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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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Interaction of HCV NS2 protein with SPCS1 in mammalian cells.(A) Expression constructs of SPCS1-myc and FLAG-NS2 used in this study. TM regions are represented as gray. Myc-tag regions are depicted by the black circles. Gray circles and bold lines indicated FLAG-tag and spacer (GGGGS) sequences, respectively. Adaptive mutations are indicated as asterisks. Positions of the aa resides are indicated above the boxes. (B) 293T cells were co-transfected with a FLAG-tagged NS2 expression plasmid in the presence of a SPCS1-myc expression plasmid. Cell lysates of the transfected cells were immunoprecipitated with anti-myc antibody. The resulting precipitates and whole cell lysates used in immunoprecipitation (IP) were examined by immunoblotting using anti-FLAG- or anti-myc antibody. An empty plasmid was used as a negative control. (C) HCVcc infected Huh-7 cells were transfected with a SPCS1-myc expression plasmid. Cell lysates of the transfected cells were immunoprecipitated with anti-myc antibody. The resulting precipitates and whole cell lysates used in immunoprecipitation (IP) were examined by immunoblotting using anti-NS2 or anti-SPCS1 antibody. (D) Expression of SPCS1-myc and its deletion mutants. 293T cells were transfected with indicated plasmids. The cell lysates were examined by immunoblotting using anti-myc or anti-actin antibody. (E) Cells were co-transfected with indicated plasmids, and then lysates of transfected cells were immunoprecipitated with anti-myc antibody. The resulting precipitates and whole cell lysates used in IP were examined by immunoblotting using anti-FLAG- or anti-myc antibody. (F) Lysates of the transfected cells were immunoprecipitated with anti-myc antibody. The resulting precipitates (right panel) and whole cell lysates used in IP (left panel) were examined by immunoblotting using anti-FLAG or anti-myc antibody. (G) 293T cells were transfected with indicated plasmids. 2 days posttransfection, cells were fixed and permeabilized with Triton X-100, then subjected to in situ PLA (Upper) or immunofluorescence staining (Lower) using anti-FLAG and anti-V5 antibodies. (H) Detection of the SPCS1-NS2 interaction in transfected cells using the mKG system. 293T cells were transfected by indicated pair of mKG fusion constructs. Twenty-four hours after transfection, cell were fixed and stained with DAPI, and observed under a confocal microscope.
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ppat-1003589-g001: Interaction of HCV NS2 protein with SPCS1 in mammalian cells.(A) Expression constructs of SPCS1-myc and FLAG-NS2 used in this study. TM regions are represented as gray. Myc-tag regions are depicted by the black circles. Gray circles and bold lines indicated FLAG-tag and spacer (GGGGS) sequences, respectively. Adaptive mutations are indicated as asterisks. Positions of the aa resides are indicated above the boxes. (B) 293T cells were co-transfected with a FLAG-tagged NS2 expression plasmid in the presence of a SPCS1-myc expression plasmid. Cell lysates of the transfected cells were immunoprecipitated with anti-myc antibody. The resulting precipitates and whole cell lysates used in immunoprecipitation (IP) were examined by immunoblotting using anti-FLAG- or anti-myc antibody. An empty plasmid was used as a negative control. (C) HCVcc infected Huh-7 cells were transfected with a SPCS1-myc expression plasmid. Cell lysates of the transfected cells were immunoprecipitated with anti-myc antibody. The resulting precipitates and whole cell lysates used in immunoprecipitation (IP) were examined by immunoblotting using anti-NS2 or anti-SPCS1 antibody. (D) Expression of SPCS1-myc and its deletion mutants. 293T cells were transfected with indicated plasmids. The cell lysates were examined by immunoblotting using anti-myc or anti-actin antibody. (E) Cells were co-transfected with indicated plasmids, and then lysates of transfected cells were immunoprecipitated with anti-myc antibody. The resulting precipitates and whole cell lysates used in IP were examined by immunoblotting using anti-FLAG- or anti-myc antibody. (F) Lysates of the transfected cells were immunoprecipitated with anti-myc antibody. The resulting precipitates (right panel) and whole cell lysates used in IP (left panel) were examined by immunoblotting using anti-FLAG or anti-myc antibody. (G) 293T cells were transfected with indicated plasmids. 2 days posttransfection, cells were fixed and permeabilized with Triton X-100, then subjected to in situ PLA (Upper) or immunofluorescence staining (Lower) using anti-FLAG and anti-V5 antibodies. (H) Detection of the SPCS1-NS2 interaction in transfected cells using the mKG system. 293T cells were transfected by indicated pair of mKG fusion constructs. Twenty-four hours after transfection, cell were fixed and stained with DAPI, and observed under a confocal microscope.

Mentions: To determine the specific interaction of NS2 with SPCS1 in mammalian cells, FLAG-tagged NS2 (FLAG-NS2; Fig. 1A) was co-expressed in 293T cells with myc-tagged SPCS1 (SPCS1-myc; Fig. 1A), followed by co-immunoprecipitation and immunoblotting. SPCS1 was shown to be co-immunoprecipitated with NS2 (Fig. 1B). Co-immunoprecipitation of SPCS1-myc with NS2 was also observed in the lysate of Huh-7 cells infected with cell culture-produced HCV (HCVcc) derived from JFH-1 isolate [29] (Fig. 1C). To determine the region of SPCS1 responsible for the interaction with NS2, deletion mutants of myc-tagged SPCS1 were constructed (Fig. 1A) and co-expressed with FLAG-tagged NS2. Since the expression of C-terminal deletion mutants, d3 and d4, was difficult to detect (Fig. 1D), N-terminal deletions (d1 and d2) as well as wild-type SPCS1 were subjected to immunoprecipitation analysis. SPCS1-myc, -d1, and -d2 were co-immunoprecipitated with NS2 (Fig. 1E), suggesting that the SPCS1 region spanning amino acids (aa) 43 to 102 is involved in its interaction with NS2. Next, to identify the NS2 region responsible for its interaction with SPCS1, deletion mutants for FLAG-NS2 (Fig. 1A) were co-expressed with SPCS1-myc-d2 in cells, followed by being immunoprecipitated with anti-myc antibody. SPCS1 was co-immunoprecipitated with the NS2 deletions, except for a mutant lacking transmembrane (TM) 2 and TM3 (dTM23) domains (Fig. 1F). These finding suggests that the TM3 region of NS2 is involved in the interaction with SPCS1.


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)

Interaction of HCV NS2 protein with SPCS1 in mammalian cells.(A) Expression constructs of SPCS1-myc and FLAG-NS2 used in this study. TM regions are represented as gray. Myc-tag regions are depicted by the black circles. Gray circles and bold lines indicated FLAG-tag and spacer (GGGGS) sequences, respectively. Adaptive mutations are indicated as asterisks. Positions of the aa resides are indicated above the boxes. (B) 293T cells were co-transfected with a FLAG-tagged NS2 expression plasmid in the presence of a SPCS1-myc expression plasmid. Cell lysates of the transfected cells were immunoprecipitated with anti-myc antibody. The resulting precipitates and whole cell lysates used in immunoprecipitation (IP) were examined by immunoblotting using anti-FLAG- or anti-myc antibody. An empty plasmid was used as a negative control. (C) HCVcc infected Huh-7 cells were transfected with a SPCS1-myc expression plasmid. Cell lysates of the transfected cells were immunoprecipitated with anti-myc antibody. The resulting precipitates and whole cell lysates used in immunoprecipitation (IP) were examined by immunoblotting using anti-NS2 or anti-SPCS1 antibody. (D) Expression of SPCS1-myc and its deletion mutants. 293T cells were transfected with indicated plasmids. The cell lysates were examined by immunoblotting using anti-myc or anti-actin antibody. (E) Cells were co-transfected with indicated plasmids, and then lysates of transfected cells were immunoprecipitated with anti-myc antibody. The resulting precipitates and whole cell lysates used in IP were examined by immunoblotting using anti-FLAG- or anti-myc antibody. (F) Lysates of the transfected cells were immunoprecipitated with anti-myc antibody. The resulting precipitates (right panel) and whole cell lysates used in IP (left panel) were examined by immunoblotting using anti-FLAG or anti-myc antibody. (G) 293T cells were transfected with indicated plasmids. 2 days posttransfection, cells were fixed and permeabilized with Triton X-100, then subjected to in situ PLA (Upper) or immunofluorescence staining (Lower) using anti-FLAG and anti-V5 antibodies. (H) Detection of the SPCS1-NS2 interaction in transfected cells using the mKG system. 293T cells were transfected by indicated pair of mKG fusion constructs. Twenty-four hours after transfection, cell were fixed and stained with DAPI, and observed under a confocal microscope.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3757040&req=5

ppat-1003589-g001: Interaction of HCV NS2 protein with SPCS1 in mammalian cells.(A) Expression constructs of SPCS1-myc and FLAG-NS2 used in this study. TM regions are represented as gray. Myc-tag regions are depicted by the black circles. Gray circles and bold lines indicated FLAG-tag and spacer (GGGGS) sequences, respectively. Adaptive mutations are indicated as asterisks. Positions of the aa resides are indicated above the boxes. (B) 293T cells were co-transfected with a FLAG-tagged NS2 expression plasmid in the presence of a SPCS1-myc expression plasmid. Cell lysates of the transfected cells were immunoprecipitated with anti-myc antibody. The resulting precipitates and whole cell lysates used in immunoprecipitation (IP) were examined by immunoblotting using anti-FLAG- or anti-myc antibody. An empty plasmid was used as a negative control. (C) HCVcc infected Huh-7 cells were transfected with a SPCS1-myc expression plasmid. Cell lysates of the transfected cells were immunoprecipitated with anti-myc antibody. The resulting precipitates and whole cell lysates used in immunoprecipitation (IP) were examined by immunoblotting using anti-NS2 or anti-SPCS1 antibody. (D) Expression of SPCS1-myc and its deletion mutants. 293T cells were transfected with indicated plasmids. The cell lysates were examined by immunoblotting using anti-myc or anti-actin antibody. (E) Cells were co-transfected with indicated plasmids, and then lysates of transfected cells were immunoprecipitated with anti-myc antibody. The resulting precipitates and whole cell lysates used in IP were examined by immunoblotting using anti-FLAG- or anti-myc antibody. (F) Lysates of the transfected cells were immunoprecipitated with anti-myc antibody. The resulting precipitates (right panel) and whole cell lysates used in IP (left panel) were examined by immunoblotting using anti-FLAG or anti-myc antibody. (G) 293T cells were transfected with indicated plasmids. 2 days posttransfection, cells were fixed and permeabilized with Triton X-100, then subjected to in situ PLA (Upper) or immunofluorescence staining (Lower) using anti-FLAG and anti-V5 antibodies. (H) Detection of the SPCS1-NS2 interaction in transfected cells using the mKG system. 293T cells were transfected by indicated pair of mKG fusion constructs. Twenty-four hours after transfection, cell were fixed and stained with DAPI, and observed under a confocal microscope.
Mentions: To determine the specific interaction of NS2 with SPCS1 in mammalian cells, FLAG-tagged NS2 (FLAG-NS2; Fig. 1A) was co-expressed in 293T cells with myc-tagged SPCS1 (SPCS1-myc; Fig. 1A), followed by co-immunoprecipitation and immunoblotting. SPCS1 was shown to be co-immunoprecipitated with NS2 (Fig. 1B). Co-immunoprecipitation of SPCS1-myc with NS2 was also observed in the lysate of Huh-7 cells infected with cell culture-produced HCV (HCVcc) derived from JFH-1 isolate [29] (Fig. 1C). To determine the region of SPCS1 responsible for the interaction with NS2, deletion mutants of myc-tagged SPCS1 were constructed (Fig. 1A) and co-expressed with FLAG-tagged NS2. Since the expression of C-terminal deletion mutants, d3 and d4, was difficult to detect (Fig. 1D), N-terminal deletions (d1 and d2) as well as wild-type SPCS1 were subjected to immunoprecipitation analysis. SPCS1-myc, -d1, and -d2 were co-immunoprecipitated with NS2 (Fig. 1E), suggesting that the SPCS1 region spanning amino acids (aa) 43 to 102 is involved in its interaction with NS2. Next, to identify the NS2 region responsible for its interaction with SPCS1, deletion mutants for FLAG-NS2 (Fig. 1A) were co-expressed with SPCS1-myc-d2 in cells, followed by being immunoprecipitated with anti-myc antibody. SPCS1 was co-immunoprecipitated with the NS2 deletions, except for a mutant lacking transmembrane (TM) 2 and TM3 (dTM23) domains (Fig. 1F). These finding suggests that the TM3 region of NS2 is involved in the interaction with SPCS1.

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