Signal Peptidase Complex Subunit 1 Participates in the Assembly of Hepatitis C Virus through an Interaction with E2 and NS2

<div><p>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 <i>Flaviviridae</i> 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.</p></div

Effect of SPCS1 knockdown on the processing of HCV structural proteins and secretion of host proteins.

<p>(A) Core-NS2 polyprotein was expressed in KD#31 cells or parental Huh-7 cells. Core, NS2, SPCS1, and actin were detected by immunoblotting 2 days post-transfection. (B) Expression constructs of NS2 and NS2/3 proteins. His to Ala substitution mutation at aa 956 in NS2 is indicated by an asterisk. Gray circles and bold lines indicate FLAG-tag and the spacer sequences, respectively. Positions of the aa residues are indicated above the boxes. (C) Effect of SPCS1 knockdown on processing at the NS2/3 junction. Huh-7 cells were transfected with SPCS1 siRNA or control siRNA at a final concentration of 30 nM, and then transfected with plasmids for FLAG-NS2, F-NS2-3, or F-NS2-3 with a protease-inactive mutation (H956A). NS2 in cell lysates was detected by anti-FLAG antibody 2 days post-transfection. Arrowhead indicates unprocessed NS2-3 polyproteins. (D) Effect of SPCS1 knockdown on the secretion of apoE. Huh7.5.1 cells were transfected with SPCS1 siRNAs or control siRNA at a final concentration of 20 nM, and apoE in the supernatant and SPCS1 and actin in the cells were detected 3 days post-transfection. (E) Effect of SPCS1 knockdown on the secretion of albumin. Huh7.5.1 cells were transfected with SPCS1 siRNA or control siRNA, and albumin in the culture supernatants at 2 and 3 days post-transfection was measured by ELISA.</p

A proposed model for a complex consisting of NS2, SPCS1 and E2 associated with ER membranes.

<p>A proposed model for a complex consisting of NS2, SPCS1 and E2 associated with ER membranes.</p

Interaction of HCV NS2 protein with SPCS1 in mammalian cells.

<p>(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.</p

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.

<p>(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.</p

Effect of SPCS1 knockdown on the production of HCV.

<p>(A) Huh7.5.1 cells were transfected with four different siRNAs targeted for SPCS1 or control siRNA at a final concentration of 15 nM, and infected with HCVcc at a multiplicity of infection (MOI) of 0.05 at 24 h post-transfection. Expression levels of endogenous SPCS1 and actin in the cells were examined by immunoblotting using anti-SPCS1 and anti-actin antibodies at 3 days post-infection. (B) Infectious titers of HCVcc in the supernatant of cells infected as above were determined at 3 days postinfection. (C) Huh-7 cells were transfected with pSilencer-SPCS1, and hygromycin B-resistant cells were selected. The SPCS1-knockdown cell line established (KD#31) and parental Huh-7 cells were subjected to immunoblotting to confirm SPCS1 knockdown. (D) KD#31 cells or parental Huh-7 cells were transfected with RNA pol I-driven full-length HCV plasmid in the presence or absence of shRNA-resistant SPCS1 expression plasmid. Expression levels of SPCS1 and actin in the cells at 5 days post-transfection were examined by immunoblotting using anti-SPCS1 and anti-actin antibodies. (E) Infectious titers of HCVcc in the supernatants of transfected SPCS1-knockdown cells or parental Huh-7 cells at 5 days post-transfection were determined.</p

SPCS1 forms a complex with NS2 and E2.

<p>(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.</p

Flap endonuclease 1 is involved in cccDNA formation in the hepatitis B virus

<div><p>Hepatitis B virus (HBV) is one of the major etiological pathogens for liver cirrhosis and hepatocellular carcinoma. Chronic HBV infection is a key factor in these severe liver diseases. During infection, HBV forms a nuclear viral episome in the form of covalently closed circular DNA (cccDNA). Current therapies are not able to efficiently eliminate cccDNA from infected hepatocytes. cccDNA is a master template for viral replication that is formed by the conversion of its precursor, relaxed circular DNA (rcDNA). However, the host factors critical for cccDNA formation remain to be determined. Here, we assessed whether one potential host factor, flap structure-specific endonuclease 1 (FEN1), is involved in cleavage of the flap-like structure in rcDNA. In a cell culture HBV model (Hep38.7-Tet), expression and activity of FEN1 were reduced by siRNA, shRNA, CRISPR/Cas9-mediated genome editing, and a FEN1 inhibitor. These reductions in FEN1 expression and activity did not affect nucleocapsid DNA (NC-DNA) production, but did reduce cccDNA levels in Hep38.7-Tet cells. Exogenous overexpression of wild-type FEN1 rescued the reduced cccDNA production in FEN1-depleted Hep38.7-Tet cells. Anti-FEN1 immunoprecipitation revealed the binding of FEN1 to HBV DNA. An <i>in vitro</i> FEN activity assay demonstrated cleavage of 5′-flap from a synthesized HBV DNA substrate. Furthermore, cccDNA was generated <i>in vitro</i> when purified rcDNA was incubated with recombinant FEN1, DNA polymerase, and DNA ligase. Importantly, FEN1 was required for the <i>in vitro</i> cccDNA formation assay. These results demonstrate that FEN1 is involved in HBV cccDNA formation in cell culture system, and that FEN1, DNA polymerase, and ligase activities are sufficient to convert rcDNA into cccDNA <i>in vitro</i>.</p></div

The FEN1 inhibitor, PTPD, reduces cccDNA production.

<p>Effect of FEN1 inhibition on HBV-replicating cells. Hep38.7-Tet cells were treated with dimethylsulfoxide (DMSO) as a vehicle control, PTPD (5 μM), or 3TC (50 μM) in the absence of tetracycline for 5 days. At day 5, levels of HBV DNA, HBV RNA (pgRNA normalized by HPRT) and pre-C mRNA were analyzed. qPCR analysis of HBV DNA in (A) culture supernatant, (B) cytoplasmic NC-DNA, (C) cccDNA, and (D) pgRNA. Each result represents the mean ± SEM of three independent experiments. Asterisks indicate statistically significant differences; *<i>P</i> < 0.05, **<i>P</i> < 0.01, ***<i>P</i> < 0.001 compared with DMSO (A–D). (E) RT-PCR analysis for pre-C mRNA transcribed from cccDNA in Hep38.7-Tet cells. β-actin was used as a loading control.</p

FEN1 siRNA knockdown and CRISPR/Cas9-mediated gene editing reduce cccDNA production.

<p>(A–D) Hep38.7-Tet cells were transfected with <i>FEN1</i>-specific siRNA (siFEN1 #1 or #2) or control (siCtrl), and cultured without tetracycline. Four days after transfection, FEN1 mRNA/protein and HBV DNA were analyzed. (A) FEN1 mRNA quantified by RT-qPCR (normalized by HPRT) (upper panel) and Western blotting of FEN1 protein (lower panel). GAPDH expression is shown as a loading control. FEN1 protein expression gives rise to two bands in our study, which may be due to post-translational modification. (B–C) Levels of HBV DNA in cytoplasmic nucleocapsid (NC) (B) and cccDNA (C). (D) Efficiency of cccDNA formation (cccDNA levels normalized by cytoplasmic NC-DNA) was calculated. Each result represents the mean ± SEM of three independent experiments. Asterisks indicate statistically significant differences; *<i>P</i> < 0.05, **<i>P</i> < 0.01, ***<i>P</i> < 0.001 compared with siCtrl. (E–G) The pX330-FEN1 plasmid, which expresses Cas9 mRNA and sgRNA for exon 2 of the <i>FEN1</i> gene, was transfected with a blasticidin-resistant gene expression vector into Hep38.7-Tet cells. After drug selection, two resistant clones (#1 and #2) were analyzed. (E) RT-qPCR and Western blotting analyses of FEN1 expression levels. (F) Efficiency of cccDNA formation. Each result represents the mean ± SEM of three independent experiments. Asterisks indicate statistically significant differences; *<i>P</i> < 0.05, **<i>P</i> < 0.01 compared with parental groups. (G) Southern blot analysis of cytoplasmic NC-DNA and Hirt-extracted HBV DNA. Hirt-extracted HBV DNA was analyzed with or without heat treatment and subsequent EcoRI digestion. Densitometric analysis of the EcoRI-digested cccDNA signal is shown below the Southern blot images. The signal for parental cell line is taken as 100%.</p