Journal of Viral Hepatitis, 2014
doi:10.1111/jvh.12241
SUMOylation of nonstructural 5A protein regulates hepatitis C virus replication H. S. Lee,1 Y. S. Lim,1 E. M. Park,1 S. H. Baek2 and S. B. Hwang1
1
National Research Laboratory of
2
Hepatitis C Virus, Ilsong Institute of Life Science, Hallym University, Anyang, Korea; and Department of Biological Sciences, Creative Research Initiative Center for Chromatin Dynamics, Seoul National University, Seoul, Korea Received November 2013; accepted for publication January 2014
SUMMARY. Viruses exploit cellular SUMOylation machinery
to favour their own propagation. We show that NS5A is a target protein of small ubiquitin-like modifier (SUMO) and is SUMOylated at lysine residue 348. We demonstrated that SUMOylation increased protein stability of NS5A by inhibiting ubiquitylation, and SUMOylation was also required for protein interaction with NS5B. These data imply that SUMO modification may contribute to HCV replication. Indeed, silencing of UBC9 impaired HCV replication in Jc1-infected cells, and HCV replication level was
INTRODUCTION Hepatitis C virus (HCV) is the major causative agent of nonA, non-B hepatitis. HCV infection often leads to chronic hepatitis, liver cirrhosis and hepatocellular carcinoma (HCC). HCV has been classified as the member of the genus Hepacivirus within the Flaviviridae family [1–3]. HCV is an enveloped virus with a positive-sense, single-stranded RNA genome of ~9.6 kb in length. Its genome encodes a single polyprotein precursor of more than 3000 amino acids, which is cleaved by host and viral proteases to generate structural (core, E1 and E2) and nonstructural proteins (p7, NS2 to NS5B). Although HCV is a highly prevalent pathogen, a protective vaccine is not yet available. Current standard therapy is pegylated interferon-a combined with ribavirin. Recently, two HCV NS3/4A protease inhibitors, boceprevir and telaprevir, were approved for triple therapy in combination with peginterferon and ribavirin. However, these direct-acting antivirals accompany with some side effects and result in a sustained virological response in only certain genotype of HCV. Abbreviations: HCC, hepatitis C virus; HCV, nonstructural 5A; HCVcc, cell-culture-grown NS5A; SUMO, small ubiquitin-like modifier; HCC, hepatocellular carcinoma. Correspondence: Soon B. Hwang, PhD, National Research Laboratory of Hepatitis C Virus, Ilsong Institute of Life Science, Hallym University, 1605-4 Gwanyang-dong, Dongan-gu, Anyang 431-060, Korea. E-mail: [email protected]
© 2014 John Wiley & Sons Ltd
also significantly reduced in SUMO-defective subgenomic replicon cells. Taken together, these data indicate that HCV replication is regulated by SUMO modification of NS5A protein. We provide evidence for the first time that HCV exploits host cellular SUMO modification system to favour its own replication. Keywords: HCV replication, hepatitis C virus, NS5A, protein stability, SUMOylation.
As viruses have evolved diverse mechanisms to exploit host post-translational modifications, identification of new type of post-translational modification in HCV protein would be an alternative way to gain more insights into the mechanism of HCV replication and to develop more effective therapeutic strategies. Post-translational modifications such as phosphorylation, glycosylation, acetylation, methylation, isoprenylation, SUMOylation and ubiquitylation are widely used to dynamically regulate protein activity. Small ubiquitin-related modifier, or small ubiquitin-like modifier (SUMO), is covalently linked to a target protein at post-translational step [4]. The SUMO is a molecule of ~11.5 kDa that is covalently conjugated to lysine residues of target proteins. SUMO is conjugated to a substrate targeting lysine in the consensus sequence motif ΨKxE (where Ψ is a hydrophobic residue, and x is any residue) [5]. There are four SUMO isoforms encoded by the human genome: SUMO-1/Smt3C, SUMO-2/Smt3A, SUMO-3/Smt3B and SUMO-4 [6]. SUMOylation is carried out by an E1 activating enzyme, an E2 conjugating enzyme (Ubc9) and an E3 ligase [6]. SUMOylation is a dynamic and reversible process that can regulate various cellular functions by altering protein’s turnover, intracellular localization and protein interaction. It has been reported that only 5–10% of a protein is in a SUMOylated form at any given time [6]. The SUMOylated proteins are involved in transcriptional regulation, nuclearcytosolic transport, protein stability, stress response, signal transduction, and cancer development and progression [6].
2
H. S. Lee et al. (Invitrogen, Carlsbad, CA, USA) vector. cDNAs encoding SENP1, SENP2 and SENP3, respectively, were amplified by PCR and cloned into the pEF6/V5 expression vector. SUMO1, SUMO-2 and SUMO-3 plasmids cloned into the pCDNA4/ Xpress-His, and pFlag CMV2-UBC9 were described as we reported previously [4]. Mutagenesis for the K139R and K348R was performed using the EZ change TM site-directed mutagenesis kit (Enzynomics, Seoul, Korea) according to the manufacturer’s instructions. Mutant constructs were subcloned into the pEF6/Myc expression vector.
It has been previously reported that protein expression level of SUMO-1 was significantly high in HCC as compared to the non-neoplastic liver tissues [7]. Moreover, the genes involved in ubiquitylation and SUMOylation were also overexpressed in HCC [8]. In this study, we have shown that HCV NS5A protein, but not other HCV proteins, is SUMOylated, and that SUMOylation occurs at lysine residue 348 in NS5A protein. SUMOylation enhanced protein stability of NS5A. Importantly, silencing of SUMOylation system impaired HCV propagation. We demonstrate for the first time that HCV exploits cellular SUMOylation machinery for its own propagation.
SUMOylation assay SUMOylation assay was performed as we reported previously [4]. Briefly, either HEK293T cells or Huh7.5 cells were cotransfected with NS5A expression plasmid and Xpresstagged SUMO-1, SUMO-2 and SUMO-3, respectively. Two days after transfection, cells were lysed in buffer supplemented with protease inhibitor cocktail and were further sonicated for 20 s. The supernatant was clarified by
MATERIALS AND METHODS Plasmids and DNA transfection cDNA encoding HCV NS5A (genotype 1b) was amplified by PCR using the pFK-I389neo/NS3-30 /NK5.1 [9] as a template and cloned into Xba1 site of the pEF6/Myc-His (b) 1.2
1 0.8 0.6 0.4 0.2
0 siRNA
Neg
Pos
UBC9
(c) 120%
*
1
100%
0.8
80%
Cell viability
**
Intracellular HCV 1b RNA
Intracellular HCV RNA level
(a) 1.2
0.6 0.4
40% 20%
0.2 0 siRNA
60%
Neg
UBC9
0% siRNA
Neg
UBC9
UBC9
UBC9 β-Actin
β-actin
Jc1-infected cells
Replicon cells
Fig. 1 Silencing of UBC9 impairs HCV RNA replication. (a) Huh7.5 cells were transfected with 25 nM of the indicated siRNAs and were then infected with Jc1 at 24 h after transfection. At 2 days postinfection, intracellular HCV RNA level was determined by qPCR. (b) Replicon cells were transfected with either negative or UBC9 siRNA. At 72 h after transfection, intracellular HCV RNA level was determined by qPCR. Values are means SEM of two independent experiments. Asterisks indicate significant differences (*, P < 0.05; **, P < 0.01) from negative siRNA-treated cells. (c) Huh7.5 cells were transfected with 25 nM siRNA of either negative or UBC9. At 96 h after transfection, cell viability was assessed by an MTT assay.
Fig. 2 NS5A is SUMOylated by both SUMO-1 and SUMO-2. (a) HEK293T cells were cotransfected with the indicated plasmid (Myc-tagged core, NS3, NS4B, NS5A and NS5B) and Flag-tagged UBC9 in the absence or presence of Xpresstagged SUMO-1, SUMO-2, SUMO-3, respectively. Cells were harvested at 36 h after transfection and analysed by immunoblot analysis using the indicated antibodies. Arrowhead indicates the SUMOylated NS5A. Arrow indicates a monoSUMO protein. (b–c) HEK293T cells were cotransfected with Myc-tagged NS5A derived from either HCV genotype 1b (b) or 2a (c) and SUMO expression plasmid. At 36 h after transfection, cell lysates were immunoprecipitated with an anti-Myc antibody, and then bound proteins were immunoblotted with either an anti-Xpress or an anti-Myc antibody. (d) NS5A is deSUMOylated by both SENP1 and SENP2. Huh7.5 cells were cotransfected with the indicated plasmids and harvested at 36 h after transfection. Protein expressions were verified by immunoblot analysis using an anti-Xpress, Flag, Myc, V5 and b-actin antibody, respectively (left panels). Total cellular extracts were immunoprecipitated with an anti-Myc antibody, and bound proteins were immunoblotted with either an anti-Xpress or an anti-Myc antibody (right panels). © 2014 John Wiley & Sons Ltd
SUMOylation of NS5A protein (a)
Core-Myc Xpress-SUMO1 – + – – Xpress-SUMO2 – – + – Xpress-SUMO3 – – – +
kDa
100
NS3-Myc
NS4B-Myc
NS5B-Myc
NS5A-Myc
– + – – – – + – – – – +
– + – – – – + – – – – +
– + – – – – + – – – – +
– + – – – – + – – – – +
35
63
25
100
75
75
Myc
75
63
25
63
180 75 48 Xpress
35 28
β-actin
IP : Myc
(b) Myc-NS5A 1b FLAG-UBC9 Xpress-SUMO-1 Xpress-SUMO-2 Xpress-SUMO-3
R R T T T
R R R T T
R R T R T
R R T T T
R R T T R
IP : Myc
(c) R R R T T
R R T R T
Myc-NS5A 2a FLAG-UBC9 Xpress-SUMO-1 Xpress-SUMO-2 Xpress-SUMO-3
R R T T R
180 130 100
Xpress
Myc-NS5A FLAG-UBC9 Xpress-SUMO-1 Xpress-SUMO-2 V5-SENPs kDa 180
Xpress
100 75
R T T T T
T T R T T
R R R T T
R R R T XG
R R R T Y
RT RT RT TR ZG T
R R T R Y
R R T R ZG
Myc-NS5A FLAG-UBC9 Xpress-SUMO-1 Xpress-SUMO-2 V5-SENPs kDa 130
Xpress
75 63
100
75
Myc
63
β-Actin
© 2014 John Wiley & Sons Ltd
R R T T R
IgG Myc
IP : Myc RR RR T T RR T XG
Flag
63
R R T R T
NS5A
Xpress
35
V5
R R R T T
48
48
75
R R T T T
SUMO2 - NS5A
Input
(d)
R R T T R
75
IgG
Myc
R R T R T
63
NS5A
63
R R R T T
130 100
SUMO2 - NS5A
75
R R T T T
Myc
75 63
R T T T T
T T R T T
R R R T T
R R R T XG
R R R T Y
RT RT RT TR ZG T
RR RR T T RR T XG
R R T R Y
R R T R ZG
3
4
H. S. Lee et al.
centrifugation (15 000 g) for 30 min at 4 °C. The protein concentration was determined by the Bradford assay (BioRad Laboratories, Hercules, CA, USA). Equal amounts of proteins were immunoprecipitated with the indicated antibodies and further incubated with the protein A/G beads for 1 h. The beads were washed with TBS-containing 0.25% Tween-20. Proteins were subjected to 10% SDS-PAGE and were immunoblotted with the indicated antibodies. Proteins were detected using an ECL kit.
(GeneAll) and were reverse transcribed using cDNA synthesis kit (TOYOBO, Osaka, japan). All quantitative realtime PCR (qRT-PCR) experiments were carried out using an iQ5 multicolour real-time PCR detection system (BioRad Laboratories) under the following conditions: 3 min at 95 °C followed by 40 cycles of 95 °C for 10 s, 55 °C for 20 s and 72 °C for 30 s. Seventy-one cycles of 10 s, with 0.5 °C temperature increments from 60 to 95 °C, were used for the melting curves.
RNA interference
Statistical analysis
siRNAs targeting UBC9 (50 -GUUCUGCGCCACUUCCUUdTdT30 ) and the universal negative control were purchased from Bioneer. siRNA targeting 50 NTR of HCV (50 - CCUCAAAGAAAAACCAAACUU- 30 ) was used as a positive control. siRNA transfection was performed using a Lipofectamine RNAiMax reagent (Invitrogen) as we described previously [10].
Data are presented as means standard deviation (SD) for at least two independent experiments. Student’s t-test was used for statistical analysis. A P value of
doi:10.1111/jvh.12241
SUMOylation of nonstructural 5A protein regulates hepatitis C virus replication H. S. Lee,1 Y. S. Lim,1 E. M. Park,1 S. H. Baek2 and S. B. Hwang1
1
National Research Laboratory of
2
Hepatitis C Virus, Ilsong Institute of Life Science, Hallym University, Anyang, Korea; and Department of Biological Sciences, Creative Research Initiative Center for Chromatin Dynamics, Seoul National University, Seoul, Korea Received November 2013; accepted for publication January 2014
SUMMARY. Viruses exploit cellular SUMOylation machinery
to favour their own propagation. We show that NS5A is a target protein of small ubiquitin-like modifier (SUMO) and is SUMOylated at lysine residue 348. We demonstrated that SUMOylation increased protein stability of NS5A by inhibiting ubiquitylation, and SUMOylation was also required for protein interaction with NS5B. These data imply that SUMO modification may contribute to HCV replication. Indeed, silencing of UBC9 impaired HCV replication in Jc1-infected cells, and HCV replication level was
INTRODUCTION Hepatitis C virus (HCV) is the major causative agent of nonA, non-B hepatitis. HCV infection often leads to chronic hepatitis, liver cirrhosis and hepatocellular carcinoma (HCC). HCV has been classified as the member of the genus Hepacivirus within the Flaviviridae family [1–3]. HCV is an enveloped virus with a positive-sense, single-stranded RNA genome of ~9.6 kb in length. Its genome encodes a single polyprotein precursor of more than 3000 amino acids, which is cleaved by host and viral proteases to generate structural (core, E1 and E2) and nonstructural proteins (p7, NS2 to NS5B). Although HCV is a highly prevalent pathogen, a protective vaccine is not yet available. Current standard therapy is pegylated interferon-a combined with ribavirin. Recently, two HCV NS3/4A protease inhibitors, boceprevir and telaprevir, were approved for triple therapy in combination with peginterferon and ribavirin. However, these direct-acting antivirals accompany with some side effects and result in a sustained virological response in only certain genotype of HCV. Abbreviations: HCC, hepatitis C virus; HCV, nonstructural 5A; HCVcc, cell-culture-grown NS5A; SUMO, small ubiquitin-like modifier; HCC, hepatocellular carcinoma. Correspondence: Soon B. Hwang, PhD, National Research Laboratory of Hepatitis C Virus, Ilsong Institute of Life Science, Hallym University, 1605-4 Gwanyang-dong, Dongan-gu, Anyang 431-060, Korea. E-mail: [email protected]
© 2014 John Wiley & Sons Ltd
also significantly reduced in SUMO-defective subgenomic replicon cells. Taken together, these data indicate that HCV replication is regulated by SUMO modification of NS5A protein. We provide evidence for the first time that HCV exploits host cellular SUMO modification system to favour its own replication. Keywords: HCV replication, hepatitis C virus, NS5A, protein stability, SUMOylation.
As viruses have evolved diverse mechanisms to exploit host post-translational modifications, identification of new type of post-translational modification in HCV protein would be an alternative way to gain more insights into the mechanism of HCV replication and to develop more effective therapeutic strategies. Post-translational modifications such as phosphorylation, glycosylation, acetylation, methylation, isoprenylation, SUMOylation and ubiquitylation are widely used to dynamically regulate protein activity. Small ubiquitin-related modifier, or small ubiquitin-like modifier (SUMO), is covalently linked to a target protein at post-translational step [4]. The SUMO is a molecule of ~11.5 kDa that is covalently conjugated to lysine residues of target proteins. SUMO is conjugated to a substrate targeting lysine in the consensus sequence motif ΨKxE (where Ψ is a hydrophobic residue, and x is any residue) [5]. There are four SUMO isoforms encoded by the human genome: SUMO-1/Smt3C, SUMO-2/Smt3A, SUMO-3/Smt3B and SUMO-4 [6]. SUMOylation is carried out by an E1 activating enzyme, an E2 conjugating enzyme (Ubc9) and an E3 ligase [6]. SUMOylation is a dynamic and reversible process that can regulate various cellular functions by altering protein’s turnover, intracellular localization and protein interaction. It has been reported that only 5–10% of a protein is in a SUMOylated form at any given time [6]. The SUMOylated proteins are involved in transcriptional regulation, nuclearcytosolic transport, protein stability, stress response, signal transduction, and cancer development and progression [6].
2
H. S. Lee et al. (Invitrogen, Carlsbad, CA, USA) vector. cDNAs encoding SENP1, SENP2 and SENP3, respectively, were amplified by PCR and cloned into the pEF6/V5 expression vector. SUMO1, SUMO-2 and SUMO-3 plasmids cloned into the pCDNA4/ Xpress-His, and pFlag CMV2-UBC9 were described as we reported previously [4]. Mutagenesis for the K139R and K348R was performed using the EZ change TM site-directed mutagenesis kit (Enzynomics, Seoul, Korea) according to the manufacturer’s instructions. Mutant constructs were subcloned into the pEF6/Myc expression vector.
It has been previously reported that protein expression level of SUMO-1 was significantly high in HCC as compared to the non-neoplastic liver tissues [7]. Moreover, the genes involved in ubiquitylation and SUMOylation were also overexpressed in HCC [8]. In this study, we have shown that HCV NS5A protein, but not other HCV proteins, is SUMOylated, and that SUMOylation occurs at lysine residue 348 in NS5A protein. SUMOylation enhanced protein stability of NS5A. Importantly, silencing of SUMOylation system impaired HCV propagation. We demonstrate for the first time that HCV exploits cellular SUMOylation machinery for its own propagation.
SUMOylation assay SUMOylation assay was performed as we reported previously [4]. Briefly, either HEK293T cells or Huh7.5 cells were cotransfected with NS5A expression plasmid and Xpresstagged SUMO-1, SUMO-2 and SUMO-3, respectively. Two days after transfection, cells were lysed in buffer supplemented with protease inhibitor cocktail and were further sonicated for 20 s. The supernatant was clarified by
MATERIALS AND METHODS Plasmids and DNA transfection cDNA encoding HCV NS5A (genotype 1b) was amplified by PCR using the pFK-I389neo/NS3-30 /NK5.1 [9] as a template and cloned into Xba1 site of the pEF6/Myc-His (b) 1.2
1 0.8 0.6 0.4 0.2
0 siRNA
Neg
Pos
UBC9
(c) 120%
*
1
100%
0.8
80%
Cell viability
**
Intracellular HCV 1b RNA
Intracellular HCV RNA level
(a) 1.2
0.6 0.4
40% 20%
0.2 0 siRNA
60%
Neg
UBC9
0% siRNA
Neg
UBC9
UBC9
UBC9 β-Actin
β-actin
Jc1-infected cells
Replicon cells
Fig. 1 Silencing of UBC9 impairs HCV RNA replication. (a) Huh7.5 cells were transfected with 25 nM of the indicated siRNAs and were then infected with Jc1 at 24 h after transfection. At 2 days postinfection, intracellular HCV RNA level was determined by qPCR. (b) Replicon cells were transfected with either negative or UBC9 siRNA. At 72 h after transfection, intracellular HCV RNA level was determined by qPCR. Values are means SEM of two independent experiments. Asterisks indicate significant differences (*, P < 0.05; **, P < 0.01) from negative siRNA-treated cells. (c) Huh7.5 cells were transfected with 25 nM siRNA of either negative or UBC9. At 96 h after transfection, cell viability was assessed by an MTT assay.
Fig. 2 NS5A is SUMOylated by both SUMO-1 and SUMO-2. (a) HEK293T cells were cotransfected with the indicated plasmid (Myc-tagged core, NS3, NS4B, NS5A and NS5B) and Flag-tagged UBC9 in the absence or presence of Xpresstagged SUMO-1, SUMO-2, SUMO-3, respectively. Cells were harvested at 36 h after transfection and analysed by immunoblot analysis using the indicated antibodies. Arrowhead indicates the SUMOylated NS5A. Arrow indicates a monoSUMO protein. (b–c) HEK293T cells were cotransfected with Myc-tagged NS5A derived from either HCV genotype 1b (b) or 2a (c) and SUMO expression plasmid. At 36 h after transfection, cell lysates were immunoprecipitated with an anti-Myc antibody, and then bound proteins were immunoblotted with either an anti-Xpress or an anti-Myc antibody. (d) NS5A is deSUMOylated by both SENP1 and SENP2. Huh7.5 cells were cotransfected with the indicated plasmids and harvested at 36 h after transfection. Protein expressions were verified by immunoblot analysis using an anti-Xpress, Flag, Myc, V5 and b-actin antibody, respectively (left panels). Total cellular extracts were immunoprecipitated with an anti-Myc antibody, and bound proteins were immunoblotted with either an anti-Xpress or an anti-Myc antibody (right panels). © 2014 John Wiley & Sons Ltd
SUMOylation of NS5A protein (a)
Core-Myc Xpress-SUMO1 – + – – Xpress-SUMO2 – – + – Xpress-SUMO3 – – – +
kDa
100
NS3-Myc
NS4B-Myc
NS5B-Myc
NS5A-Myc
– + – – – – + – – – – +
– + – – – – + – – – – +
– + – – – – + – – – – +
– + – – – – + – – – – +
35
63
25
100
75
75
Myc
75
63
25
63
180 75 48 Xpress
35 28
β-actin
IP : Myc
(b) Myc-NS5A 1b FLAG-UBC9 Xpress-SUMO-1 Xpress-SUMO-2 Xpress-SUMO-3
R R T T T
R R R T T
R R T R T
R R T T T
R R T T R
IP : Myc
(c) R R R T T
R R T R T
Myc-NS5A 2a FLAG-UBC9 Xpress-SUMO-1 Xpress-SUMO-2 Xpress-SUMO-3
R R T T R
180 130 100
Xpress
Myc-NS5A FLAG-UBC9 Xpress-SUMO-1 Xpress-SUMO-2 V5-SENPs kDa 180
Xpress
100 75
R T T T T
T T R T T
R R R T T
R R R T XG
R R R T Y
RT RT RT TR ZG T
R R T R Y
R R T R ZG
Myc-NS5A FLAG-UBC9 Xpress-SUMO-1 Xpress-SUMO-2 V5-SENPs kDa 130
Xpress
75 63
100
75
Myc
63
β-Actin
© 2014 John Wiley & Sons Ltd
R R T T R
IgG Myc
IP : Myc RR RR T T RR T XG
Flag
63
R R T R T
NS5A
Xpress
35
V5
R R R T T
48
48
75
R R T T T
SUMO2 - NS5A
Input
(d)
R R T T R
75
IgG
Myc
R R T R T
63
NS5A
63
R R R T T
130 100
SUMO2 - NS5A
75
R R T T T
Myc
75 63
R T T T T
T T R T T
R R R T T
R R R T XG
R R R T Y
RT RT RT TR ZG T
RR RR T T RR T XG
R R T R Y
R R T R ZG
3
4
H. S. Lee et al.
centrifugation (15 000 g) for 30 min at 4 °C. The protein concentration was determined by the Bradford assay (BioRad Laboratories, Hercules, CA, USA). Equal amounts of proteins were immunoprecipitated with the indicated antibodies and further incubated with the protein A/G beads for 1 h. The beads were washed with TBS-containing 0.25% Tween-20. Proteins were subjected to 10% SDS-PAGE and were immunoblotted with the indicated antibodies. Proteins were detected using an ECL kit.
(GeneAll) and were reverse transcribed using cDNA synthesis kit (TOYOBO, Osaka, japan). All quantitative realtime PCR (qRT-PCR) experiments were carried out using an iQ5 multicolour real-time PCR detection system (BioRad Laboratories) under the following conditions: 3 min at 95 °C followed by 40 cycles of 95 °C for 10 s, 55 °C for 20 s and 72 °C for 30 s. Seventy-one cycles of 10 s, with 0.5 °C temperature increments from 60 to 95 °C, were used for the melting curves.
RNA interference
Statistical analysis
siRNAs targeting UBC9 (50 -GUUCUGCGCCACUUCCUUdTdT30 ) and the universal negative control were purchased from Bioneer. siRNA targeting 50 NTR of HCV (50 - CCUCAAAGAAAAACCAAACUU- 30 ) was used as a positive control. siRNA transfection was performed using a Lipofectamine RNAiMax reagent (Invitrogen) as we described previously [10].
Data are presented as means standard deviation (SD) for at least two independent experiments. Student’s t-test was used for statistical analysis. A P value of
