5′UTR and A3G or its mutants. ( E ) A3G mutants L123A, Y124A and W127A could not inhibit EV71 replication. The assay was performed as described for panel A. ( F ) Immunoprecipitation assays were performed as indicated for panel B. ( G ) A3G mutants L123A, Y124A and W127A lost the ability to interact with the 5′UTR according to RT-qPCR analysis. GAPDH was used as a control. ( H ) EMSA of the EV71 5′UTR and A3G or its mutants. (C and G) The results are the means with SD from three independent experiments. The asterisks indicate statistically significant differences between groups as assessed by Student's t -test (*** P < 0.001).

Article Snippet: The following antibodies were used in this study: polyclonal antibody (pAb) against EV71 and CA16 was obtained from rabbits immunized with EV71 and CA16 whole viruses in our laboratory respectively, anti-hemagglutinin (anti-HA) monoclonal antibody (mAb, Covance, Princeton, NJ, USA, MMS-101R-10000), anti-tubulin mAb (Abcam, Cambridge, MA, USA, ab11323,), anti-V5 mAb (Invitrogen, R960-25), anti-myc mAb (Millipore, Billerica, MA, USA), anti-GFP pAb (Invitrogen, A-21311), anti-A3G (Cell signaling technology, 43584), anti-flag mAb (Sigma, F1804), anti-APOBEC3C rabbit pAb (Proteintech, 10591-1-AP), anti-PPIA rabbit pAb (Sangon Biotec, Shanghai, CHN, D122908), anti-ElonginC rabbit pAb (Sangon Biotec, D123299), goat anti-Mouse IgG (H+L) Highly Cross Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Invitrogen, A32723).

Techniques: RNA Binding Assay, Binding Assay, Inhibition, Transfection, Infection, Western Blot, Immunoprecipitation, Expressing, Plasmid Preparation, Quantitative RT-PCR

Journal: Nucleic Acids Research

Article Title: Enterovirus 71 antagonizes the inhibition of the host intrinsic antiviral factor A3G

doi: 10.1093/nar/gky840

Figure Lengend Snippet: ( A ) The secondary structure of the 5′UTR was predicted by MFold. (B–D) The interactions between A3G and the 5′UTR truncated mutants. A3G or VR1012 and the WT 5′UTR or indicated 5′UTR truncations were transfected into HEK293T cells; then, the cells were harvested for IP and RT-PCR analysis at 48 h post-transfection. ( B ) A3G protein levels in the cell lysates and co-IP elutes were confirmed by immunoblotting analysis. ( C ) 5′UTR RNA input in the cell lysates was detected by RT-qPCR. ( D ) Loop I and loop II in the EV71 5′UTR maintained binding ability with A3G according to RT-qPCR detection. The RNA level of the WT 5′UTR binding to A3G was set as 100%. The results are the means with SD from three independent experiments. The asterisks indicate statistically significant differences between groups as assessed by Student's t-test (* P < 0.05, ** P < 0.01, *** P < 0.001). ( E ) EMSA of the EV71 5′UTR or its truncated mutants with A3G.

Article Snippet: The following antibodies were used in this study: polyclonal antibody (pAb) against EV71 and CA16 was obtained from rabbits immunized with EV71 and CA16 whole viruses in our laboratory respectively, anti-hemagglutinin (anti-HA) monoclonal antibody (mAb, Covance, Princeton, NJ, USA, MMS-101R-10000), anti-tubulin mAb (Abcam, Cambridge, MA, USA, ab11323,), anti-V5 mAb (Invitrogen, R960-25), anti-myc mAb (Millipore, Billerica, MA, USA), anti-GFP pAb (Invitrogen, A-21311), anti-A3G (Cell signaling technology, 43584), anti-flag mAb (Sigma, F1804), anti-APOBEC3C rabbit pAb (Proteintech, 10591-1-AP), anti-PPIA rabbit pAb (Sangon Biotec, Shanghai, CHN, D122908), anti-ElonginC rabbit pAb (Sangon Biotec, D123299), goat anti-Mouse IgG (H+L) Highly Cross Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Invitrogen, A32723).

Techniques: Transfection, Reverse Transcription Polymerase Chain Reaction, Co-Immunoprecipitation Assay, Western Blot, Quantitative RT-PCR, Binding Assay

Journal: Nucleic Acids Research

Article Title: Enterovirus 71 antagonizes the inhibition of the host intrinsic antiviral factor A3G

doi: 10.1093/nar/gky840

Figure Lengend Snippet: EV71 2C antagonizes A3G via the autophagy–lysosome degradation pathway but not the proteasome pathway. ( A ) 2C reduced the expression of A3G. A3G-HA plus VR1012 (negative control) or the indicated EV71 non-structural proteins were co-transfected into H9 cells. The cells were harvested for immunoblotting analysis at 48 h post-transfection. ( B ) The MG132 inhibitor could not rescue EV71 2C-mediated A3G degradation. The cells were treated for another 12 h with 10 μM DMSO or MG132 prior to harvest for immunoblotting analysis at 36 h post-transfection. ( C ) An autophagy inhibitor could rescue EV71 2C-mediated A3G degradation. The cells were treated for another 12 h with 3 μM DMSO, 3 μM thapsigargin or 10 nM Baf-A1 prior to harvest for immunoblotting analysis at 36 h post-transfection. ( D ) EV71 2C interacted with A3G according to immunoprecipitation assay. HIV-1 Vif-HA was used as a positive control. The cells were treated for another 12 h with 10 μM DMSO, 10 μM MG132 or 10 nM Baf-A1 to avoid A3G degradation as indicated prior to harvest. ( E ) A3G was ubiquitinated by EV71 2C. ( F ) Co-localization of EV71 2C and A3G in HeLa cells. 2C formed autophagic puncta and co-localized with A3G. ( G ) Co-localization of A3G and p62 in the presence or absence of EV71 2C. (F and G) Images were taken under a Zeiss LZM710 confocal microscope.

Article Snippet: The following antibodies were used in this study: polyclonal antibody (pAb) against EV71 and CA16 was obtained from rabbits immunized with EV71 and CA16 whole viruses in our laboratory respectively, anti-hemagglutinin (anti-HA) monoclonal antibody (mAb, Covance, Princeton, NJ, USA, MMS-101R-10000), anti-tubulin mAb (Abcam, Cambridge, MA, USA, ab11323,), anti-V5 mAb (Invitrogen, R960-25), anti-myc mAb (Millipore, Billerica, MA, USA), anti-GFP pAb (Invitrogen, A-21311), anti-A3G (Cell signaling technology, 43584), anti-flag mAb (Sigma, F1804), anti-APOBEC3C rabbit pAb (Proteintech, 10591-1-AP), anti-PPIA rabbit pAb (Sangon Biotec, Shanghai, CHN, D122908), anti-ElonginC rabbit pAb (Sangon Biotec, D123299), goat anti-Mouse IgG (H+L) Highly Cross Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Invitrogen, A32723).

Techniques: Expressing, Negative Control, Transfection, Western Blot, Immunoprecipitation, Positive Control, Microscopy

Journal: Nucleic Acids Research

Article Title: Enterovirus 71 antagonizes the inhibition of the host intrinsic antiviral factor A3G

doi: 10.1093/nar/gky840

Figure Lengend Snippet: The functional domain of EV71 2C is required for A3G degradation. ( A ) EV71 2C mutant construction. ( B and C ) The amino-terminal domain but not the carboxy-terminal domain of EV71 2C is required for 2C-mediated degradation. HEK293T cells were co-transfected as indicated and then harvested for immunoblotting analysis at 48 h post-transfection. ( D ) Truncated mutants in the amino-terminal domain of EV712C were constructed. ( E ) Amino acids 26–40 were required for the 2C-mediated degradation of A3G.

Article Snippet: The following antibodies were used in this study: polyclonal antibody (pAb) against EV71 and CA16 was obtained from rabbits immunized with EV71 and CA16 whole viruses in our laboratory respectively, anti-hemagglutinin (anti-HA) monoclonal antibody (mAb, Covance, Princeton, NJ, USA, MMS-101R-10000), anti-tubulin mAb (Abcam, Cambridge, MA, USA, ab11323,), anti-V5 mAb (Invitrogen, R960-25), anti-myc mAb (Millipore, Billerica, MA, USA), anti-GFP pAb (Invitrogen, A-21311), anti-A3G (Cell signaling technology, 43584), anti-flag mAb (Sigma, F1804), anti-APOBEC3C rabbit pAb (Proteintech, 10591-1-AP), anti-PPIA rabbit pAb (Sangon Biotec, Shanghai, CHN, D122908), anti-ElonginC rabbit pAb (Sangon Biotec, D123299), goat anti-Mouse IgG (H+L) Highly Cross Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Invitrogen, A32723).

Techniques: Functional Assay, Mutagenesis, Transfection, Western Blot, Construct

Journal: Frontiers in Immunology

Article Title: APOBEC3G/3A Expression in Human Immunodeficiency Virus Type 1-Infected Individuals Following Initiation of Antiretroviral Therapy Containing Cenicriviroc or Efavirenz

doi: 10.3389/fimmu.2018.01839

Figure Lengend Snippet: A3G expression in cenicriviroc (CVC) and efavirenz (EFV) arms and correlation with surrogate markers of disease progression. (A) Expression of A3G in peripheral blood mononuclear cells of subjects treated with CVC (circles) or EFV (triangles). The dot plots show the ratios of A3G to actin OD determined by densitometry for each time point of the patients analyzed (CVC: n = 21 baseline, n = 19 week 4, n = 18 week 12, n = 20 week 24, n = 23 week 48; EFV: n = 9 baseline, n = 10 week 4, n = 9 week 12, n = 10 week 24, n = 9 week 48). Median values (50th percentiles) are shown by the horizontal bars. (B) Correlation between baseline A3G levels and CD4 + cell counts ( n = 30). (C) Correlation between baseline A3G levels and week 48 CD4 + /CD8 + cell ratio ( n = 29; CVC: n = 21; EFV: n = 8). Gray circles and black triangles, CVC- and EFV-treated subjects, respectively. Statistical analysis was done using the Wilcoxon signed-rank test (A) and the non-parametric one-tailed Spearman’s test to determine correlation coefficients (B,C) .

Article Snippet: Membranes were incubated with 4–5% fat-free milk dissolved in PBS-T (PBS 1×, 0.05%) to block non-specific binding and then probed with the following Abs: anti-human A3A (rabbit polyclonal D23, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-human A3G (rabbit polyclonal D9C6Z, Cell Signaling Technology, Beverly, MA, USA), and anti-actin (mouse monoclonal Abs-5, BD Biosciences, San Diego, CA, USA) as gel loading control.

Techniques: Expressing, One-tailed Test