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Signal peptidase complex subunit 1 participates in the assembly of hepatitis C virus through an interaction with E2 and NS2.

Suzuki R, Matsuda M, Watashi K, Aizaki H, Matsuura Y, Wakita T, Suzuki T - PLoS Pathog. (2013)

Bottom Line: Silencing of endogenous SPCS1 resulted in markedly reduced production of infectious HCV, whereas neither processing of structural proteins, cell entry, RNA replication, nor release of virus from the cells was impaired.SPCS1 was found to interact with both NS2 and E2.Our findings suggest that SPCS1 plays a key role in the formation of the membrane-associated NS2-E2 complex via its interaction with NS2 and E2, which leads to a coordinating interaction between the structural and non-structural proteins and facilitates the early step of assembly of infectious particles.

View Article: PubMed Central - PubMed

Affiliation: Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan. ryosuke@nih.go.jp

ABSTRACT
Hepatitis C virus (HCV) nonstructural protein 2 (NS2) is a hydrophobic, transmembrane protein that is required not only for NS2-NS3 cleavage, but also for infectious virus production. To identify cellular factors that interact with NS2 and are important for HCV propagation, we screened a human liver cDNA library by split-ubiquitin membrane yeast two-hybrid assay using full-length NS2 as a bait, and identified signal peptidase complex subunit 1 (SPCS1), which is a component of the microsomal signal peptidase complex. Silencing of endogenous SPCS1 resulted in markedly reduced production of infectious HCV, whereas neither processing of structural proteins, cell entry, RNA replication, nor release of virus from the cells was impaired. Propagation of Japanese encephalitis virus was not affected by knockdown of SPCS1, suggesting that SPCS1 does not widely modulate the viral lifecycles of the Flaviviridae family. SPCS1 was found to interact with both NS2 and E2. A complex of NS2, E2, and SPCS1 was formed in cells as demonstrated by co-immunoprecipitation assays. Knockdown of SPCS1 impaired interaction of NS2 with E2. Our findings suggest that SPCS1 plays a key role in the formation of the membrane-associated NS2-E2 complex via its interaction with NS2 and E2, which leads to a coordinating interaction between the structural and non-structural proteins and facilitates the early step of assembly of infectious particles.

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SPCS1 forms a complex with NS2 and E2.(A) Lysates of cells, which were co-transfected with Core-p7, FLAG-NS2, and SPCS1-myc expression plasmids, were immunoprecipitated with anti-myc or anti-FLAG antibody. The resulting precipitates and whole cell lysates used in IP were examined by immunoblotting using anti-E2, anti-FLAG, or anti-myc antibody. An empty plasmid was used as a negative control. (B) Cells were transfected with Core-p7 expression plasmid in the presence or absence of SPCS1-myc expression plasmid. The cell lysates of the transfected cells were immunoprecipitated with anti-myc antibody. The resulting precipitates and whole cell lysates used in IP were examined by immunoblotting using anti-E2 or anti-myc antibody. An empty plasmid was used as a negative control. The bands corresponding to immunoglobulin heavy chain are marked by an asterisk. (C) Cells were co-transfected with Core-p7 and SPCS1-myc expression plasmids. The cell lysates of the transfected cells were immunoprecipitated with anti-myc antibody. The resulting precipitates and whole cell lysates used in IP were examined by immunoblotting using anti-E2 or anti-myc antibody. (D) Huh7.5.1 cells were transfected with SPCS1 siRNA or control siRNA at a final concentration of 20 nM. After 24 h, Huh7.5.1 cells were then co-transfected with FLAG-NS2 and Core-p7 expression plasmids. The lysates of transfected cells were immunoprecipitated with anti-FLAG antibody, followed by immunoblotting with anti-FLAG and anti-E2 antibodies. Immunoblot analysis of whole cell lysates was also performed. Intensity of E2 bands was quantified, and the ratio of immunoprecipitated E2 to E2 in cell lysate was shown. Similar results were obtained in 2 independent experiments. (E) KD#31 cells and parental Huh-7 cells were co-transfected with FLAG-NS2, Core-p7, and NS3 expression plasmids. The lysates of transfected cells were immunoprecipitated with anti-FLAG antibody followed by immunoblotting with anti-FLAG, anti-E2, and anti-NS3 antibodies. Immunoblot analysis of whole cell lysates was also performed. The ratio of immunoprecipitated E2 or NS3 to E2 or NS3 in cell lysate, respectively, were shown.
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ppat-1003589-g006: SPCS1 forms a complex with NS2 and E2.(A) Lysates of cells, which were co-transfected with Core-p7, FLAG-NS2, and SPCS1-myc expression plasmids, were immunoprecipitated with anti-myc or anti-FLAG antibody. The resulting precipitates and whole cell lysates used in IP were examined by immunoblotting using anti-E2, anti-FLAG, or anti-myc antibody. An empty plasmid was used as a negative control. (B) Cells were transfected with Core-p7 expression plasmid in the presence or absence of SPCS1-myc expression plasmid. The cell lysates of the transfected cells were immunoprecipitated with anti-myc antibody. The resulting precipitates and whole cell lysates used in IP were examined by immunoblotting using anti-E2 or anti-myc antibody. An empty plasmid was used as a negative control. The bands corresponding to immunoglobulin heavy chain are marked by an asterisk. (C) Cells were co-transfected with Core-p7 and SPCS1-myc expression plasmids. The cell lysates of the transfected cells were immunoprecipitated with anti-myc antibody. The resulting precipitates and whole cell lysates used in IP were examined by immunoblotting using anti-E2 or anti-myc antibody. (D) Huh7.5.1 cells were transfected with SPCS1 siRNA or control siRNA at a final concentration of 20 nM. After 24 h, Huh7.5.1 cells were then co-transfected with FLAG-NS2 and Core-p7 expression plasmids. The lysates of transfected cells were immunoprecipitated with anti-FLAG antibody, followed by immunoblotting with anti-FLAG and anti-E2 antibodies. Immunoblot analysis of whole cell lysates was also performed. Intensity of E2 bands was quantified, and the ratio of immunoprecipitated E2 to E2 in cell lysate was shown. Similar results were obtained in 2 independent experiments. (E) KD#31 cells and parental Huh-7 cells were co-transfected with FLAG-NS2, Core-p7, and NS3 expression plasmids. The lysates of transfected cells were immunoprecipitated with anti-FLAG antibody followed by immunoblotting with anti-FLAG, anti-E2, and anti-NS3 antibodies. Immunoblot analysis of whole cell lysates was also performed. The ratio of immunoprecipitated E2 or NS3 to E2 or NS3 in cell lysate, respectively, were shown.

Mentions: It has been shown that HCV NS2 interacts with the viral structural and NS proteins in virus-producing cells [18]–[21], and that some of the interactions, especially the NS2-E2 interaction, are important for the assembly of infectious HCV particles. However, the functional role of NS2 in the HCV assembly process has not been fully elucidated. To test whether SPCS1 is involved in the interaction between NS2 and E2, cells were co-transfected with expression plasmids for E2, FLAG-NS2, and SPCS1-myc. E2 and NS2 were co-immunoprecipitated with SPCS1-myc, and E2 and SPCS1-myc were co-immunoprecipitated with FLAG-NS2 (Fig. 6A), suggesting the formation of an E2-NS2-SPCS1 complex in cells. To investigate the interaction of SPCS1 with E2 in the absence of NS2, HCV Core-p7 polyprotein or E2 protein were co-expressed with SPCS1-myc in cells, followed by immunoprecipitation with anti-myc antibody. As shown in Fig. 6B and Fig. S2, E2 was co-immunoprecipitated with SPCS1-myc. The interaction between SPCS1 and E2 was further analyzed in situ by PLA and mKG system. Specific signals indicating formation of the SPCS1-E2 complex were detected in both assays (Fig. S3), suggesting physical interaction between SPCS1 and E2 in cells.


Signal peptidase complex subunit 1 participates in the assembly of hepatitis C virus through an interaction with E2 and NS2.

Suzuki R, Matsuda M, Watashi K, Aizaki H, Matsuura Y, Wakita T, Suzuki T - PLoS Pathog. (2013)

SPCS1 forms a complex with NS2 and E2.(A) Lysates of cells, which were co-transfected with Core-p7, FLAG-NS2, and SPCS1-myc expression plasmids, were immunoprecipitated with anti-myc or anti-FLAG antibody. The resulting precipitates and whole cell lysates used in IP were examined by immunoblotting using anti-E2, anti-FLAG, or anti-myc antibody. An empty plasmid was used as a negative control. (B) Cells were transfected with Core-p7 expression plasmid in the presence or absence of SPCS1-myc expression plasmid. The cell lysates of the transfected cells were immunoprecipitated with anti-myc antibody. The resulting precipitates and whole cell lysates used in IP were examined by immunoblotting using anti-E2 or anti-myc antibody. An empty plasmid was used as a negative control. The bands corresponding to immunoglobulin heavy chain are marked by an asterisk. (C) Cells were co-transfected with Core-p7 and SPCS1-myc expression plasmids. The cell lysates of the transfected cells were immunoprecipitated with anti-myc antibody. The resulting precipitates and whole cell lysates used in IP were examined by immunoblotting using anti-E2 or anti-myc antibody. (D) Huh7.5.1 cells were transfected with SPCS1 siRNA or control siRNA at a final concentration of 20 nM. After 24 h, Huh7.5.1 cells were then co-transfected with FLAG-NS2 and Core-p7 expression plasmids. The lysates of transfected cells were immunoprecipitated with anti-FLAG antibody, followed by immunoblotting with anti-FLAG and anti-E2 antibodies. Immunoblot analysis of whole cell lysates was also performed. Intensity of E2 bands was quantified, and the ratio of immunoprecipitated E2 to E2 in cell lysate was shown. Similar results were obtained in 2 independent experiments. (E) KD#31 cells and parental Huh-7 cells were co-transfected with FLAG-NS2, Core-p7, and NS3 expression plasmids. The lysates of transfected cells were immunoprecipitated with anti-FLAG antibody followed by immunoblotting with anti-FLAG, anti-E2, and anti-NS3 antibodies. Immunoblot analysis of whole cell lysates was also performed. The ratio of immunoprecipitated E2 or NS3 to E2 or NS3 in cell lysate, respectively, were shown.
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ppat-1003589-g006: SPCS1 forms a complex with NS2 and E2.(A) Lysates of cells, which were co-transfected with Core-p7, FLAG-NS2, and SPCS1-myc expression plasmids, were immunoprecipitated with anti-myc or anti-FLAG antibody. The resulting precipitates and whole cell lysates used in IP were examined by immunoblotting using anti-E2, anti-FLAG, or anti-myc antibody. An empty plasmid was used as a negative control. (B) Cells were transfected with Core-p7 expression plasmid in the presence or absence of SPCS1-myc expression plasmid. The cell lysates of the transfected cells were immunoprecipitated with anti-myc antibody. The resulting precipitates and whole cell lysates used in IP were examined by immunoblotting using anti-E2 or anti-myc antibody. An empty plasmid was used as a negative control. The bands corresponding to immunoglobulin heavy chain are marked by an asterisk. (C) Cells were co-transfected with Core-p7 and SPCS1-myc expression plasmids. The cell lysates of the transfected cells were immunoprecipitated with anti-myc antibody. The resulting precipitates and whole cell lysates used in IP were examined by immunoblotting using anti-E2 or anti-myc antibody. (D) Huh7.5.1 cells were transfected with SPCS1 siRNA or control siRNA at a final concentration of 20 nM. After 24 h, Huh7.5.1 cells were then co-transfected with FLAG-NS2 and Core-p7 expression plasmids. The lysates of transfected cells were immunoprecipitated with anti-FLAG antibody, followed by immunoblotting with anti-FLAG and anti-E2 antibodies. Immunoblot analysis of whole cell lysates was also performed. Intensity of E2 bands was quantified, and the ratio of immunoprecipitated E2 to E2 in cell lysate was shown. Similar results were obtained in 2 independent experiments. (E) KD#31 cells and parental Huh-7 cells were co-transfected with FLAG-NS2, Core-p7, and NS3 expression plasmids. The lysates of transfected cells were immunoprecipitated with anti-FLAG antibody followed by immunoblotting with anti-FLAG, anti-E2, and anti-NS3 antibodies. Immunoblot analysis of whole cell lysates was also performed. The ratio of immunoprecipitated E2 or NS3 to E2 or NS3 in cell lysate, respectively, were shown.
Mentions: It has been shown that HCV NS2 interacts with the viral structural and NS proteins in virus-producing cells [18]–[21], and that some of the interactions, especially the NS2-E2 interaction, are important for the assembly of infectious HCV particles. However, the functional role of NS2 in the HCV assembly process has not been fully elucidated. To test whether SPCS1 is involved in the interaction between NS2 and E2, cells were co-transfected with expression plasmids for E2, FLAG-NS2, and SPCS1-myc. E2 and NS2 were co-immunoprecipitated with SPCS1-myc, and E2 and SPCS1-myc were co-immunoprecipitated with FLAG-NS2 (Fig. 6A), suggesting the formation of an E2-NS2-SPCS1 complex in cells. To investigate the interaction of SPCS1 with E2 in the absence of NS2, HCV Core-p7 polyprotein or E2 protein were co-expressed with SPCS1-myc in cells, followed by immunoprecipitation with anti-myc antibody. As shown in Fig. 6B and Fig. S2, E2 was co-immunoprecipitated with SPCS1-myc. The interaction between SPCS1 and E2 was further analyzed in situ by PLA and mKG system. Specific signals indicating formation of the SPCS1-E2 complex were detected in both assays (Fig. S3), suggesting physical interaction between SPCS1 and E2 in cells.

Bottom Line: Silencing of endogenous SPCS1 resulted in markedly reduced production of infectious HCV, whereas neither processing of structural proteins, cell entry, RNA replication, nor release of virus from the cells was impaired.SPCS1 was found to interact with both NS2 and E2.Our findings suggest that SPCS1 plays a key role in the formation of the membrane-associated NS2-E2 complex via its interaction with NS2 and E2, which leads to a coordinating interaction between the structural and non-structural proteins and facilitates the early step of assembly of infectious particles.

View Article: PubMed Central - PubMed

Affiliation: Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan. ryosuke@nih.go.jp

ABSTRACT
Hepatitis C virus (HCV) nonstructural protein 2 (NS2) is a hydrophobic, transmembrane protein that is required not only for NS2-NS3 cleavage, but also for infectious virus production. To identify cellular factors that interact with NS2 and are important for HCV propagation, we screened a human liver cDNA library by split-ubiquitin membrane yeast two-hybrid assay using full-length NS2 as a bait, and identified signal peptidase complex subunit 1 (SPCS1), which is a component of the microsomal signal peptidase complex. Silencing of endogenous SPCS1 resulted in markedly reduced production of infectious HCV, whereas neither processing of structural proteins, cell entry, RNA replication, nor release of virus from the cells was impaired. Propagation of Japanese encephalitis virus was not affected by knockdown of SPCS1, suggesting that SPCS1 does not widely modulate the viral lifecycles of the Flaviviridae family. SPCS1 was found to interact with both NS2 and E2. A complex of NS2, E2, and SPCS1 was formed in cells as demonstrated by co-immunoprecipitation assays. Knockdown of SPCS1 impaired interaction of NS2 with E2. Our findings suggest that SPCS1 plays a key role in the formation of the membrane-associated NS2-E2 complex via its interaction with NS2 and E2, which leads to a coordinating interaction between the structural and non-structural proteins and facilitates the early step of assembly of infectious particles.

Show MeSH
Related in: MedlinePlus