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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 the propagation of JEV.Huh7.5.1 cells were transfected with SPCS1 siRNA or control siRNA at a final concentration of 10 nM, and infected with JEV or HCVcc at an MOI of 0.05 at 24 h post-transfection.(A) Infectious titers of HCVcc in the supernatant at 3 days post-infection were determined. (B) Infectious titers of JEV in the supernatant at indicated time points were determined. (C) Expression levels of endogenous SPCS1 and actin as well as viral proteins in the cells were determined by immunoblotting using anti-SPCS1, anti-actin, anti-HCV core, and anti-JEV antibodies 3 days post-infection.
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ppat-1003589-g003: Effect of SPCS1 knockdown on the propagation of JEV.Huh7.5.1 cells were transfected with SPCS1 siRNA or control siRNA at a final concentration of 10 nM, and infected with JEV or HCVcc at an MOI of 0.05 at 24 h post-transfection.(A) Infectious titers of HCVcc in the supernatant at 3 days post-infection were determined. (B) Infectious titers of JEV in the supernatant at indicated time points were determined. (C) Expression levels of endogenous SPCS1 and actin as well as viral proteins in the cells were determined by immunoblotting using anti-SPCS1, anti-actin, anti-HCV core, and anti-JEV antibodies 3 days post-infection.

Mentions: A typical feature of the Flaviviridae family is that their precursor polyprotein is processed into individual mature proteins mediated by host ER-resident peptidase(s) and viral-encoded protease(s). We therefore next examined the role of SPCS1 in the propagation of Japanese encephalitis virus (JEV), another member of the Flaviviridae family. SPCS1 siRNAs or control siRNA were transfected into Huh7.5.1 cells followed by infection with JEV or HCVcc. Although knockdown of SPCS1 severely impaired HCV production (Fig. 3A), the propagation of JEV was not affected under the SPCS1-knockdown condition (Fig. 3B). Expression of the viral proteins as well as knockdown of SPCS1 were confirmed (Fig. 3C). This suggests that SPCS1 is not a broadly active modulator of the flavivirus lifecycle, but rather is involved specifically in the production of certain virus(es) such as HCV.


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 the propagation of JEV.Huh7.5.1 cells were transfected with SPCS1 siRNA or control siRNA at a final concentration of 10 nM, and infected with JEV or HCVcc at an MOI of 0.05 at 24 h post-transfection.(A) Infectious titers of HCVcc in the supernatant at 3 days post-infection were determined. (B) Infectious titers of JEV in the supernatant at indicated time points were determined. (C) Expression levels of endogenous SPCS1 and actin as well as viral proteins in the cells were determined by immunoblotting using anti-SPCS1, anti-actin, anti-HCV core, and anti-JEV antibodies 3 days post-infection.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3757040&req=5

ppat-1003589-g003: Effect of SPCS1 knockdown on the propagation of JEV.Huh7.5.1 cells were transfected with SPCS1 siRNA or control siRNA at a final concentration of 10 nM, and infected with JEV or HCVcc at an MOI of 0.05 at 24 h post-transfection.(A) Infectious titers of HCVcc in the supernatant at 3 days post-infection were determined. (B) Infectious titers of JEV in the supernatant at indicated time points were determined. (C) Expression levels of endogenous SPCS1 and actin as well as viral proteins in the cells were determined by immunoblotting using anti-SPCS1, anti-actin, anti-HCV core, and anti-JEV antibodies 3 days post-infection.
Mentions: A typical feature of the Flaviviridae family is that their precursor polyprotein is processed into individual mature proteins mediated by host ER-resident peptidase(s) and viral-encoded protease(s). We therefore next examined the role of SPCS1 in the propagation of Japanese encephalitis virus (JEV), another member of the Flaviviridae family. SPCS1 siRNAs or control siRNA were transfected into Huh7.5.1 cells followed by infection with JEV or HCVcc. Although knockdown of SPCS1 severely impaired HCV production (Fig. 3A), the propagation of JEV was not affected under the SPCS1-knockdown condition (Fig. 3B). Expression of the viral proteins as well as knockdown of SPCS1 were confirmed (Fig. 3C). This suggests that SPCS1 is not a broadly active modulator of the flavivirus lifecycle, but rather is involved specifically in the production of certain virus(es) such as HCV.

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