Journal: Cell Death & Disease

Article Title: Covalent ISG15 conjugation to CHIP promotes its ubiquitin E3 ligase activity and inhibits lung cancer cell growth in response to type I interferon

doi: 10.1038/s41419-017-0138-9

Figure Lengend Snippet: a – e All samples were transfected with plasmids encoding UBE1L and Myc-tagged UbcH8. a HEK293 cells were transfected for 24 h with plasmid encoding Xpress-His-CHIP and/or FLAG-HERC5. Cell lysates were immunoprecipitated with anti-Xpress antibody, followed by immunoblotting with anti-Xpress or anti-HERC5 antibody. b Cells were transfected for 36 h with plasmid encoding Xpress-His-CHIP, FLAG-ISG15, or Myc-EFP, alone or in combination. Cell lysates were immunoprecipitated with anti-Xpress antibody, followed by western blotting with anti-Myc or anti-Xpress antibody. c HEK293 cells were transfected for 24 h with plasmid encoding Xpress-His-CHIP, FLAG-ISG15, FLAG-HERC5-WT, or its catalytically inactive mutant FLAG-HERC5-CA, alone or in combination. Cell lysates were subjected to NTA pull-down (PD: NTA) under denaturing conditions followed by western blotting with anti-FLAG or anti-Xpress antibody. d Cells were transfected for 24 h with plasmid encoding Xpress-His-CHIP, FLAG-ISG15, Myc-EFP-WT, or Myc-tagged catalytically inactive EFP mutant Myc-EFP-CS, alone or in combination. Cell lysates were subjected to NTA pull-down, as in c . e HEK293 cells were transfected for 48 h with nonspecific control scrambled siRNA (NC), HERC5 -siRNA (H5), or plasmid encoding Xpress-His-CHIP or FLAG-ISG15, alone or in combination. Cell lysates were subjected to NTA pull-down as in b . f Cells were transfected for 36 h with plasmid encoding Xpress-His-CHIP, FLAG-ISG15, or Flag-HHARI, alone or in combination. Cell lysates were immunoprecipitated with anti-Xpress antibody, followed by western blotting with anti-Flag or anti-Xpress antibody. Asterisk indicates IgG heavy chains, and closed arrowhead indicates predicted or right size band

Article Snippet: Polyclonal anti-HERC5 antibodies were purchased from Novus Biologicals (Littleton, CO, USA).

Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot, Mutagenesis, Control

Journal: Cell Death & Disease

Article Title: Covalent ISG15 conjugation to CHIP promotes its ubiquitin E3 ligase activity and inhibits lung cancer cell growth in response to type I interferon

doi: 10.1038/s41419-017-0138-9

Figure Lengend Snippet: a , b All samples were transfected with plasmids encoding UBE1L and Myc-UbcH8. a HEK293 cells were transfected for 24 h with plasmid encoding HA-CHIP, FLAG-ISG15-GG, or FLAG-ISG15-AA, alone or in combination. Cells were treated for an additional 6 h with 10 μM MG132. Cell lysates were immunoprecipitated with anti-HA antibody, followed by western blotting with anti-ubiquitin antibody. Tubulin served as a loading control. b HEK293 cells were transfected for 24 h with plasmid encoding Xpress-His-CHIP, FLAG-ISG15, FLAG-HERC5-WT, or FLAG-HERC5-CA, alone or in combination, and treated for an additional 6 h with 10 μM MG132. Cell lysates were immunoprecipitated with anti-Xpress antibody, followed by western blotting with anti-ubiquitin antibody. Actin served a as loading control. c A549 cells were treated for 48 h with vehicle (−) or IFN-α (1000 U/ml). Immunoprecipitation of cell lysates was performed with preimmune IgG or anti-CHIP antibody, followed by western blotting with anti-ubiquitin or anti-CHIP antibody. Tubulin served as a loading control

Article Snippet: Polyclonal anti-HERC5 antibodies were purchased from Novus Biologicals (Littleton, CO, USA).

Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot, Ubiquitin Proteomics, Control

Journal: bioRxiv

Article Title: The interferon-stimulated gene product HERC5 inhibits human LINE-1 retrotransposition with an ISGylation-independent mechanism

doi: 10.1101/2025.09.09.675047

Figure Lengend Snippet: ( A ) Schematic of HERC5 and its mutants used in this study. HERC5 contains an N-terminal RLD domain (pink) and a C-terminal HECT domain (light green). Numbers shown above each domain indicate amino acid (a.a.) positions. A C-terminal 3×MYC epitope tag was fused to HERC5 WT, ΔRLD, ΔHECT, and C994A expression vectors. ( B ) Schematic of the L1 retrotransposition assay. Left: the retrotransposition-competent L1.3-expressing vector (cepB-gfp-L1.3) contains an enhanced green fluorescent protein retrotransposition indicator cassette ( mEGFPI ) in the 3′ UTR of L1 in the opposite orientation relative to the sense strand of L1 transcription. The mEGFPI cassette is interrupted by an intron in the same orientation relative to L1 transcription. This ensures that EGFP is expressed only after successful retrotransposition. Right: HEK293T cells were transfected with the L1-expressing plasmid containing the mEGFPI indicator cassette. The EGFP-positive cells were quantified using flow cytometry. The figure was created with BioRender. Bottom: retrotransposition efficiency was calculated by normalizing the percentage of EGFP-positive cells obtained in transfection with L1-expressing plasmid (cepB-gfp-L1.3) to that obtained in transfection with an RT-deficient L1-expressing plasmid lacking the intron in mEGFPI (cepB-gfp-L1.3RT[-] intronless). ( C ) L1 retrotransposition assay in HEK293T cells using the pCMV-3Tag-9 vector system. Top: timeline of the assay. HEK293T cells were co-transfected with the L1-expressing vector (cepB-gfp-L1.3) and either pCMV-3Tag-9 (control), HERC5 WT, HERC5 mutants, or MOV10 (positive control). Cells independently co-transfected with cepB-gfp-L1.3RT(-) intronless served as transfection-normalization controls. The transfected cells were selected with blasticidin (10 µg/mL), and the percentage of EGFP-positive cells was determined by flow cytometry. Bottom: L1 retrotransposition assay with HERC5 overexpression using pCMV-3Tag-9 in HEK293T. MOV10 and RT-deficient L1 (cepB-gfp-L1.3RT[-]) served as controls. X-axis, name of the transfected constructs. Y-axis, relative L1 retrotransposition efficiency compared to the control (pCMV-3Tag-9, set to 1.0). The error bars represent the mean ± the standard error of the mean (SEM) of at least three independent biological replicates. Each dot represents an independent biological replicate. The p -values were calculated using a one-way ANOVA followed by Bonferroni-Holm post-hoc tests; * p < 0.05, ** p < 0.01, *** p < 0.001; n.s.: not significant. ( D ) Protein expression levels of HERC5 WT and mutants in HEK293T. Cells were transfected with HERC5 constructs (pCMV-3Tag-9 vector system). HERC5 and GAPDH proteins were detected by western blot using anti-MYC and anti-GAPDH antibodies, respectively. GAPDH served as a loading control. The predicted molecular weights of the proteins are indicated on the right of the blots. ΔRLD showed two bands, likely due to multiple N-terminal ATG start codons. Although the predicted molecular weight of ΔRLD is ∼80 kDa, it migrated below 75 kDa, probably because of its high glutamic acid and aspartic acid content, consistent with a previous observation . ( E ) L1 retrotransposition assay with IFN-α and HERC5 knockdown in HEK293T cells. Top: timeline of the assay. HEK293T cells were treated with IFN-α (100 U/mL) (day −2) and siRNA (day −1) and then transfected with the WT L1-expressing construct (cepB-gfp-L1.3) (day 0). The RT-deficient L1 (cepB-gfp-L1.3RT[-]) served as a negative control. After blasticidin selection (10 µg/mL), the percentage of EGFP-positive cells was measured. Bottom left: L1 retrotransposition assay with IFN-α and siRNA treatments in HEK293T cells. X-axis, siRNA and IFN-α treatments. Y-axis, relative L1 retrotransposition efficiency to the non-targeting siRNA control (siControl) in the absence of IFN-α (set to 1.0). The error bars and p -values were calculated as in (C). Each dot represents an independent biological replicate. Bottom right: the relative retrotransposition ratio calculated by normalizing values obtained with IFN-α to those without IFN-α in the left panel. The ratio of siControl is set to 1.0 to compare with that of siHERC5. The p -values were calculated using a two-tailed, unpaired Student’s t-test; ** p < 0.01. Each dot represents an independent biological replicate. ( F ) Endogenous HERC5 protein expression levels with IFN-α and siRNA treatments in HEK293T cells. HERC5 and GAPDH proteins were detected by western blot using anti-HERC5 and anti-GAPDH antibodies, respectively. GAPDH served as a loading control.

Article Snippet: Human ARRB2 cDNA was cloned into pCMV-3Tag-8 (Agilent Technologies), enabling its expression with three copies of a FLAG tag at the C-terminus driven by the CMV promoter. pKN002_hHERC5-3×FLAG : Human HERC5 cDNA was cloned into pCMV-3Tag-8, enabling its expression with three copies of a FLAG tag at the C-terminus driven by the CMV promoter. pKN003_hHERC5-3×FLAG_ΔRLD : This plasmid is similar to pKN002_hHERC5-3×FLAG, but lacks the RLD domain. pKN004_hHERC5-3×FLAG_ΔHECT : This plasmid is similar to pKN002_hHERC5-3×FLAG, but lacks the HECT domain. pKN005_hHERC5-3×FLAG_C994A : This plasmid is similar to pKN002_hHERC5-3×FLAG, but contains the C994A mutation, therefore expressing ISGylation-deficient HERC5. pKN024_pJM101ORFeus : This plasmid is derived from pJM101/L1.3, but the ORF1- and ORF2 -coding sequences were replaced with the codon-optimized human L1 ORFeus sequence from pDA093 , which was generously provided by Dr. Kathleen Burns (Addgene plasmid #131390). pKN025_hHERC5-3×MYC_pEBNA : Human HERC5 cDNA fused with three copies of a MYC tag sequence in frame to the 3′ end was cloned into pEBNA, enabling its expression driven by the EF1α promoter. pKN026_hHERC5-3×MYC_ΔRLD_pEBNA : This plasmid is similar to pKN025_hHERC5-3×MYC_pEBNA, but lacks the RLD domain. pKN027_hHERC5-3×MYC_ΔHECT_pEBNA : This plasmid is similar to pKN025_hHERC5-3×MYC_pEBNA, but lacks the HECT domain. pKN028_hHERC5-3×MYC_C994A_pEBNA : This plasmid is similar to pKN025_hHERC5-3×MYC_pEBNA, but contains the C994A mutation, therefore expressing ISGylation activity-deficient HERC5. pKN031_hHERC4-3×MYC : Human HERC4 cDNA was cloned into pCMV-3Tag-9, enabling its expression with three copies of a MYC tag at the C-terminus driven by the CMV promoter. pKN032_hHERC6-3×MYC : Human HERC6 cDNA was cloned into pCMV-3Tag-9, enabling its expression with three copies of a MYC tag at the C-terminus driven by the CMV promoter. pVan583 : was previously described in ( , ).

Techniques: Expressing, Plasmid Preparation, Transfection, Flow Cytometry, Control, Positive Control, Over Expression, Construct, Western Blot, Molecular Weight, Knockdown, Negative Control, Selection, Two Tailed Test

Journal: bioRxiv

Article Title: The interferon-stimulated gene product HERC5 inhibits human LINE-1 retrotransposition with an ISGylation-independent mechanism

doi: 10.1101/2025.09.09.675047

Figure Lengend Snippet: ( A – C ) Protein levels of modified L1 constructs with HERC5. HEK293T cells were co-transfected with modified L1 and HERC5-expressing vectors. The cells were harvested 3 days post-transfection, and protein levels were assessed by western blotting. Top: schematic of modified L1 vectors. Left: schematic of pKN035 (A), which expresses monocistronic ORF1p tagged with a T7 gene 10 epitope. Middle: schematic of pTMO2F3 (B), which expresses monocistronic ORF2p with a 3×FLAG epitope. Right: schematic of pTMF3 (C), which expresses ORF1p tagged with a T7 gene 10 epitope and ORF2p tagged with a 3×FLAG epitope at their carboxyl termini. Bottom: the protein expression levels were assessed by western blotting. HERC5 and MOV10 were detected by an anti-MYC antibody. ORF1p, ORF2p, and GAPDH were detected by anti-T7, anti-FLAG, and anti-GAPDH antibodies, respectively. GAPDH served as a loading control. ( D ) Alu retrotransposition assay with full-length L1 in HeLa-HA. Top: HeLa-HA cells were co-transfected with the Alu and full-length L1-expressing construct (pKN040) and either pCMV-3Tag-8-Barr (control), a HERC5-expressing construct, or a MOV10-expressing construct. Cells were selected with G418 (500 µg/mL), stained with crystal violet, and the resulting colonies were counted. The representative images of stained G418-resistant colonies are shown below each condition. The colony numbers of pKN040 were normalized to transfection efficiency and determined as retrotransposition efficiency. MOV10 and the RT mutant served as controls. X-axis, name of the transfected constructs. Y-axis, relative Alu retrotransposition efficiency compared to the control (pCMV-3Tag-8-Barr, set to 1.0). The error bars represent the mean ± the standard error of the mean (SEM) of four independent biological replicates. Each dot represents an independent biological replicate. The p -values were calculated using a one-way ANOVA followed by Bonferroni-Holm post-hoc tests; *** p < 0.001; n.s.: not significant. Bottom: schematic of pKN040. The Alu sequence contains the neomycin-resistant gene cassette in the opposite direction of Alu transcription, and the self-splicing intron was inserted into the neomycin-resistant gene in the same direction as the Alu sequence, together with the full-length L1 sequence. S.D.: splice donor site, S.A.: splice acceptor site. The plasmid figure was created with BioRender. ( E ) Alu retrotransposition assay with monocistronic ORF2 in HeLa-HA. Top: HeLa-HA cells were co-transfected with the Alu and monocistronic ORF2-expressing construct (pTM489) and either pCMV-3Tag-8-Barr, a HERC5-expressing construct, or a MOV10-expressing construct. The assay was conducted as noted in (D). X-axis, name of the transfected constructs. Y-axis, relative retrotransposition efficiency compared to the control (pCMV-3Tag-8-Barr, set to 1.0). MOV10 and the EN/RT mutant served as controls. The error bars and p -values were calculated as in (D). Bottom: schematic of pTM489, which is similar to pKN040, but does not contain the ORF1 sequence.

Article Snippet: Human ARRB2 cDNA was cloned into pCMV-3Tag-8 (Agilent Technologies), enabling its expression with three copies of a FLAG tag at the C-terminus driven by the CMV promoter. pKN002_hHERC5-3×FLAG : Human HERC5 cDNA was cloned into pCMV-3Tag-8, enabling its expression with three copies of a FLAG tag at the C-terminus driven by the CMV promoter. pKN003_hHERC5-3×FLAG_ΔRLD : This plasmid is similar to pKN002_hHERC5-3×FLAG, but lacks the RLD domain. pKN004_hHERC5-3×FLAG_ΔHECT : This plasmid is similar to pKN002_hHERC5-3×FLAG, but lacks the HECT domain. pKN005_hHERC5-3×FLAG_C994A : This plasmid is similar to pKN002_hHERC5-3×FLAG, but contains the C994A mutation, therefore expressing ISGylation-deficient HERC5. pKN024_pJM101ORFeus : This plasmid is derived from pJM101/L1.3, but the ORF1- and ORF2 -coding sequences were replaced with the codon-optimized human L1 ORFeus sequence from pDA093 , which was generously provided by Dr. Kathleen Burns (Addgene plasmid #131390). pKN025_hHERC5-3×MYC_pEBNA : Human HERC5 cDNA fused with three copies of a MYC tag sequence in frame to the 3′ end was cloned into pEBNA, enabling its expression driven by the EF1α promoter. pKN026_hHERC5-3×MYC_ΔRLD_pEBNA : This plasmid is similar to pKN025_hHERC5-3×MYC_pEBNA, but lacks the RLD domain. pKN027_hHERC5-3×MYC_ΔHECT_pEBNA : This plasmid is similar to pKN025_hHERC5-3×MYC_pEBNA, but lacks the HECT domain. pKN028_hHERC5-3×MYC_C994A_pEBNA : This plasmid is similar to pKN025_hHERC5-3×MYC_pEBNA, but contains the C994A mutation, therefore expressing ISGylation activity-deficient HERC5. pKN031_hHERC4-3×MYC : Human HERC4 cDNA was cloned into pCMV-3Tag-9, enabling its expression with three copies of a MYC tag at the C-terminus driven by the CMV promoter. pKN032_hHERC6-3×MYC : Human HERC6 cDNA was cloned into pCMV-3Tag-9, enabling its expression with three copies of a MYC tag at the C-terminus driven by the CMV promoter. pVan583 : was previously described in ( , ).

Techniques: Modification, Construct, Transfection, Expressing, Western Blot, Control, Staining, Mutagenesis, Sequencing, Plasmid Preparation

Journal: bioRxiv

Article Title: The interferon-stimulated gene product HERC5 inhibits human LINE-1 retrotransposition with an ISGylation-independent mechanism

doi: 10.1101/2025.09.09.675047

Figure Lengend Snippet: ( A–C ) Differential effects of HERC5 on non-human LINE retrotransposition. To measure retrotransposition efficiency, HEK293T cells were co-transfected with either mouse TG F 21 (A), mouse ORFeus-Mm (B), or zebrafish L2-2 (C) together with either the control vector, a HERC5-expressing vector, or a MOV10-expressing vector. Cells were selected with puromycin (1 µg/mL), and the percentage of EGFP-positive cells was determined by flow cytometry. Cells co-transfected with cep99-gfp-L1.3RT(-) intronless served as transfection-normalization controls. X-axis, name of the transfected constructs. Y-axis, relative retrotransposition efficiency compared to the control (pCMV-3Tag-8-Barr, set to 1.0). Each dot represents an independent biological replicate. The p -values were calculated using a one-way ANOVA followed by Bonferroni-Holm post-hoc tests; *** p < 0.001; n.s.: not significant. ( D ) Summary of HERC5 effects on retrotransposition. A schematic of retrotransposon structures is shown. Requirements for ORF1 and ORF2p, and whether HERC5 inhibits each element, are indicated for different retrotransposons (human L1.3, mouse L1, zebrafish L2-2, and human Alu ). ( E ) Schematic timeline of the estimated divergence times of small HERC paralogs. HERC4 is predicted to have emerged earliest (>595 million years ago, Mya), HERC3 to have arisen >∼476 Mya, HERC6 ∼430 Mya, and the HERC5 paralog ∼413 Mya. Divergence times are based on the phylogenetic analysis in . The silhouette images of organisms were downloaded from PHYLOPIC version 2.0 ( https://www.phylopic.org/ ). Mice lack HERC5 but have HERC6, which has the E3 ligase activity of ISGylation. Humans have both HERC5 and HERC6; only HERC5 has the activity. ( F ) L1 retrotransposition assay with the small HERC family. HEK293T cells were co-transfected with an L1-expressing construct (cepB-gfp-L1.3) and a member of the small HERC family (HERC4, HERC5, hHERC6, and mHERC6). Cells were selected with blasticidin (10 µg/mL), and the percentage of EGFP-positive cells was measured by flow cytometry. The RT mutant served as a control. X-axis, name of the transfected constructs. Y-axis, relative L1 retrotransposition efficiency compared to the control (pCMV-3Tag-9, set to 1.0). The error bars and p -values were calculated as noted in (A).

Article Snippet: Human ARRB2 cDNA was cloned into pCMV-3Tag-8 (Agilent Technologies), enabling its expression with three copies of a FLAG tag at the C-terminus driven by the CMV promoter. pKN002_hHERC5-3×FLAG : Human HERC5 cDNA was cloned into pCMV-3Tag-8, enabling its expression with three copies of a FLAG tag at the C-terminus driven by the CMV promoter. pKN003_hHERC5-3×FLAG_ΔRLD : This plasmid is similar to pKN002_hHERC5-3×FLAG, but lacks the RLD domain. pKN004_hHERC5-3×FLAG_ΔHECT : This plasmid is similar to pKN002_hHERC5-3×FLAG, but lacks the HECT domain. pKN005_hHERC5-3×FLAG_C994A : This plasmid is similar to pKN002_hHERC5-3×FLAG, but contains the C994A mutation, therefore expressing ISGylation-deficient HERC5. pKN024_pJM101ORFeus : This plasmid is derived from pJM101/L1.3, but the ORF1- and ORF2 -coding sequences were replaced with the codon-optimized human L1 ORFeus sequence from pDA093 , which was generously provided by Dr. Kathleen Burns (Addgene plasmid #131390). pKN025_hHERC5-3×MYC_pEBNA : Human HERC5 cDNA fused with three copies of a MYC tag sequence in frame to the 3′ end was cloned into pEBNA, enabling its expression driven by the EF1α promoter. pKN026_hHERC5-3×MYC_ΔRLD_pEBNA : This plasmid is similar to pKN025_hHERC5-3×MYC_pEBNA, but lacks the RLD domain. pKN027_hHERC5-3×MYC_ΔHECT_pEBNA : This plasmid is similar to pKN025_hHERC5-3×MYC_pEBNA, but lacks the HECT domain. pKN028_hHERC5-3×MYC_C994A_pEBNA : This plasmid is similar to pKN025_hHERC5-3×MYC_pEBNA, but contains the C994A mutation, therefore expressing ISGylation activity-deficient HERC5. pKN031_hHERC4-3×MYC : Human HERC4 cDNA was cloned into pCMV-3Tag-9, enabling its expression with three copies of a MYC tag at the C-terminus driven by the CMV promoter. pKN032_hHERC6-3×MYC : Human HERC6 cDNA was cloned into pCMV-3Tag-9, enabling its expression with three copies of a MYC tag at the C-terminus driven by the CMV promoter. pVan583 : was previously described in ( , ).

Techniques: Transfection, Control, Plasmid Preparation, Expressing, Flow Cytometry, Construct, Activity Assay, Mutagenesis

Journal: bioRxiv

Article Title: The interferon-stimulated gene product HERC5 inhibits human LINE-1 retrotransposition with an ISGylation-independent mechanism

doi: 10.1101/2025.09.09.675047

Figure Lengend Snippet: ( A ) Interaction of ORF1p with HERC5 WT and its mutants. Top left: timeline of the experiment. HEK293T cells were co-transfected with L1 and HERC5 expression vectors. Cells were harvested on 4 days post-transfection, and ORF1p-FLAG complexes were immunoprecipitated. Top right: schematic of L1-expressing plasmids. The pJM101/L1.3 plasmid contains the full-length L1.3, and pJM101/L1.3FLAG expresses ORF1p tagged with a FLAG epitope at the carboxyl terminus. Bottom: the input and anti-FLAG IP reactions were analyzed by western blotting. pJM101/L1.3 served as a negative control. HERC5 and its mutants expressed from pEBNA were detected by an anti-MYC antibody, and ORF1p was detected by an anti-FLAG antibody. ( B ) Co-immunoprecipitation of ORF1p and HERC5 with RNase treatment. HEK293T cells were co-transfected with L1 and HERC5 expression vectors. ORF1p-FLAG complexes were purified as in (A) and were treated in the presence or absence of RNase A. The rightmost lane shows the RNase A-treated ORF1p-FLAG complex. HERC5 and ORF1p were detected by anti-MYC and anti-FLAG antibodies, respectively. ( C ) Interaction of the ORF1p RNA-binding mutant (RBM) with HERC5. HEK293T cells were co-transfected with HERC5 and either L1 WT or RBM expression vectors. Top: schematic diagram of L1 RNP with ORF1p WT (yellow) and RBM (purple). Bottom: the input and anti-FLAG IP reactions were analyzed by western blotting. FLAG-tagged ORF1p WT and RBM were immunoprecipitated. HERC5 and ORF1p were detected by anti-MYC and anti-FLAG antibodies, respectively. ( D ) Retrotransposition assay of L1 ORFeus and L1.3. Top: schematic of the pKN039 plasmid, which expresses codon-optimized ORF1p and ORF2p. L1 ORFeus is driven by the CMV promoter and the L1.3 5′ UTR promoter and terminates with the SV40 poly(A) signal sequence. The mEGFPI retrotransposition indicator cassette was inserted into the L1 3′ UTR. Bottom: the RT-deficient L1 (cepB-gfp-L1.3RT[-]) served as a negative control. X-axis, name of the transfected constructs. Y-axis, relative retrotransposition efficiency compared to the control (pCMV-3Tag-9 was set to 1.0 for each L1 construct). The error bars represent the mean ± the standard error of the mean (SEM) of four independent biological replicates. Each dot represents an independent biological replicate. The p -values were calculated using a one-way ANOVA followed by Bonferroni-Holm post-hoc tests; *** p < 0.001; n.s.: not significant. ( E ) ORF1p levels from L1.3 and L1 ORFeus with HERC5. HEK293T cells were transfected with L1 ORFeus and either the empty vector (pCMV-3Tag-9) or a HERC5-expressing vector. HERC5, ORF1p, and GAPDH were detected by anti-MYC, anti-ORF1p, and anti-GAPDH antibodies, respectively. ORF1p and GAPDH band signal intensities were measured with Empiria Studio. ORF1p signal intensities were normalized to GAPDH intensities to calculate the ORF1p ratio. The signal intensity of L1.3 ORF1p in the control condition was set to 1.0. GAPDH served as a loading control.

Article Snippet: Human ARRB2 cDNA was cloned into pCMV-3Tag-8 (Agilent Technologies), enabling its expression with three copies of a FLAG tag at the C-terminus driven by the CMV promoter. pKN002_hHERC5-3×FLAG : Human HERC5 cDNA was cloned into pCMV-3Tag-8, enabling its expression with three copies of a FLAG tag at the C-terminus driven by the CMV promoter. pKN003_hHERC5-3×FLAG_ΔRLD : This plasmid is similar to pKN002_hHERC5-3×FLAG, but lacks the RLD domain. pKN004_hHERC5-3×FLAG_ΔHECT : This plasmid is similar to pKN002_hHERC5-3×FLAG, but lacks the HECT domain. pKN005_hHERC5-3×FLAG_C994A : This plasmid is similar to pKN002_hHERC5-3×FLAG, but contains the C994A mutation, therefore expressing ISGylation-deficient HERC5. pKN024_pJM101ORFeus : This plasmid is derived from pJM101/L1.3, but the ORF1- and ORF2 -coding sequences were replaced with the codon-optimized human L1 ORFeus sequence from pDA093 , which was generously provided by Dr. Kathleen Burns (Addgene plasmid #131390). pKN025_hHERC5-3×MYC_pEBNA : Human HERC5 cDNA fused with three copies of a MYC tag sequence in frame to the 3′ end was cloned into pEBNA, enabling its expression driven by the EF1α promoter. pKN026_hHERC5-3×MYC_ΔRLD_pEBNA : This plasmid is similar to pKN025_hHERC5-3×MYC_pEBNA, but lacks the RLD domain. pKN027_hHERC5-3×MYC_ΔHECT_pEBNA : This plasmid is similar to pKN025_hHERC5-3×MYC_pEBNA, but lacks the HECT domain. pKN028_hHERC5-3×MYC_C994A_pEBNA : This plasmid is similar to pKN025_hHERC5-3×MYC_pEBNA, but contains the C994A mutation, therefore expressing ISGylation activity-deficient HERC5. pKN031_hHERC4-3×MYC : Human HERC4 cDNA was cloned into pCMV-3Tag-9, enabling its expression with three copies of a MYC tag at the C-terminus driven by the CMV promoter. pKN032_hHERC6-3×MYC : Human HERC6 cDNA was cloned into pCMV-3Tag-9, enabling its expression with three copies of a MYC tag at the C-terminus driven by the CMV promoter. pVan583 : was previously described in ( , ).

Techniques: Transfection, Expressing, Immunoprecipitation, Plasmid Preparation, FLAG-tag, Western Blot, Negative Control, Purification, RNA Binding Assay, Mutagenesis, Sequencing, Construct, Control

Journal: bioRxiv

Article Title: The interferon-stimulated gene product HERC5 inhibits human LINE-1 retrotransposition with an ISGylation-independent mechanism

doi: 10.1101/2025.09.09.675047

Figure Lengend Snippet: ( A ) Experimental design for checking L1 RNA and ORF1p levels. Top: schematic of the full-length L1 construct (pTMF3), which expresses ORF1p tagged with a T7 gene 10 epitope and ORF2p tagged with a 3×FLAG epitope at their carboxyl termini. The red bar indicates the amplified region for the RT-qPCR primer pair used to measure L1 RNA levels (primer sequences are described in Materials and Methods). Bottom: timeline of the experiment. HEK293T cells were co-transfected with L1 expression constructs and either pCMV-3Tag-9 (control), HERC5, HELZ2 (positive control for RNA levels) or MOV10 (positive control for protein levels). The cells were harvested 3 days post-transfection. L1 RNA levels were measured by RT-qPCR, and ORF1p levels were measured by western blot or flow cytometry. ( B ) L1 RNA levels with HERC5. The T7 primer pair was used to quantify L1 RNA levels, which were normalized to GAPDH RNA levels. X-axis, name of the transfected constructs. Y-axis, relative L1 RNA level compared to the control (pCMV-3Tag-9, set to 1.0). The error bars represent the mean ± the standard error of the mean (SEM) of three independent biological replicates. Each dot represents an independent biological replicate. The p -values were calculated using a one-way ANOVA followed by Bonferroni-Holm post-hoc tests; * p < 0.05, ** p < 0.01, *** p < 0.001; n.s.: not significant. ( C ) ORF1p levels with HERC5. HERC5 and MOV10 were detected by an anti-MYC antibody. ORF1p and GAPDH were detected by anti-T7 and anti-GAPDH antibodies, respectively. GAPDH served as a loading control. ( D ) Flow cytometry analysis of ORF1p-EGFP expression. HEK293T cells were co-transfected with pVan583 and either pCMV-3Tag-9, HERC5, or MOV10 expression vector. Top: schematic of the pVan583 plasmid, which expresses EGFP-tagged ORF1p and mCherry-tagged ORF2p and contains the CMV promoter, the 5′ UTR promoter, and the SV40 poly(A) signal sequence for L1 expression. Bottom left and middle panels: relative percentages and median intensities (FITC-A) of ORF1p-EGFP-positive cells, respectively. X-axis, name of the transfected constructs. Y-axis, relative percentages or median intensities compared to the control (pCMV-3Tag-9, set to 1.0). The error bars and p -values were calculated as in (B). Bottom right: overlaid frequency distribution plot of FITC-A intensity of control (black), HERC5 (red), or MOV10 (green). X-axis, log-scaled fluorescence intensity. Y-axis, cell count. ( E ) Flow cytometry analysis measuring EGFP expression. HEK293T cells were co-transfected with pKN033 and either pCMV-3Tag-9, HERC5, or MOV10. Top: schematic of the pKN033 plasmid, which is derived from pVan583 and expresses EGFP alone. Bottom left and middle panels: relative percentages and median intensities of EGFP-positive cells, respectively. X-axis, name of the transfected constructs. Y-axis, relative percentages or median intensities compared to the control (pCMV-3Tag-9, set to 1.0). The error bars and p -values were calculated as in (B). Bottom right: overlaid frequency distribution plot of FITC-A intensity of control (black), HERC5 (red), or MOV10 (green). X-axis, log-scaled fluorescence intensity. Y-axis, cell count. ( F ) ORF1p-EGFP levels with HERC5 WT and its mutants. HEK293T cells were co-transfected with pVan583 and either pEBNA, HERC5 WT, or its mutants. The relative cell percentages and median intensities of ORF1p-EGFP-positive cells are shown in the left and middle panels, respectively. X-axis, name of the transfected constructs. Y-axis, relative percentages or median intensities compared to the control (pEBNA, set to 1.0). The error bars and p -values were calculated as in (B).

Article Snippet: Human ARRB2 cDNA was cloned into pCMV-3Tag-8 (Agilent Technologies), enabling its expression with three copies of a FLAG tag at the C-terminus driven by the CMV promoter. pKN002_hHERC5-3×FLAG : Human HERC5 cDNA was cloned into pCMV-3Tag-8, enabling its expression with three copies of a FLAG tag at the C-terminus driven by the CMV promoter. pKN003_hHERC5-3×FLAG_ΔRLD : This plasmid is similar to pKN002_hHERC5-3×FLAG, but lacks the RLD domain. pKN004_hHERC5-3×FLAG_ΔHECT : This plasmid is similar to pKN002_hHERC5-3×FLAG, but lacks the HECT domain. pKN005_hHERC5-3×FLAG_C994A : This plasmid is similar to pKN002_hHERC5-3×FLAG, but contains the C994A mutation, therefore expressing ISGylation-deficient HERC5. pKN024_pJM101ORFeus : This plasmid is derived from pJM101/L1.3, but the ORF1- and ORF2 -coding sequences were replaced with the codon-optimized human L1 ORFeus sequence from pDA093 , which was generously provided by Dr. Kathleen Burns (Addgene plasmid #131390). pKN025_hHERC5-3×MYC_pEBNA : Human HERC5 cDNA fused with three copies of a MYC tag sequence in frame to the 3′ end was cloned into pEBNA, enabling its expression driven by the EF1α promoter. pKN026_hHERC5-3×MYC_ΔRLD_pEBNA : This plasmid is similar to pKN025_hHERC5-3×MYC_pEBNA, but lacks the RLD domain. pKN027_hHERC5-3×MYC_ΔHECT_pEBNA : This plasmid is similar to pKN025_hHERC5-3×MYC_pEBNA, but lacks the HECT domain. pKN028_hHERC5-3×MYC_C994A_pEBNA : This plasmid is similar to pKN025_hHERC5-3×MYC_pEBNA, but contains the C994A mutation, therefore expressing ISGylation activity-deficient HERC5. pKN031_hHERC4-3×MYC : Human HERC4 cDNA was cloned into pCMV-3Tag-9, enabling its expression with three copies of a MYC tag at the C-terminus driven by the CMV promoter. pKN032_hHERC6-3×MYC : Human HERC6 cDNA was cloned into pCMV-3Tag-9, enabling its expression with three copies of a MYC tag at the C-terminus driven by the CMV promoter. pVan583 : was previously described in ( , ).

Techniques: Construct, Amplification, Quantitative RT-PCR, Transfection, Expressing, Control, Positive Control, Western Blot, Flow Cytometry, Plasmid Preparation, Sequencing, Fluorescence, Cell Counting, Derivative Assay

Journal: bioRxiv

Article Title: The interferon-stimulated gene product HERC5 inhibits human LINE-1 retrotransposition with an ISGylation-independent mechanism

doi: 10.1101/2025.09.09.675047

Figure Lengend Snippet: ( A ) Rationale for the cycloheximide (CHX) treatment experiment. CHX treatment stops de novo protein synthesis. Unstable proteins (colored magenta) are reduced by CHX treatment due to protein degradation, while stable proteins (colored green) are not. The figure was created with BioRender. ( B ) Protein levels following CHX treatment. HEK293T cells were co-transfected with pTMF3 and either pCMV-3Tag-9 or a HERC5-expressing vector and treated with 50 µg/mL CHX for the indicated times (0, 2, 4, and 8 h). HERC5, ORF1p, α-tubulin, and RNF138 were detected by anti-MYC, anti-T7, anti-α-tubulin, and anti-RNF138 antibodies, respectively. α-tubulin served as a stable protein control, whereas RNF138 served as an unstable protein control. N.T., non-transfected cells. ( C ) Quantification of the remaining ORF1p levels in the control (blue line) and HERC5 (red line) from (B). ORF1p and GAPDH band signal intensities were measured with Empiria Studio. The ORF1p intensities were normalized to GAPDH intensities, and the relative ORF1p levels were calculated. X-axis, CHX treatment time. Y-axis, the relative ORF1p level compared to the ORF1p level at 0 h (both control and HERC5 were set to 1.0, respectively). Each dot represents an independent biological replicate. The p -values were calculated using a one-way ANOVA followed by Bonferroni-Holm post-hoc tests; * p < 0.05, ** p < 0.01, *** p < 0.001; n.s.: not significant. ( D ) Polysome profiling analysis of control (blue line) and HERC5 (red line). HEK293T cells were co-transfected with L1 and either pCMV-3Tag-9 (control) or a HERC5 expression vector. Cell lysates were subjected to sucrose gradient centrifugation and separated into the indicated fractions. X-axis, fraction number. Y-axis, UV absorbance at 280 nm, showing the distribution of ribosomal subunits (40S and 60S), monosomes (80S), and polysomes. ( E ) RNA enrichment ratio in polysome fractions. L1 (normalized to GAPDH) and GAPDH RNA levels in each sucrose fraction were measured by RT-qPCR, and the ratio of RNA enrichment (HERC5/control) was calculated. The green and orange lines show L1 and GAPDH RNA ratios, respectively. X-axis, ribosome fraction. Y-axis, ratio of RNA enrichment (HERC5/control). The error bars and p -values were calculated as noted in (C). ( F ) RC-L1 RNP formation efficiency with HERC5. HEK293T cells were co-transfected with pTMF3 and either pCMV-3Tag-9, a HERC5-expressing vector, or a MOV10 expression vector. Cells were harvested on 4 days post-transfection, and ORF2p-3×FLAG complexes were immunoprecipitated. Top left: schematic of full-length L1 constructs (pTMF3 and pTMH3). pTMF3 expresses ORF1p tagged with a T7 gene 10 epitope and ORF2p tagged with a 3×FLAG epitope at their carboxyl termini. pTMH3 is similar to pTMF3 but expresses 3×HA epitope-tagged ORF2p and served as a negative control. Bottom left: the input and anti-FLAG IP reactions were analyzed by western blotting. ORF2p and ORF1p were detected by anti-FLAG and anti-T7 antibodies, respectively. HERC5 and MOV10 were detected by an anti-MYC antibody. Right: the input ORF1p/ORF2p ratios (top) and immunoprecipitated ORF1p/ORF2p ratios (bottom), which indicate RC-L1 RNP formation efficiencies. The protein band signal intensities were measured with Empiria Studio. Each ORF1p signal intensity was divided by the respective ORF2p signal intensity, and the resulting ORF1p/ORF2p ratio was calculated. X-axis, name of the transfected constructs. Y-axis, relative ORF1p/ORF2p ratio of input compared to the control (pCMV-3Tag-9, set to 1.0). The error bars and p -values were calculated as noted in (C).

Article Snippet: Human ARRB2 cDNA was cloned into pCMV-3Tag-8 (Agilent Technologies), enabling its expression with three copies of a FLAG tag at the C-terminus driven by the CMV promoter. pKN002_hHERC5-3×FLAG : Human HERC5 cDNA was cloned into pCMV-3Tag-8, enabling its expression with three copies of a FLAG tag at the C-terminus driven by the CMV promoter. pKN003_hHERC5-3×FLAG_ΔRLD : This plasmid is similar to pKN002_hHERC5-3×FLAG, but lacks the RLD domain. pKN004_hHERC5-3×FLAG_ΔHECT : This plasmid is similar to pKN002_hHERC5-3×FLAG, but lacks the HECT domain. pKN005_hHERC5-3×FLAG_C994A : This plasmid is similar to pKN002_hHERC5-3×FLAG, but contains the C994A mutation, therefore expressing ISGylation-deficient HERC5. pKN024_pJM101ORFeus : This plasmid is derived from pJM101/L1.3, but the ORF1- and ORF2 -coding sequences were replaced with the codon-optimized human L1 ORFeus sequence from pDA093 , which was generously provided by Dr. Kathleen Burns (Addgene plasmid #131390). pKN025_hHERC5-3×MYC_pEBNA : Human HERC5 cDNA fused with three copies of a MYC tag sequence in frame to the 3′ end was cloned into pEBNA, enabling its expression driven by the EF1α promoter. pKN026_hHERC5-3×MYC_ΔRLD_pEBNA : This plasmid is similar to pKN025_hHERC5-3×MYC_pEBNA, but lacks the RLD domain. pKN027_hHERC5-3×MYC_ΔHECT_pEBNA : This plasmid is similar to pKN025_hHERC5-3×MYC_pEBNA, but lacks the HECT domain. pKN028_hHERC5-3×MYC_C994A_pEBNA : This plasmid is similar to pKN025_hHERC5-3×MYC_pEBNA, but contains the C994A mutation, therefore expressing ISGylation activity-deficient HERC5. pKN031_hHERC4-3×MYC : Human HERC4 cDNA was cloned into pCMV-3Tag-9, enabling its expression with three copies of a MYC tag at the C-terminus driven by the CMV promoter. pKN032_hHERC6-3×MYC : Human HERC6 cDNA was cloned into pCMV-3Tag-9, enabling its expression with three copies of a MYC tag at the C-terminus driven by the CMV promoter. pVan583 : was previously described in ( , ).

Techniques: Transfection, Expressing, Plasmid Preparation, Control, Gradient Centrifugation, Quantitative RT-PCR, Immunoprecipitation, Construct, Negative Control, Western Blot

Journal: Journal of Virology

Article Title: ISGylation of Hepatitis C Virus NS5A Protein Promotes Viral RNA Replication via Recruitment of Cyclophilin A

doi: 10.1128/JVI.00532-20

Figure Lengend Snippet: HCV NS5A is a target substrate of ISGylation. (A to D) Expression vector encoding HA-NS5A (1b, Con1) was coexpressed with FLAG-ISG15 together with E1 (UBE1L), E2 (UbcH8), and E3 ligase (HERC5) in 293T cells, followed by immunoprecipitation with anti-FLAG mouse MAb or anti-HA rabbit PAb and detection with anti-HA rabbit PAb (A), anti-FLAG mouse MAb (B), and specific antibodies against NS5A (C) and ISG15 (D), respectively. (E) Schematic diagrams of ISG15 and ISG15 mutant ISG15-AA. ISG15 was mutated by replacing the amino acid glycine (G) with alanine (A) within the C-terminal LRLRGG motif sequence. UBL1, ubiquitin-like domain 1; LRLRGG, Leu-Arg-Leu-Arg-Gly-Gly. (F) Expression vector encoding HA-NS5A was coexpressed with FLAG-ISG15 or FLAG-ISG15-AA together with E1, E2, and E3 ligase in 293T cells, followed by immunoprecipitation with anti-HA rabbit PAb and detection with anti-ISG15 mouse MAb or anti-HA rabbit PAb. Input samples (Lysate) were detected by anti-HA rabbit PAb, anti-FLAG mouse MAb, anti-NS5A mouse MAb, or anti-ISG15 mouse MAb. The asterisks indicate the various ISG15-conjugated NS5A proteins. IP, immunoprecipitation; IB, immunoblotting; HC, immunoglobulin heavy chain.

Article Snippet: The rabbit polyclonal antibodies (PAbs) used in this study were anti-HA PAb (H-6908; Sigma-Aldrich) and anti-Herc5 PAb (BML-PW0920; Enzo Life Sciences, New York, NY).

Techniques: Expressing, Plasmid Preparation, Immunoprecipitation, Mutagenesis, Sequencing, Western Blot

Journal: Journal of Virology

Article Title: ISGylation of Hepatitis C Virus NS5A Protein Promotes Viral RNA Replication via Recruitment of Cyclophilin A

doi: 10.1128/JVI.00532-20

Figure Lengend Snippet: Identification of ISGylation sites on NS5A protein. (A) Schematic diagram of HCV NS5A (1b, Con1). NS5A possesses 14 Lys residues within the coding sequence. (B, D) Expression vector encoding HA-NS5A or HA-NS5A mutants in which all Lys residues except one (K44, K68, K166, K215, and K308) are mutated to Ala and HA-NS5A mutants containing a point mutation of Lys to Arg at corresponding Lys residues (K44R, K68R, K166R, K215R, and K308R) was coexpressed with FLAG-ISG15 together with E1 (UBE1L), E2 (UbcH8), and E3 ligase (HERC5) in 293T cells, followed by immunoprecipitation with anti-FLAG mouse MAb and detection with anti-HA rabbit PAb. Null indicates a mutant with mutation of all of the Lys residues on NS5A. (C) Expression vector encoding either HA-NS5A (WT) or HA-NS5A mutants (K44, K68, K166, K215 and K308) was coexpressed with FLAG-ISG15 or FLAG-ISG15-AA together with E1, E2, and E3 ligase (HERC5) in 293T cells, followed by immunoprecipitation with anti-HA rabbit PAb and detection with anti-FLAG mouse MAb. (E) Expression vector encoding either HA-NS5A (WT) or NS5A Lys double mutants (K44/68R) was coexpressed with FLAG-ISG15 together with E1, E2, and E3 ligase (HERC5) in 293T cells, followed by immunoprecipitation with anti-FLAG mouse MAb and detection with anti-HA rabbit PAb. Input samples (Lysate) were detected with anti-HA rabbit PAb and anti-FLAG mouse MAb. The asterisks indicate the various ISG15-conjugated NS5A proteins.

Article Snippet: The rabbit polyclonal antibodies (PAbs) used in this study were anti-HA PAb (H-6908; Sigma-Aldrich) and anti-Herc5 PAb (BML-PW0920; Enzo Life Sciences, New York, NY).

Techniques: Sequencing, Expressing, Plasmid Preparation, Mutagenesis, Immunoprecipitation

Journal: Journal of Virology

Article Title: ISGylation of Hepatitis C Virus NS5A Protein Promotes Viral RNA Replication via Recruitment of Cyclophilin A

doi: 10.1128/JVI.00532-20

Figure Lengend Snippet: Detection of NS5A ISGylation in the HCV-replicating cells. Huh-7.5 cells were electroporated with in vitro-transcribed chimeric JFH1/5A-Con1 RNA together with E1 (UBE1L), E2 (UbcH8), and E3 ligase (FLAG-tagged HERC5), followed by immunoprecipitation analysis coupled with immunoblotting using NS5A-specific antibody. Input samples (Lysate) were detected with anti-NS5A mouse MAb, anti-FLAG mouse MAb, or anti-ISG15 mouse MAb. The asterisk indicates the 100-kDa conjugates of NS5A ISGylation. IP, immunoprecipitation; IB, immunoblotting.

Article Snippet: The rabbit polyclonal antibodies (PAbs) used in this study were anti-HA PAb (H-6908; Sigma-Aldrich) and anti-Herc5 PAb (BML-PW0920; Enzo Life Sciences, New York, NY).

Techniques: In Vitro, Immunoprecipitation, Western Blot

Journal: Journal of Virology

Article Title: ISGylation of Hepatitis C Virus NS5A Protein Promotes Viral RNA Replication via Recruitment of Cyclophilin A

doi: 10.1128/JVI.00532-20

Figure Lengend Snippet: HERC5 is an E3 ligase that is specific for NS5A ISGylation. (A) Expression vector encoding NS5A-Myc-His6 was coexpressed with FLAG-ISG15 and each of three HA-tagged E3 ligases, including (a) HERC5, (b) TRIM25, and (c) HHARI, together with E1 (UBE1L) and E2 (UbcH8) in 293T cells, followed by detection with anti-NS5A antibody. (B) Expression vector encoding HA-NS5A (1b, Con1) was coexpressed with FLAG-ISG15 together with E1, E2, and E3 ligase (HERC5) or without each of the enzymes, as indicated, in 293T cells, followed by immunoprecipitation with anti-HA rabbit PAb and detection with anti-ISG15 mouse MAb or anti-HA rabbit PAb. (C) Expression vector encoding HA-NS5A was coexpressed with FLAG-ISG15 together with E1, E2, and E3 ligase (HERC5) or an inactive C994A HERC5 mutant in 293T cells, followed by immunoprecipitation with anti-HA rabbit PAb and detection with anti-ISG15 mouse MAb and anti-HA rabbit PAb. Input samples (Lysate) were detected with anti-HA rabbit PAb, anti-FLAG mouse MAb, or anti-HERC5 rabbit PAb. The asterisks indicate the various ISG15-conjugated NS5A proteins.

Article Snippet: The rabbit polyclonal antibodies (PAbs) used in this study were anti-HA PAb (H-6908; Sigma-Aldrich) and anti-Herc5 PAb (BML-PW0920; Enzo Life Sciences, New York, NY).

Techniques: Expressing, Plasmid Preparation, Immunoprecipitation, Mutagenesis

Journal: Journal of Virology

Article Title: ISGylation of Hepatitis C Virus NS5A Protein Promotes Viral RNA Replication via Recruitment of Cyclophilin A

doi: 10.1128/JVI.00532-20

Figure Lengend Snippet: NS5A ISGylation in HCV genotypes. (A) Schematic diagrams of the locations of Lys (K) residues on HCV NS5A of different genotypes (1b, Con1; 1a, H77c; 2a, JFH1; 3a, S52; 4a, ED43; 5a, SA1). (B) Expression vector encoding HA-NS5A of different genotypes (1a [H77c], 2a [JFH1], 3a [S52], 4a [ED43], and 5a [SA1]) was coexpressed with FLAG-ISG15 together with E1 (UBE1L), E2 (UbcH8), and E3 ligase (HERC5) in 293T cells, followed by immunoprecipitation with anti-HA rabbit PAb and detection with anti-ISG15 mouse MAb and anti-HA antibody. Input samples (Lysate) were detected with anti-HA rabbit PAb and anti-FLAG mouse MAb. The slowly migrating bands indicate various ISG15-conjugated NS5A proteins.

Article Snippet: The rabbit polyclonal antibodies (PAbs) used in this study were anti-HA PAb (H-6908; Sigma-Aldrich) and anti-Herc5 PAb (BML-PW0920; Enzo Life Sciences, New York, NY).

Techniques: Expressing, Plasmid Preparation, Immunoprecipitation

Journal: Journal of Virology

Article Title: ISGylation of Hepatitis C Virus NS5A Protein Promotes Viral RNA Replication via Recruitment of Cyclophilin A

doi: 10.1128/JVI.00532-20

Figure Lengend Snippet: Reduced expression of NS5A ISGylation in the ISG15 knockdown cells. Huh7/scr cells expressing shRNA targeted to ISG15 and sc-ISG15 were electroporated with the in vitro-transcribed chimeric JFH1/5A-Con1 RNA together with E1 (UBE1L), E2 (UbcH8), and E3 ligase (HA-tagged HERC5), followed by immunoprecipitation analysis coupled with immunoblotting using NS5A-specific antibody. Input samples (Lysate) were detected with anti-NS5A mouse MAb, anti-HA rabbit PAb, anti-GAPDH mouse MAb, or anti-ISG15 mouse MAb. The asterisk indicates the 100-kDa conjugates of NS5A ISGylation. NS, nonspecific bands.

Article Snippet: The rabbit polyclonal antibodies (PAbs) used in this study were anti-HA PAb (H-6908; Sigma-Aldrich) and anti-Herc5 PAb (BML-PW0920; Enzo Life Sciences, New York, NY).

Techniques: Expressing, shRNA, In Vitro, Immunoprecipitation, Western Blot

Journal: Journal of Virology

Article Title: ISGylation of Hepatitis C Virus NS5A Protein Promotes Viral RNA Replication via Recruitment of Cyclophilin A

doi: 10.1128/JVI.00532-20

Figure Lengend Snippet: NS5A ISGylation promotes the association of NS5A with cyclophilin A (CypA). (A) The CypA-binding region on NS5A (1b, Con1) domain II. The amino acid regions from 305 to 311, 308 to 324, and 308 to 329 comprising the RNA-binding motif, the CypA-binding region (highlighted in pink), and the NS5B-binding region, respectively, are indicated. W316 is one of the key residues on NS5A for the protein-protein interactions with CypA and NS5B. (B) Expression vector encoding HA-NS5A (1b, Con1) was coexpressed with C-terminal Myc-His6-CypA in the presence or absence of FLAG-ISG15 together with E1 (UBE1L), E2 (UbcH8), and E3 ligase (HERC5) in 293T cells, followed by immunoprecipitation with anti-Myc mouse MAb or anti-HA rabbit PAb and detection with anti-HA rabbit PAb and anti-Myc mouse MAb, respectively. (C) HA-NS5A was coexpressed with CypA-Myc-His6 in the presence or absence of FLAG-ISG15 together with E1, E2, and E3 ligase in 293T cells. At 24 h posttransfection, the cells were treated with 5 μg/ml of CsA for 24 h, followed by immunoprecipitation with anti-Myc mouse MAb and detection with anti-HA rabbit PAb and anti-Myc mouse MAb. Input samples (Lysate) were detected with anti-Myc mouse MAb, anti-HA rabbit PAb, and anti-FLAG mouse MAb. NS, nonspecific bands. The binding ratio of HA-NS5A and CypA-Myc-His6 was determined to measure the band intensities (HA-NS5A [first panel]/CypA-Myc-His6 [second panel], lanes 3 and 6), using ImageQuant TL software (version 7). Results are the mean values from triplicates ± SE. *, P < 0.05 versus the results for the cells treated with dimethyl sulfoxide (DMSO). HC, immunoglobulin heavy chain. (D) The proximity ligation assay (PLA). Huh-7.5 cells were coexpressed with HA-NS5A and CypA-Myc-His6 together with pEGFP or pEGFP-ISG15 plus E1, E2, and E3 ligase, respectively. Cells were fixed, permeabilized, and incubated with anti-HA rabbit PAb and anti-Myc mouse MAb, followed by staining with PLA probes. Red fluorescent spots indicate an interaction with NS5A and CypA. Nuclei were stained with Hoechst 33342. Scale bars, 10 μm.

Article Snippet: The rabbit polyclonal antibodies (PAbs) used in this study were anti-HA PAb (H-6908; Sigma-Aldrich) and anti-Herc5 PAb (BML-PW0920; Enzo Life Sciences, New York, NY).

Techniques: Binding Assay, RNA Binding Assay, Expressing, Plasmid Preparation, Immunoprecipitation, Software, Proximity Ligation Assay, Incubation, Staining

Journal: Journal of Virology

Article Title: ISGylation of Hepatitis C Virus NS5A Protein Promotes Viral RNA Replication via Recruitment of Cyclophilin A

doi: 10.1128/JVI.00532-20

Figure Lengend Snippet: NS5A, but not CypA, associates with ISG15. Expression vector encoding either CypA-Myc-His6 or NS5A-Myc-His6 was coexpressed in the presence or absence of FLAG-ISG15 together with or without E1 (UBE1L), E2 (UbcH8), and E3 ligase (HERC5) in 293T cells, followed by immunoprecipitation with anti-FLAG mouse MAb and detection with anti-Myc mouse MAb or anti-FLAG mouse MAb. Input samples (Lysate) were detected by anti-Myc mouse MAb and anti-FLAG mouse MAb. HC, immunoglobulin heavy chain; LC, immunoglobulin light chain.

Article Snippet: The rabbit polyclonal antibodies (PAbs) used in this study were anti-HA PAb (H-6908; Sigma-Aldrich) and anti-Herc5 PAb (BML-PW0920; Enzo Life Sciences, New York, NY).

Techniques: Expressing, Plasmid Preparation, Immunoprecipitation

Journal: Journal of Virology

Article Title: ISGylation of Hepatitis C Virus NS5A Protein Promotes Viral RNA Replication via Recruitment of Cyclophilin A

doi: 10.1128/JVI.00532-20

Figure Lengend Snippet: Enhancement of the interaction between NS5A and CypA by ISGylation in an HCV genotype-dependent manner. Expression vector encoding each of the HA-NS5A genotypes (1a [H77c], 2a [JFH1], 3a [S52], 4a [ED43], and 5a [SA1]) was coexpressed with CypA-Myc-His6 in the presence or absence of FLAG-ISG15 together with E1 (UBE1L), E2 (UbcH8), and E3 ligase (HERC5) in 293T cells, followed by immunoprecipitation with anti-Myc mouse MAb and detection with anti-HA rabbit PAb and anti-Myc mouse MAb. Input samples (Lysate) were detected with anti-Myc mouse MAb, anti-HA rabbit PAb, and anti-ISG15 mouse MAb.

Article Snippet: The rabbit polyclonal antibodies (PAbs) used in this study were anti-HA PAb (H-6908; Sigma-Aldrich) and anti-Herc5 PAb (BML-PW0920; Enzo Life Sciences, New York, NY).

Techniques: Expressing, Plasmid Preparation, Immunoprecipitation

Journal: Journal of Virology

Article Title: ISGylation of Hepatitis C Virus NS5A Protein Promotes Viral RNA Replication via Recruitment of Cyclophilin A

doi: 10.1128/JVI.00532-20

Figure Lengend Snippet: CsA interferes with the ISGylation-promoted association of NS5A with CypA in a dose-dependent manner. Expression vector encoding HA-NS5A was coexpressed with CypA-Myc-His6 in the presence or absence of FLAG-ISG15 together with E1 (UBE1L), E2 (UbcH8), and E3 ligase (HERC5) in 293T cells. At 24 h posttransfection, the cells were treated with 5, 0.5, or 0.05 μg/ml of CsA for 24 h, followed by immunoprecipitation with anti-Myc mouse MAb and detection with anti-HA rabbit PAb and anti-Myc mouse MAb. Input samples (Lysate) were detected with anti-Myc mouse MAb, anti-HA rabbit PAb, and anti-FLAG mouse MAb. NS, nonspecific bands.

Article Snippet: The rabbit polyclonal antibodies (PAbs) used in this study were anti-HA PAb (H-6908; Sigma-Aldrich) and anti-Herc5 PAb (BML-PW0920; Enzo Life Sciences, New York, NY).

Techniques: Expressing, Plasmid Preparation, Immunoprecipitation

Journal: Journal of Virology

Article Title: ISGylation of Hepatitis C Virus NS5A Protein Promotes Viral RNA Replication via Recruitment of Cyclophilin A

doi: 10.1128/JVI.00532-20

Figure Lengend Snippet: Enhancement of the interaction between NS5A and CypA in an ISGylation-dependent manner. (A) Expression vector encoding HA-NS5A (1b, Con1) was coexpressed with CypA-Myc-His6 in the presence or absence of FLAG-ISG15 or its conjugation-defective mutant (FLAG-ISG15-AA) together with E1 (UBE1L), E2 (UbcH8), and E3 ligase (HERC5) in 293T cells, followed by immunoprecipitation with anti-Myc mouse MAb and detection with anti-HA rabbit PAb and anti-Myc mouse MAb. (B, C) Expression vector encoding either HA-NS5A (1b, Con1) or NS5A Lys mutant K-Null (B) or K308R (C) was coexpressed with CypA-Myc-His6 in the presence or absence of FLAG-ISG15 together with E1, E2, and E3 ligase in 293T cells, followed by immunoprecipitation with anti-Myc mouse MAb or anti-HA rabbit PAb and detection with anti-HA rabbit PAb or anti-Myc mouse MAb, respectively. Input samples (Lysate) were detected by anti-Myc mouse MAb, anti-HA rabbit PAb, and anti-FLAG mouse MAb. NS, nonspecific bands. The binding ratios of HA-NS5A and CypA-Myc-His6 were determined to measure the band intensities of HA-NS5A (first panel)/CypA-Myc-His6 (second panel), lanes 3 and 4 (A), HA-NS5A (first panel)/CypA-Myc-His6 (second panel), lanes 3 and 6 (B), and CypA-Myc-His6 (first panel)/HA-NS5A (second panel), lanes 3 and 4 (C), using ImageQuant TL software (version 7). Results are the mean values from triplicates ± SE. *, P < 0.05, and **, P < 0.01, versus the results for the cells transduced with WT NS5A.

Article Snippet: The rabbit polyclonal antibodies (PAbs) used in this study were anti-HA PAb (H-6908; Sigma-Aldrich) and anti-Herc5 PAb (BML-PW0920; Enzo Life Sciences, New York, NY).

Techniques: Expressing, Plasmid Preparation, Conjugation Assay, Mutagenesis, Immunoprecipitation, Binding Assay, Software, Transduction

Journal: Journal of Virology

Article Title: ISGylation of Hepatitis C Virus NS5A Protein Promotes Viral RNA Replication via Recruitment of Cyclophilin A

doi: 10.1128/JVI.00532-20

Figure Lengend Snippet: The K44 and K166 residues on NS5A are not involved in the enhanced association with CypA. Expression vector encoding HA-NS5A (1b, Con1) or an NS5A Lys mutant (K44R or K166R) was coexpressed with CypA-Myc-His6 in the presence or absence of FLAG-ISG15 together with E1 (UBE1L), E2 (UbcH8), and E3 ligase (HERC5) in the 293T cells, followed by immunoprecipitation with anti-HA rabbit PAb and detection with anti-Myc mouse MAb and anti-HA rabbit PAb. Input samples (Lysate) were detected by anti-Myc mouse MAb, anti-HA rabbit PAb, and anti-FLAG mouse MAb.

Article Snippet: The rabbit polyclonal antibodies (PAbs) used in this study were anti-HA PAb (H-6908; Sigma-Aldrich) and anti-Herc5 PAb (BML-PW0920; Enzo Life Sciences, New York, NY).

Techniques: Expressing, Plasmid Preparation, Mutagenesis, Immunoprecipitation

Journal: Journal of Virology

Article Title: ISGylation of Hepatitis C Virus NS5A Protein Promotes Viral RNA Replication via Recruitment of Cyclophilin A

doi: 10.1128/JVI.00532-20

Figure Lengend Snippet: A model of the functional role of NS5A ISGylation in efficient HCV RNA replication. An intracellular ISG15 is covalently conjugated to HCV NS5A protein by the action of three enzymes, such as E1 (UBE1L), E2 (UbcH8) and E3 ligase (HERC5). The K308 residue within domain II (DII) of NS5A (genotype 1b, Con1) is the acceptor site for ISGylation. The ISGylated NS5A DII may allow its association with CypA, facilitating efficient HCV RNA replication.

Article Snippet: The rabbit polyclonal antibodies (PAbs) used in this study were anti-HA PAb (H-6908; Sigma-Aldrich) and anti-Herc5 PAb (BML-PW0920; Enzo Life Sciences, New York, NY).

Techniques: Functional Assay