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