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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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Effect of SPCS1 knockdown on entry into cells, genome replication, and assembly or release of infectious virus.(A) Huh7.5.1 cells were transfected with siRNA for SPCS1 or claudin1, or control siRNA at a final concentration of 30 nM. Expression levels of endogenous SPCS1, claudin-1, and actin in the cells at 2 days post-transfection were examined by immunoblotting using anti-SPCS1, anti-actin, and anti-claudin-1 antibodies. (B) Huh7.5.1 cells transfected with indicated siRNAs were infected with HCVtcp at 2 days post-transfection. Luciferase activity in the cells was subsequently determined at 2 days post-infection. Data are averages of triplicate values with error bars showing standard deviations. (C) Effect of SPCS1 knockdown on replication of HCV genome. HCV-infected Huh-7 cells transfected with siRNA for SPCS1, PI4K or claudin1, or control siRNA at a final concentration of 30 nM. Expression levels of HCV proteins as well as endogenous SPCS1, PI4K, claudin-1, and actin in the cells at 3 days post-transfection were examined by immunoblotting. (D) HCV infectivity in Huh7.5.1 cells inoculated with culture supernatant and cell lysate from Huh7-25 cells transfected with pSilencer-SPCS1 or control vector along with pHH/JFH1am at 5 days post-transfection. Statistical differences between Control and SPCS1 knockdown were evaluated using Student's t-test. *p<0.005 vs. Control.
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ppat-1003589-g005: Effect of SPCS1 knockdown on entry into cells, genome replication, and assembly or release of infectious virus.(A) Huh7.5.1 cells were transfected with siRNA for SPCS1 or claudin1, or control siRNA at a final concentration of 30 nM. Expression levels of endogenous SPCS1, claudin-1, and actin in the cells at 2 days post-transfection were examined by immunoblotting using anti-SPCS1, anti-actin, and anti-claudin-1 antibodies. (B) Huh7.5.1 cells transfected with indicated siRNAs were infected with HCVtcp at 2 days post-transfection. Luciferase activity in the cells was subsequently determined at 2 days post-infection. Data are averages of triplicate values with error bars showing standard deviations. (C) Effect of SPCS1 knockdown on replication of HCV genome. HCV-infected Huh-7 cells transfected with siRNA for SPCS1, PI4K or claudin1, or control siRNA at a final concentration of 30 nM. Expression levels of HCV proteins as well as endogenous SPCS1, PI4K, claudin-1, and actin in the cells at 3 days post-transfection were examined by immunoblotting. (D) HCV infectivity in Huh7.5.1 cells inoculated with culture supernatant and cell lysate from Huh7-25 cells transfected with pSilencer-SPCS1 or control vector along with pHH/JFH1am at 5 days post-transfection. Statistical differences between Control and SPCS1 knockdown were evaluated using Student's t-test. *p<0.005 vs. Control.

Mentions: To further address the molecular mechanism(s) of the HCV lifecycle mediated by SPCS1, we examined the effect of SPCS1 knockdown on viral entry and genome replication using single-round infectious trans-complemented HCV particles (HCVtcp) [33], of which the packaged genome is a subgenomic replicon containing a luciferase reporter gene. This assay system allows us to evaluate viral entry and replication without the influence of reinfection. Despite efficient knockdown of SPCS1 (Fig. 5A), luciferase activity expressed from HCVtcp in SPCS1-knockdown cells was comparable to that in control or non-siRNA-transfected cells (Fig. 5B), suggesting that SPCS1 is not involved in viral entry into cells and subgenomic RNA replication. As a positive control, knockdown of claudin-1, a cell surface protein required for HCV entry, reduced the luciferase activity. We also examined the effect of SPCS1 knockdown on full-genome replication using HCVcc-infected cells. Despite efficient knockdown of SPCS1, expression of HCV proteins was comparable to that in control cells (Fig. 5C). By contrast, knockdown of PI4 Kinase (PI4K), which is required for replication of HCV genome, led to decrease in expression of HCV proteins. As cells that had already been infected with HCV were used, knockdown of claudin-1 had no effect on HCV protein levels. These data suggest that SPCS1 is not involved in viral entry into cells and the viral genome replication. We also observed properly processed Core, E2, NS2 and NS5B in SPCS1-knockdown cells in consistent with the result as shown in Fig. 4A, indicating no effect of SPCS1 on HCV polyprotein processing.


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)

Effect of SPCS1 knockdown on entry into cells, genome replication, and assembly or release of infectious virus.(A) Huh7.5.1 cells were transfected with siRNA for SPCS1 or claudin1, or control siRNA at a final concentration of 30 nM. Expression levels of endogenous SPCS1, claudin-1, and actin in the cells at 2 days post-transfection were examined by immunoblotting using anti-SPCS1, anti-actin, and anti-claudin-1 antibodies. (B) Huh7.5.1 cells transfected with indicated siRNAs were infected with HCVtcp at 2 days post-transfection. Luciferase activity in the cells was subsequently determined at 2 days post-infection. Data are averages of triplicate values with error bars showing standard deviations. (C) Effect of SPCS1 knockdown on replication of HCV genome. HCV-infected Huh-7 cells transfected with siRNA for SPCS1, PI4K or claudin1, or control siRNA at a final concentration of 30 nM. Expression levels of HCV proteins as well as endogenous SPCS1, PI4K, claudin-1, and actin in the cells at 3 days post-transfection were examined by immunoblotting. (D) HCV infectivity in Huh7.5.1 cells inoculated with culture supernatant and cell lysate from Huh7-25 cells transfected with pSilencer-SPCS1 or control vector along with pHH/JFH1am at 5 days post-transfection. Statistical differences between Control and SPCS1 knockdown were evaluated using Student's t-test. *p<0.005 vs. Control.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3757040&req=5

ppat-1003589-g005: Effect of SPCS1 knockdown on entry into cells, genome replication, and assembly or release of infectious virus.(A) Huh7.5.1 cells were transfected with siRNA for SPCS1 or claudin1, or control siRNA at a final concentration of 30 nM. Expression levels of endogenous SPCS1, claudin-1, and actin in the cells at 2 days post-transfection were examined by immunoblotting using anti-SPCS1, anti-actin, and anti-claudin-1 antibodies. (B) Huh7.5.1 cells transfected with indicated siRNAs were infected with HCVtcp at 2 days post-transfection. Luciferase activity in the cells was subsequently determined at 2 days post-infection. Data are averages of triplicate values with error bars showing standard deviations. (C) Effect of SPCS1 knockdown on replication of HCV genome. HCV-infected Huh-7 cells transfected with siRNA for SPCS1, PI4K or claudin1, or control siRNA at a final concentration of 30 nM. Expression levels of HCV proteins as well as endogenous SPCS1, PI4K, claudin-1, and actin in the cells at 3 days post-transfection were examined by immunoblotting. (D) HCV infectivity in Huh7.5.1 cells inoculated with culture supernatant and cell lysate from Huh7-25 cells transfected with pSilencer-SPCS1 or control vector along with pHH/JFH1am at 5 days post-transfection. Statistical differences between Control and SPCS1 knockdown were evaluated using Student's t-test. *p<0.005 vs. Control.
Mentions: To further address the molecular mechanism(s) of the HCV lifecycle mediated by SPCS1, we examined the effect of SPCS1 knockdown on viral entry and genome replication using single-round infectious trans-complemented HCV particles (HCVtcp) [33], of which the packaged genome is a subgenomic replicon containing a luciferase reporter gene. This assay system allows us to evaluate viral entry and replication without the influence of reinfection. Despite efficient knockdown of SPCS1 (Fig. 5A), luciferase activity expressed from HCVtcp in SPCS1-knockdown cells was comparable to that in control or non-siRNA-transfected cells (Fig. 5B), suggesting that SPCS1 is not involved in viral entry into cells and subgenomic RNA replication. As a positive control, knockdown of claudin-1, a cell surface protein required for HCV entry, reduced the luciferase activity. We also examined the effect of SPCS1 knockdown on full-genome replication using HCVcc-infected cells. Despite efficient knockdown of SPCS1, expression of HCV proteins was comparable to that in control cells (Fig. 5C). By contrast, knockdown of PI4 Kinase (PI4K), which is required for replication of HCV genome, led to decrease in expression of HCV proteins. As cells that had already been infected with HCV were used, knockdown of claudin-1 had no effect on HCV protein levels. These data suggest that SPCS1 is not involved in viral entry into cells and the viral genome replication. We also observed properly processed Core, E2, NS2 and NS5B in SPCS1-knockdown cells in consistent with the result as shown in Fig. 4A, indicating no effect of SPCS1 on HCV polyprotein processing.

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