Journal: Viruses

Article Title: A Dengue Virus Type 2 (DENV-2) NS4B-Interacting Host Factor, SERP1, Reduces DENV-2 Production by Suppressing Viral RNA Replication

doi: 10.3390/v11090787

Figure Lengend Snippet: Protein–protein interactions between stress-associated endoplasmic reticulum protein 1 (SERP1) and nonstructural protein (NS)4B. ( A ) NS4B interacts with SERP1, as shown in a membrane-base split ubiquitin yeast-two-hybrid assay. Yeast was co-transformed with the baits p-BT3 NS2B, p-BT3 NS4A, p-BT3 NS4B, and p-BT3 (vector only), and the prey pPR3-SERP1. Ten-fold serial yeast dilutions were spotted onto nonselective plates (-Trp–Leu; lacking tryptophan and leucine) and selective plates (-Trp–Leu–His–Ade; lacking tryptophan, leucine, histidine, and adenine) for the detection of protein–protein interactions. ( B ) Dengue virus type 2 (DENV-2) NS4B colocalized with SERP1 and the endoplasmic reticulum (ER) marker calnexin. The Flag-SERP1-overexpressing Huh7.5 cells were infected with DENV-2 (multiplicity of infection (MOI) = 5) and subjected to co-stain with antibodies raised against Flag, NS4B, or ER-located calnexin. At 72 h post-infection, the subcellular distributions of SERP1, NS4B, or calnexin were examined by indirect immunofluorescence assay using the corresponding antibodies. NS4B and SERP1 showed a strong colocalization (upper panel). ER-located calnexin and SERP1 (middle panel) and NS4B (lower panel) also showed colocalization. The nuclei were stained with DAPI (blue). Scale bar, 25 μm. ( C ) The colocalization analysis between SERP1, NS4B, and calnexin was quantified using Pearson’s correlation coefficients. Each counterstain was determined for 10 Huh7.5 cells in three independent experiments using the colocalization tool provided, with Leica-SP5 software. The values are shown as the average of the Pearson’s correlation coefficients in 10 cells. Error bars indicate the means ± standard errors of the mean (SEMs). ( D ) The topological scheme of the SERP1, NS4B, and NS2B complexes with selected interaction pairs in a Nano-Luc Binary Technology (NanoBiT) protein–protein interactions (PPIs) assay. ( E ) The interactions of SEPR1-NS4B and SEPR1-NS2B were determined in the HEK-293 cells using a NanoBiT complementation assay. Luminescence is expressed as the means ± SEMs from three independent experiments ( n = 3). ***, p < 0.001 (Student’s t -test). ( F ) Schematic diagram of the NS2B-HA (pLKO-AS2-NS2B-HA), NS4B-HA (pLKO-AS2-NS4B-HA), and Flag-SERP1 (pLKO-AS2-Flag-SERP1) fusion constructs. ( G ) The interaction of SEPR1-NS4B was determined in the Huh7.5 cells by co-immunoprecipitation (co-IP). The Huh7.5 cells were transfected or co-transfected with pLKO-AS2-Flag-SERP1, pLKO-AS2-NS2B-HA, and pLKO-AS2-NS4B-HA. Equal amounts of protein complexes were analyzed by immunoprecipitation using anti-Flag M2 affinity gel or anti-HA magnetic beads. The co-IP was performed with the anti-Flag antibody and anti-HA antibody.

Article Snippet: To further measure the protein level of SERP1 in the DENV-2 infected cells, we failed to detect the SERP1 protein expression in virus infected cells by either in-house mouse, rabbit, or commercial anti-SERP1 antibodies (Genetex, Abcam, Proteintech Group, Atlas antibodies), possibly because of the poor antigenicity of SERP1 (data not shown).

Techniques: Protein-Protein interactions, Membrane, Ubiquitin Proteomics, Y2H Assay, Transformation Assay, Plasmid Preparation, Virus, Marker, Infection, Staining, Immunofluorescence, Software, Construct, Immunoprecipitation, Co-Immunoprecipitation Assay, Transfection, Magnetic Beads

Journal: Viruses

Article Title: A Dengue Virus Type 2 (DENV-2) NS4B-Interacting Host Factor, SERP1, Reduces DENV-2 Production by Suppressing Viral RNA Replication

doi: 10.3390/v11090787

Figure Lengend Snippet: SERP1 expressions were induced in Huh7.5 cells upon DENV-2 infection and wild-type (WT) replicon transfection. ( A ) The Huh7.5 cells were uninfected or infected with DENV-2 at an MOI = 1. The quantification of SERP1 transcripts was performed by RT-qPCR at 0, 1, 2, 3, 4, and 5 d.p.i. The relative quantitative values of the SERP1 gene were normalized to the level of β-actin. *, p < 0.05; ***, p < 0.001 (Student’s t -test). ( B ) Schematic representation of the DNA-launched DENV-2 reporter replicon pCMV-DV2Rep, which was used in a transient replicon assay. The transcriptional expression of the replicon RNA is under the control of the CMVmin promoter, and the 3′ terminus of the transcript is processed by hepatitis delta virus (HDV) ribozyme sequences. The N-terminal 102 amino acids of the C protein (C102), the Renilla luciferase gene (Rluc), the FMDV2A cleavage site, a neomycin resistance gene (Neo), an internal ribosome entry site (IRES) element, the C-terminal 24 amino acids of E (E24), the entire NS protein region (NS1 to NS5), the HDV ribozyme sequence, and the SV40 poly(A) signal sequence are indicated. The star represents the replication-defective mutant—a replicon with an inactivating mutation (Gly–Asp–Asp (GDD) to Gly–Ala–Ala (GAA) at the amino acids 662–664 of NS5) in the catalytic site of the NS5 RNA-dependent RNA polymerase (RdRp) was used as a negative control. ( C ) The Huh7.5 cells were transfected with the WT replicon or mutant replicon. The quantification of the SERP1 transcripts was performed by RT-qPCR at 0, 1, 2, 3, and 4 d.p.t. The relative quantitative values of the SERP1 gene were normalized to the level of β-actin. **, p < 0.01; ***, p < 0.001 (Student’s t -test).

Article Snippet: To further measure the protein level of SERP1 in the DENV-2 infected cells, we failed to detect the SERP1 protein expression in virus infected cells by either in-house mouse, rabbit, or commercial anti-SERP1 antibodies (Genetex, Abcam, Proteintech Group, Atlas antibodies), possibly because of the poor antigenicity of SERP1 (data not shown).

Techniques: Infection, Transfection, Quantitative RT-PCR, Expressing, Control, Virus, Luciferase, Sequencing, Mutagenesis, Negative Control

Journal: Viruses

Article Title: A Dengue Virus Type 2 (DENV-2) NS4B-Interacting Host Factor, SERP1, Reduces DENV-2 Production by Suppressing Viral RNA Replication

doi: 10.3390/v11090787

Figure Lengend Snippet: The SERP1 overexpression inhibited DENV-2 infection, and SERP1 knockdown increased DENV-2 infection. ( A ) Western blot analysis of Flag-tagged SERP1 proteins in Huh7.5 cells expressing exogenous SERP1 and empty-vector cells. Equal amounts of lysates were incubated with anti-Flag M2 affinity gel, and the precipitates were analyzed by Western blot using the anti-Flag antibody and anti-SERP1 antibody. ( B ) The reduction in DENV-2 yields in SERP1-overexpressing cells infected with DENV-2. We established Huh7.5 cells with a stable expression of SERP1 and empty-vector cells. The cells were infected with DENV-2 at MOI = 1. The virus yields were determined at the indicated times. Infectious virus yields in the BHK21 clone 15 cells were quantified by plaque assay. Tde differences in the virus yields between the Huh7.5 cells stably expressing SERP1 and empty-vector cells at 3 or 4 d.p.i. were analyzed using Student’s t -test. *, p < 0.05. ( C ) A RT-qPCR analysis of the SERP1 mRNA expression in the empty vector HEK-293 cells and knockdown cells. shRNA-mediated knockdown of SERP1 reduces the mRNA expression in the HEK-293 uninfected cells. The expression values were normalized to the β-actin expression. The values are the means ± standard errors of the means (SEMs). ***, p < 0.001 (Student’s t -test). ( D ) The knockdown of the SERP1 cells increased the viral yields of DENV-2. We established HEK-293 cells with a SERP1 knockdown by a specific shRNA. The HEK-293 cells stably expressing shSEPR1 were infected with DENV-2 at MOI = 1. The virus yields were determined at 3 d.p.i.

Article Snippet: To further measure the protein level of SERP1 in the DENV-2 infected cells, we failed to detect the SERP1 protein expression in virus infected cells by either in-house mouse, rabbit, or commercial anti-SERP1 antibodies (Genetex, Abcam, Proteintech Group, Atlas antibodies), possibly because of the poor antigenicity of SERP1 (data not shown).

Techniques: Over Expression, Infection, Knockdown, Western Blot, Expressing, Plasmid Preparation, Incubation, Virus, Plaque Assay, Stable Transfection, Quantitative RT-PCR, shRNA

Journal: Viruses

Article Title: A Dengue Virus Type 2 (DENV-2) NS4B-Interacting Host Factor, SERP1, Reduces DENV-2 Production by Suppressing Viral RNA Replication

doi: 10.3390/v11090787

Figure Lengend Snippet: Knockout of SERP1 by the II clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system in Huh7.5 cells decreased SERP1 mRNA levels, and viral yields were significantly enhanced in the SERP1 knockout cells. ( A ) SERP1 RNA expression patterns in the SERP1 knockout sublines. The products of RT-PCR performed on RNA isolated from the parental Huh7.5 cells (SERP1 +/+ ) and SERP1 knockout sublines (SERP1 +/− and SERP1 −/− ) using the primers in SERP1 exon 1 and exon 3, which generate a 697 bp product. The SERP1 mutant allele was amplified as a 563 bp product, where the SERP1 exon 1 region was deleted. ( B ) Sequencing analysis of the parental Huh7.5 cells and SERP1 knockout cell lines from the RT-PCR product. ( C ) Detection of SERP1 mRNA levels in the parental Huh7.5 cells and SERP1 knockout sublines by qRT-PCR. The expression values are normalized to β-actin expression. ***, p < 0.001 (Student’s t -test). ( D ) Kinetics of DENV-2 replication in the parental cells and SERP1 knockout sublines. The parental Huh7.5 cells and SERP1 knockout sublines were infected with DENV-2 (MOI = 1) at the indicated times. Infectious virus yield in the BHK21 clone 15 cells was quantified by plaque assay. The differences in virus yields between the parental cells (SERP1 +/+ ) and SERP1 knockout cells (SERP1 −/− ) at 2 or 3 d.p.i. were analyzed using Student’s t -test. *, p < 0.05.

Article Snippet: To further measure the protein level of SERP1 in the DENV-2 infected cells, we failed to detect the SERP1 protein expression in virus infected cells by either in-house mouse, rabbit, or commercial anti-SERP1 antibodies (Genetex, Abcam, Proteintech Group, Atlas antibodies), possibly because of the poor antigenicity of SERP1 (data not shown).

Techniques: Knock-Out, CRISPR, RNA Expression, Reverse Transcription Polymerase Chain Reaction, Isolation, Mutagenesis, Amplification, Sequencing, Quantitative RT-PCR, Expressing, Infection, Virus, Plaque Assay

Journal: Viruses

Article Title: A Dengue Virus Type 2 (DENV-2) NS4B-Interacting Host Factor, SERP1, Reduces DENV-2 Production by Suppressing Viral RNA Replication

doi: 10.3390/v11090787

Figure Lengend Snippet: The effect of SERP1 on the DENV-2 replicon capacity in the Huh7.5 cells. The replication activities of the transient expression of DNA-launched mutant replicon ( A ) or WT replicon ( B ) plasmids were detected in the parental cells, stable cells overexpressing SERP1, and SERP1 knockout cells. The luciferase activity of the transfected cells was measured at 1, 2, 3, and 4 d.p.t. The error bars represent the SEMs from three independent experiments. The differences in the luciferase activity between the transfected cells at 3 or 4 d.p.t. were analyzed using Student’s t-test. ***, p < 0.001 (relative to the parental cells).

Article Snippet: To further measure the protein level of SERP1 in the DENV-2 infected cells, we failed to detect the SERP1 protein expression in virus infected cells by either in-house mouse, rabbit, or commercial anti-SERP1 antibodies (Genetex, Abcam, Proteintech Group, Atlas antibodies), possibly because of the poor antigenicity of SERP1 (data not shown).

Techniques: Expressing, Mutagenesis, Knock-Out, Luciferase, Activity Assay, Transfection

Journal: Viruses

Article Title: A Dengue Virus Type 2 (DENV-2) NS4B-Interacting Host Factor, SERP1, Reduces DENV-2 Production by Suppressing Viral RNA Replication

doi: 10.3390/v11090787

Figure Lengend Snippet: Overexpression of NS4B improves the virus replication in the Huh7.5 cells overexpressing SERP1. ( A ) Schematic diagram of HA C-terminal fusion constructs of NS2B and NS4B. The N-terminal 2K signal peptide was deleted (△2K-NS4B-HA). ( B ) Immunoblot analysis of HA-tagged NS2B and NS4B proteins in the parental cells and SERP1-overexpressing Huh7.5 cells. All of the fragments represented in panel ( A ) were cloned in pLKO-AS2 to tag the C-terminal end of each protein. Equal amounts of lysates were incubated with anti-HA magnetic beads, and the precipitates were analyzed by Western blot using an anti-HA antibody. The parental cells ( C ) and SERP1-overexpressing Huh7.5 cells ( D ) were co-transfected with WT replicon and plasmids encoding individual viral proteins, as indicated. The cell lysates were harvested 24, 48, 72, and 96 h after transfection, and the luciferase activity of the transfected cells was measured. The replication efficiency was calculated by determining the ratio of luciferase activity obtained at 48, 72, and 96 h, to the average value obtained from all of the replicon constructs at 24 h post-transfection. The error bars represent the standard errors of the means (SEMs) from three independent experiments. *, p < 0.05; ***, p < 0.001 (Student’s t -test).

Article Snippet: To further measure the protein level of SERP1 in the DENV-2 infected cells, we failed to detect the SERP1 protein expression in virus infected cells by either in-house mouse, rabbit, or commercial anti-SERP1 antibodies (Genetex, Abcam, Proteintech Group, Atlas antibodies), possibly because of the poor antigenicity of SERP1 (data not shown).

Techniques: Over Expression, Virus, Construct, Western Blot, Clone Assay, Incubation, Magnetic Beads, Transfection, Luciferase, Activity Assay

Journal: Viruses

Article Title: A Dengue Virus Type 2 (DENV-2) NS4B-Interacting Host Factor, SERP1, Reduces DENV-2 Production by Suppressing Viral RNA Replication

doi: 10.3390/v11090787

Figure Lengend Snippet: Hypothetical model of SERP1-mediated DENV-2 infection. ( A ) SERP1 overexpression inhibits DENV-2 viral RNA replication and titers. ( B ) DENV-2 NS4B interacts with SERP1. DENV-2 NS4B may alleviate the inhibitory effect of SERP1 on DENV-2 viral replication via the interaction of NS4B with SERP1.

Article Snippet: To further measure the protein level of SERP1 in the DENV-2 infected cells, we failed to detect the SERP1 protein expression in virus infected cells by either in-house mouse, rabbit, or commercial anti-SERP1 antibodies (Genetex, Abcam, Proteintech Group, Atlas antibodies), possibly because of the poor antigenicity of SERP1 (data not shown).

Techniques: Infection, Over Expression

Journal: Molecular Medicine Reports

Article Title: SERP1 reduces inchoate acute hepatic injury through regulation of endoplasmic reticulum stress via the GSK3β/β-catenin/TCF/LEF signaling pathway

doi: 10.3892/mmr.2022.12709

Figure Lengend Snippet: Establishment of an inchoate acute hepatic injury model and detection of ERS-related proteins. (A) Pathological changes of tissues of mice in the model group detected by H&E staining. (B) Levels of alanine aminotransferase and aspartate aminotransferase in the serum. (C) ERS-related protein expression detected by western blotting. SERP1 expression in acute hepatic injury tissues detected by (D) immunohistochemistry and (E) western blotting. (F) SERP1 expression in acute hepatic injury cells detected by western blotting and reverse transcription-quantitative PCR. *P<0.05, **P<0.01 and ***P<0.001 vs. the control; n≥3. ERS, endoplasmic reticulum stress; SERP1, stress-associated endoplasmic reticulum protein 1; ALT, alanine aminotransferase; AST, aspartate aminotransferase; LPS, lipopolysaccharide; D-GalN, D-galactosamine; GRP78, glucose-regulated protein 78; GRP94, glucose-regulated protein 94.

Article Snippet: Subsequently, paraffin sections were washed with xylene, rehydrated with descending alcohol and antigen retrieval was performed at 95°C for 20 min. Then, 5% goat serum (Beijing Solarbio Technology Co. Ltd.) was used for blocking for 10 min at room temperature and sections were then incubated with the SERP1 primary antibody (1:200; cat. no. DF9873; Affinity Biosciences) at 4°C overnight.

Techniques: Staining, Expressing, Western Blot, Immunohistochemistry, Reverse Transcription, Real-time Polymerase Chain Reaction, Control

Journal: Molecular Medicine Reports

Article Title: SERP1 reduces inchoate acute hepatic injury through regulation of endoplasmic reticulum stress via the GSK3β/β-catenin/TCF/LEF signaling pathway

doi: 10.3892/mmr.2022.12709

Figure Lengend Snippet: Effect of SERP1 overexpression on the expression of inflammatory factors in hepatocytes. Expression levels of SERP1 following transfection with SERP1 plasmid detected by (A) western blotting and (B) reverse transcription-quantitative PCR. **P<0.01, ***P<0.001 vs. OE-NC. (C) Expression levels of inflammatory factors (TNF-α, IL-18, IL-6 and IL-1β) detected by ELISA. (D) NLRP3 inflammasome (NLRP3, ASC and caspase-1) expression detected by western blotting. ***P<0.001 vs. the control; # P<0.05, ### P<0.001 vs. LPS + NC; Δ P<0.05, ΔΔ P<0.01 vs. LPS + SERP1; n≥3. SERP1, stress-associated endoplasmic reticulum protein 1; OE, overexpression; NC, negative control; NLRP3, NLR family pyrin domain containing 3; ASC, apoptosis-associated speck-like protein containing a CARD; LPS, lipopolysaccharide.

Article Snippet: Subsequently, paraffin sections were washed with xylene, rehydrated with descending alcohol and antigen retrieval was performed at 95°C for 20 min. Then, 5% goat serum (Beijing Solarbio Technology Co. Ltd.) was used for blocking for 10 min at room temperature and sections were then incubated with the SERP1 primary antibody (1:200; cat. no. DF9873; Affinity Biosciences) at 4°C overnight.

Techniques: Over Expression, Expressing, Transfection, Plasmid Preparation, Western Blot, Reverse Transcription, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Control, Negative Control

Journal: Molecular Medicine Reports

Article Title: SERP1 reduces inchoate acute hepatic injury through regulation of endoplasmic reticulum stress via the GSK3β/β-catenin/TCF/LEF signaling pathway

doi: 10.3892/mmr.2022.12709

Figure Lengend Snippet: Effect of SERP1 overexpression on apoptosis of hepatocytes. (A) A TUNEL assay was employed to detect cell apoptosis. (B) Apoptosis-related protein expression was detected by western blotting. ***P<0.001 vs. the control; # P<0.05, ## P<0.01, ### P<0.001 vs. LPS + NC; Δ P<0.05 vs. LPS + SERP1; n≥3. SERP1, stress-associated endoplasmic reticulum protein 1; LPS, lipopolysaccharide; NC, negative control.

Article Snippet: Subsequently, paraffin sections were washed with xylene, rehydrated with descending alcohol and antigen retrieval was performed at 95°C for 20 min. Then, 5% goat serum (Beijing Solarbio Technology Co. Ltd.) was used for blocking for 10 min at room temperature and sections were then incubated with the SERP1 primary antibody (1:200; cat. no. DF9873; Affinity Biosciences) at 4°C overnight.

Techniques: Over Expression, TUNEL Assay, Expressing, Western Blot, Control, Negative Control

Journal: Molecular Medicine Reports

Article Title: SERP1 reduces inchoate acute hepatic injury through regulation of endoplasmic reticulum stress via the GSK3β/β-catenin/TCF/LEF signaling pathway

doi: 10.3892/mmr.2022.12709

Figure Lengend Snippet: Effect of SERP1 overexpression on endoplasmic reticulum stress-related protein expression and GSK3β/β-catenin/TCF/LEF signaling in hepatocytes. (A) Expression levels of GRP78, GRP94 and CHOP in hepatocytes detected by western blotting. (B) TOP Flash/FOP Flash fluorescent gene reporter assay to detect TCF/LEF activity. (C) GSK3β/β-catenin expression was detected by western blotting. ***P<0.001 vs. the control; # P<0.05, ## P<0.01, ### P<0.001 vs. LPS + NC; ΔΔΔ P<0.001 vs. LPS + SERP1; n≥3. SERP1, stress-associated endoplasmic reticulum protein 1; GRP78, glucose-regulated protein 78; GRP94, glucose-regulated protein 94; TCF, T-cell factor; LEF, lymphoid enhancing factor; LPS, lipopolysaccharide; NC, negative control.

Article Snippet: Subsequently, paraffin sections were washed with xylene, rehydrated with descending alcohol and antigen retrieval was performed at 95°C for 20 min. Then, 5% goat serum (Beijing Solarbio Technology Co. Ltd.) was used for blocking for 10 min at room temperature and sections were then incubated with the SERP1 primary antibody (1:200; cat. no. DF9873; Affinity Biosciences) at 4°C overnight.

Techniques: Over Expression, Expressing, Western Blot, Reporter Assay, Activity Assay, Control, Negative Control

Journal: Molecular Medicine Reports

Article Title: SERP1 reduces inchoate acute hepatic injury through regulation of endoplasmic reticulum stress via the GSK3β/β-catenin/TCF/LEF signaling pathway

doi: 10.3892/mmr.2022.12709

Figure Lengend Snippet: GSK3β/β-catenin signaling activation reverses the effect of SERP1 overexpression on ERS and cell apoptosis. (A) Overexpression was verified by reverse transcription-quantitative PCR and western blotting. ***P<0.001 vs. OE-NC; n≥3. (B) TCF/LEF activity was detected using a luciferase assay. (C) GSK3β/β-catenin expression was detected by western blotting. (D) Western blotting was applied to detect ERS-related protein expression. (E) A TUNEL assay was employed to detect cell apoptosis. (F) Apoptosis-related protein expression was detected by western blotting. *P<0.05, **P<0.01, ***P<0.001 vs. LPS; # P<0.05, ## P<0.01, ### P<0.001 vs. LPS + SERP1 + NC; n≥3. SERP1, stress-associated endoplasmic reticulum protein 1; ERS, endoplasmic reticulum stress; OE, overexpression; TCF, T-cell factor; LEF, lymphoid enhancing factor; LPS, lipopolysaccharide; NC, negative control.

Article Snippet: Subsequently, paraffin sections were washed with xylene, rehydrated with descending alcohol and antigen retrieval was performed at 95°C for 20 min. Then, 5% goat serum (Beijing Solarbio Technology Co. Ltd.) was used for blocking for 10 min at room temperature and sections were then incubated with the SERP1 primary antibody (1:200; cat. no. DF9873; Affinity Biosciences) at 4°C overnight.

Techniques: Activation Assay, Over Expression, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot, Activity Assay, Luciferase, Expressing, TUNEL Assay, Negative Control