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  • 93
    Sino Biological his tagged recombinant np
    His Tagged Recombinant Np, supplied by Sino Biological, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ATCC htnv strain
    Htnv Strain, supplied by ATCC, used in various techniques. Bioz Stars score: 96/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Progen Biotechnik mouse anti n protein htnv
    Mouse Anti N Protein Htnv, supplied by Progen Biotechnik, used in various techniques. Bioz Stars score: 93/100, based on 23 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    BEI Resources anti htnv gn mouse mab 3d5
    Anti Htnv Gn Mouse Mab 3d5, supplied by BEI Resources, used in various techniques. Bioz Stars score: 93/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Austral Biologicals anti htnv gn mab 1f5 g2
    Anti Htnv Gn Mab 1f5 G2, supplied by Austral Biologicals, used in various techniques. Bioz Stars score: 93/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher htnv np
    Htnv Np, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    bei resources anti htnv gc mab 3g1
    Anti Htnv Gc Mab 3g1, supplied by bei resources, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam monoclonal antibody against htnv 76 118
    HTNV induces TRAIL-dependent apoptosis in primary HUVECs. HUVECs were infected with/without HTNV 76-118 (MOI = 1) for 2 h and followed by qRT-PCR and Western blot at different days to measure TRAIL-related apoptosis factors.  (A)  HTNV S, TRAIL, DcR1, DcR2, caspase-8, and caspase-3 mRNA in HTNV-infected HUVECs.  (B)  HTNV NP and TRAIL-related apoptosis proteins expression in HTNV-infected HUVECs.  (C)  TRAIL-DR4/DR5 and  (D)  caspase-9 mRNA and protein expression during HTNV infection. The mRNA results shown are the average of three replicates; values represent the mean ± SD ( * p
    Monoclonal Antibody Against Htnv 76 118, supplied by Abcam, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abnova monoclonal anti htnv 76 118 strain antibody
    Identification of dysregulated mRNAs.  (A)  Heatmap and clustering analysis of DE mRNAs. Each row represents one mRNA and each column represents one sample; −2, −1, 0, 1, and 2 represent fold change. Red indicates high expression and blue represents low expression. CON-1, CON-2, and CON-3 represent three mock-infected samples; HTNV-1, HTNV-2, and HTNV-3 represent three HTNV-infected samples.  (B)  DE mRNAs count.  (C)  Verification of dysregulated mRNAs. HUVECs were infected with HTNV 76-118 for 3 days (MOI = 1). Then the total RNA was extracted and expression levels of the selected mRNAs were measured by RT-qPCR. Student's  t -test, mean ± SD, *** P
    Monoclonal Anti Htnv 76 118 Strain Antibody, supplied by Abnova, used in various techniques. Bioz Stars score: 94/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    BEI Resources hantaan virus htnv strain fojnica
    Identification of dysregulated mRNAs.  (A)  Heatmap and clustering analysis of DE mRNAs. Each row represents one mRNA and each column represents one sample; −2, −1, 0, 1, and 2 represent fold change. Red indicates high expression and blue represents low expression. CON-1, CON-2, and CON-3 represent three mock-infected samples; HTNV-1, HTNV-2, and HTNV-3 represent three HTNV-infected samples.  (B)  DE mRNAs count.  (C)  Verification of dysregulated mRNAs. HUVECs were infected with HTNV 76-118 for 3 days (MOI = 1). Then the total RNA was extracted and expression levels of the selected mRNAs were measured by RT-qPCR. Student's  t -test, mean ± SD, *** P
    Hantaan Virus Htnv Strain Fojnica, supplied by BEI Resources, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Progen Biotechnik htnv infected vero e6 cells
    Identification of dysregulated mRNAs.  (A)  Heatmap and clustering analysis of DE mRNAs. Each row represents one mRNA and each column represents one sample; −2, −1, 0, 1, and 2 represent fold change. Red indicates high expression and blue represents low expression. CON-1, CON-2, and CON-3 represent three mock-infected samples; HTNV-1, HTNV-2, and HTNV-3 represent three HTNV-infected samples.  (B)  DE mRNAs count.  (C)  Verification of dysregulated mRNAs. HUVECs were infected with HTNV 76-118 for 3 days (MOI = 1). Then the total RNA was extracted and expression levels of the selected mRNAs were measured by RT-qPCR. Student's  t -test, mean ± SD, *** P
    Htnv Infected Vero E6 Cells, supplied by Progen Biotechnik, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Progen Biotechnik htnv antigens
    Identification of dysregulated mRNAs.  (A)  Heatmap and clustering analysis of DE mRNAs. Each row represents one mRNA and each column represents one sample; −2, −1, 0, 1, and 2 represent fold change. Red indicates high expression and blue represents low expression. CON-1, CON-2, and CON-3 represent three mock-infected samples; HTNV-1, HTNV-2, and HTNV-3 represent three HTNV-infected samples.  (B)  DE mRNAs count.  (C)  Verification of dysregulated mRNAs. HUVECs were infected with HTNV 76-118 for 3 days (MOI = 1). Then the total RNA was extracted and expression levels of the selected mRNAs were measured by RT-qPCR. Student's  t -test, mean ± SD, *** P
    Htnv Antigens, supplied by Progen Biotechnik, used in various techniques. Bioz Stars score: 85/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam htnv strain 76 118
    Identification of dysregulated mRNAs.  (A)  Heatmap and clustering analysis of DE mRNAs. Each row represents one mRNA and each column represents one sample; −2, −1, 0, 1, and 2 represent fold change. Red indicates high expression and blue represents low expression. CON-1, CON-2, and CON-3 represent three mock-infected samples; HTNV-1, HTNV-2, and HTNV-3 represent three HTNV-infected samples.  (B)  DE mRNAs count.  (C)  Verification of dysregulated mRNAs. HUVECs were infected with HTNV 76-118 for 3 days (MOI = 1). Then the total RNA was extracted and expression levels of the selected mRNAs were measured by RT-qPCR. Student's  t -test, mean ± SD, *** P
    Htnv Strain 76 118, supplied by Abcam, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    BEI Resources htnv nucleocapsid 76 118
    Identification of dysregulated mRNAs.  (A)  Heatmap and clustering analysis of DE mRNAs. Each row represents one mRNA and each column represents one sample; −2, −1, 0, 1, and 2 represent fold change. Red indicates high expression and blue represents low expression. CON-1, CON-2, and CON-3 represent three mock-infected samples; HTNV-1, HTNV-2, and HTNV-3 represent three HTNV-infected samples.  (B)  DE mRNAs count.  (C)  Verification of dysregulated mRNAs. HUVECs were infected with HTNV 76-118 for 3 days (MOI = 1). Then the total RNA was extracted and expression levels of the selected mRNAs were measured by RT-qPCR. Student's  t -test, mean ± SD, *** P
    Htnv Nucleocapsid 76 118, supplied by BEI Resources, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ChinaPeptides htnv gc peptides sixty eight peptides
    Identification of dysregulated mRNAs.  (A)  Heatmap and clustering analysis of DE mRNAs. Each row represents one mRNA and each column represents one sample; −2, −1, 0, 1, and 2 represent fold change. Red indicates high expression and blue represents low expression. CON-1, CON-2, and CON-3 represent three mock-infected samples; HTNV-1, HTNV-2, and HTNV-3 represent three HTNV-infected samples.  (B)  DE mRNAs count.  (C)  Verification of dysregulated mRNAs. HUVECs were infected with HTNV 76-118 for 3 days (MOI = 1). Then the total RNA was extracted and expression levels of the selected mRNAs were measured by RT-qPCR. Student's  t -test, mean ± SD, *** P
    Htnv Gc Peptides Sixty Eight Peptides, supplied by ChinaPeptides, used in various techniques. Bioz Stars score: 91/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    bei resources htnv gn gc expressing vectors
    The <t>HTNV</t> Gn/Gc I532K S1094L mutations together enhance plasma membrane localization of Gn/Gc. (A) U2OS cells, co-transfected with plasmids expressing eGFP and wild type, I532K, S1094L or I532K/S1094L forms of HTNV Gn/Gc, were stained for cell surface expression of HTNV Gn or Gc at 48 h post-transfection. (B) Primary human endothelial cells (HUVECs), nucleofected with plasmids encoding eGFP and wild type, I532K, S1094L, or I532K/S1094L versions of HTNV Gn/Gc, were fixed 72 h later, permeabilized, and stained with HTNV Gc-specific antibody. Representative images from a single experiment, illustrating at least 3 independent experiments, are shown for each panel A and B. EV, empty vector. Scale bars, 20 μm. (C) U2OS cells, transfected as described in panel A, were immunostained for cell surface expression of HTNV Gc and analyzed using flow cytometry. Data from 3 independent experiments are shown as mean ± SD. Groups were compared by one-way ANOVA with Tukey’s correction for multiple comparisons. ns, P > 0.05; *, P
    Htnv Gn Gc Expressing Vectors, supplied by bei resources, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    QuantoBio htnv gp nonapeptides
    High binding affinity of eight Hantaan virus <t>(HTNV)</t> GP <t>nonapeptides</t> to HLA-A*0201 molecules. The T2 cell binding assay was used to quantify the peptide-binding affinity of 34 predicted HTNV GP nonapeptides to HLA-A*0201 molecules. T2 cells incubated with each peptide and β2-microglobulin were then stained with phycoerythrin-labeled HLA-A*02 monoclonal antibody and detected by flow cytometry. The orange curves indicate HLA-A*0201 stabilization with HLA-A*0201-restricted HTNV NP FA9 (aa129–aa137, FVVPILLKA) peptides serving as positive controls. The red curves indicate T2 cells incubated without peptide serving as negative controls. The blue curves indicate T2 cells incubated with each HTNV GP nonapeptide. The overlay of the three conditions in histograms clearly show that the curve of T2 cells incubated with each of the eight HTNV GP nonapeptides was shifted more to the right with a higher fluorescence intensity of HLA-A*0201 molecules than those incubated without peptide, indicating that these nonapeptides have a high binding affinity to the HLA-A*0201 molecule. GP, glycoprotein; NP, nucleoprotein.
    Htnv Gp Nonapeptides, supplied by QuantoBio, used in various techniques. Bioz Stars score: 91/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ATUM htnv l protein
    Puumala virus (PUUV) L protein possesses a functional endonuclease domain. ( A ) Sequence alignment of the N-terminal domains of <t>HTNV</t> and PUUV L protein. ( B ) BSR T7/5 cells were transfected with wt or D97A PUUV and <t>HTNV</t> L protein N-terminal constructs along with an NLuc reporter control plasmid. Cells were lyzed 48 hours post-transfection. Proteins were separated by SDS-PAGE and transferred on membranes for immunoblotting. ( C ) NLuc activity was measured using the Nano Glow ® Luciferase Assay System (Promega). Data are expressed as mean ± SD ( n = 3) and were analyzed by one-way ANOVA with p-values indicated.
    Htnv L Protein, supplied by ATUM, used in various techniques. Bioz Stars score: 90/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Essentials Inc htnv n antigen
    Colocalization of <t>HTNV</t> N with ERGIC-53 and redistribution of N with BFA. Vero E6 cells were infected with HTNV at an MOI of 0.1, and after 3 days slides were acetone fixed (except for Mann II staining, in which case paraformaldehyde was used). Prior to
    Htnv N Antigen, supplied by Cell Essentials Inc, used in various techniques. Bioz Stars score: 85/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam htnv n
    Examination of TNF-α-induced NF-κB <t>p65</t> nuclear translocation and degradation of IκBα in HTNV-infected cells. A549 cells were mock infected (M) or infected with HTNV (V) at an MOI of 5. On day 5, mock- and HTNV-infected cells were left untreated (−) or treated (+) with 50 ng/ml of TNF-α for 15 min. (A) After fixation, cells were stained with antibodies against NF-κB p65 (green) and <t>HTNV</t> N protein (red) and stained with DAPI to highlight nuclei (blue). (B) Cell lysates were separated into cytoplasmic and nuclear fractions. (C) Total cell lysates were prepared for immunoblotting, and proteins were transferred onto PVDF membranes. Blots were probed with antibodies against IκBα, p50, p65, HTNV N protein, GAPDH, or histone H2B.
    Htnv N, supplied by Abcam, used in various techniques. Bioz Stars score: 85/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Essentials Inc htnv n
    Examination of TNF-α-induced NF-κB <t>p65</t> nuclear translocation and degradation of IκBα in HTNV-infected cells. A549 cells were mock infected (M) or infected with HTNV (V) at an MOI of 5. On day 5, mock- and HTNV-infected cells were left untreated (−) or treated (+) with 50 ng/ml of TNF-α for 15 min. (A) After fixation, cells were stained with antibodies against NF-κB p65 (green) and <t>HTNV</t> N protein (red) and stained with DAPI to highlight nuclei (blue). (B) Cell lysates were separated into cytoplasmic and nuclear fractions. (C) Total cell lysates were prepared for immunoblotting, and proteins were transferred onto PVDF membranes. Blots were probed with antibodies against IκBα, p50, p65, HTNV N protein, GAPDH, or histone H2B.
    Htnv N, supplied by Cell Essentials Inc, used in various techniques. Bioz Stars score: 85/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    htnv  (Abcam)
    93
    Abcam htnv
    Examination of TNF-α-induced NF-κB <t>p65</t> nuclear translocation and degradation of IκBα in HTNV-infected cells. A549 cells were mock infected (M) or infected with HTNV (V) at an MOI of 5. On day 5, mock- and HTNV-infected cells were left untreated (−) or treated (+) with 50 ng/ml of TNF-α for 15 min. (A) After fixation, cells were stained with antibodies against NF-κB p65 (green) and <t>HTNV</t> N protein (red) and stained with DAPI to highlight nuclei (blue). (B) Cell lysates were separated into cytoplasmic and nuclear fractions. (C) Total cell lysates were prepared for immunoblotting, and proteins were transferred onto PVDF membranes. Blots were probed with antibodies against IκBα, p50, p65, HTNV N protein, GAPDH, or histone H2B.
    Htnv, supplied by Abcam, used in various techniques. Bioz Stars score: 93/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    HTNV induces TRAIL-dependent apoptosis in primary HUVECs. HUVECs were infected with/without HTNV 76-118 (MOI = 1) for 2 h and followed by qRT-PCR and Western blot at different days to measure TRAIL-related apoptosis factors.  (A)  HTNV S, TRAIL, DcR1, DcR2, caspase-8, and caspase-3 mRNA in HTNV-infected HUVECs.  (B)  HTNV NP and TRAIL-related apoptosis proteins expression in HTNV-infected HUVECs.  (C)  TRAIL-DR4/DR5 and  (D)  caspase-9 mRNA and protein expression during HTNV infection. The mRNA results shown are the average of three replicates; values represent the mean ± SD ( * p

    Journal: Frontiers in Immunology

    Article Title: HTNV Sensitizes Host Toward TRAIL-Mediated Apoptosis—A Pivotal Anti-hantaviral Role of TRAIL

    doi: 10.3389/fimmu.2020.01072

    Figure Lengend Snippet: HTNV induces TRAIL-dependent apoptosis in primary HUVECs. HUVECs were infected with/without HTNV 76-118 (MOI = 1) for 2 h and followed by qRT-PCR and Western blot at different days to measure TRAIL-related apoptosis factors. (A) HTNV S, TRAIL, DcR1, DcR2, caspase-8, and caspase-3 mRNA in HTNV-infected HUVECs. (B) HTNV NP and TRAIL-related apoptosis proteins expression in HTNV-infected HUVECs. (C) TRAIL-DR4/DR5 and (D) caspase-9 mRNA and protein expression during HTNV infection. The mRNA results shown are the average of three replicates; values represent the mean ± SD ( * p

    Article Snippet: Cells were then incubated with monoclonal antibody against HTNV/76-118 (ab20309, 1:100, Abcam) for 1.5 h at room temperature or 4°C overnight.

    Techniques: Infection, Quantitative RT-PCR, Western Blot, Expressing

    Identification of dysregulated mRNAs.  (A)  Heatmap and clustering analysis of DE mRNAs. Each row represents one mRNA and each column represents one sample; −2, −1, 0, 1, and 2 represent fold change. Red indicates high expression and blue represents low expression. CON-1, CON-2, and CON-3 represent three mock-infected samples; HTNV-1, HTNV-2, and HTNV-3 represent three HTNV-infected samples.  (B)  DE mRNAs count.  (C)  Verification of dysregulated mRNAs. HUVECs were infected with HTNV 76-118 for 3 days (MOI = 1). Then the total RNA was extracted and expression levels of the selected mRNAs were measured by RT-qPCR. Student's  t -test, mean ± SD, *** P

    Journal: Frontiers in Cellular and Infection Microbiology

    Article Title: RNA-Seq Revealed a Circular RNA-microRNA-mRNA Regulatory Network in Hantaan Virus Infection

    doi: 10.3389/fcimb.2020.00097

    Figure Lengend Snippet: Identification of dysregulated mRNAs. (A) Heatmap and clustering analysis of DE mRNAs. Each row represents one mRNA and each column represents one sample; −2, −1, 0, 1, and 2 represent fold change. Red indicates high expression and blue represents low expression. CON-1, CON-2, and CON-3 represent three mock-infected samples; HTNV-1, HTNV-2, and HTNV-3 represent three HTNV-infected samples. (B) DE mRNAs count. (C) Verification of dysregulated mRNAs. HUVECs were infected with HTNV 76-118 for 3 days (MOI = 1). Then the total RNA was extracted and expression levels of the selected mRNAs were measured by RT-qPCR. Student's t -test, mean ± SD, *** P

    Article Snippet: The primary monoclonal anti-HTNV 76-118 strain antibody was purchased from Abnova (MAB5482, Taiwan).

    Techniques: Expressing, Infection, Quantitative RT-PCR

    Identification of dysregulated miRNAs.  (A)  Heatmap and clustering analysis of DE miRNAs. Each row represents one miRNA and each column represents one sample; −2, −1, 0, 1, and 2 represent fold change. Red indicates high expression and blue represents low expression. CON-1, CON-2, and CON-3 represent three mock-infected samples; HTNV-1, HTNV-2, and HTNV-3 represent three HTNV-infected samples.  (B)  DE miRNAs count.  (C)  Verification of DE miRNAs upon HTNV infection. HUVECs were infected with HTNV 76-118 (MOI = 1) for 3 days. Then the total RNA was extracted and the expression levels of miRNAs were measured by RT-qPCR. Student's  t -test, mean ± SD, *** P

    Journal: Frontiers in Cellular and Infection Microbiology

    Article Title: RNA-Seq Revealed a Circular RNA-microRNA-mRNA Regulatory Network in Hantaan Virus Infection

    doi: 10.3389/fcimb.2020.00097

    Figure Lengend Snippet: Identification of dysregulated miRNAs. (A) Heatmap and clustering analysis of DE miRNAs. Each row represents one miRNA and each column represents one sample; −2, −1, 0, 1, and 2 represent fold change. Red indicates high expression and blue represents low expression. CON-1, CON-2, and CON-3 represent three mock-infected samples; HTNV-1, HTNV-2, and HTNV-3 represent three HTNV-infected samples. (B) DE miRNAs count. (C) Verification of DE miRNAs upon HTNV infection. HUVECs were infected with HTNV 76-118 (MOI = 1) for 3 days. Then the total RNA was extracted and the expression levels of miRNAs were measured by RT-qPCR. Student's t -test, mean ± SD, *** P

    Article Snippet: The primary monoclonal anti-HTNV 76-118 strain antibody was purchased from Abnova (MAB5482, Taiwan).

    Techniques: Expressing, Infection, Quantitative RT-PCR

    circRNA expression overview.  (A)  circRNAs category chart.  (B)  circRNAs length distribution.  (C)  Volcano plot of DE circRNAs upon HTNV infection in HUVECs. Red dots represent up-regulated circRNAs and green dots represent down-regulated circRNAs.  (D)  Chromosome distribution of DE circRNAs.  (E)  Heatmap and clustering analysis of DE circRNAs. Each row represents one circRNA and each column represents one sample; −2, −1, 0, 1, and 2 represent fold change. Red indicates high expression and blue represents low expression. CON-1, CON-2 and CON-3 represent three mock-infected samples; HTNV-1, HTNV-2, and HTNV-3 represent three HTNV-infected samples.  (F)  Verification of dysregulated circRNAs. HUVECs were infected with HTNV 76-118 (MOI = 1) for 3 days. Then the total RNA was extracted and the expression levels of circRNAs were measured by RT-qPCR. Student's  t -test, mean ± standard deviation (SD), * P

    Journal: Frontiers in Cellular and Infection Microbiology

    Article Title: RNA-Seq Revealed a Circular RNA-microRNA-mRNA Regulatory Network in Hantaan Virus Infection

    doi: 10.3389/fcimb.2020.00097

    Figure Lengend Snippet: circRNA expression overview. (A) circRNAs category chart. (B) circRNAs length distribution. (C) Volcano plot of DE circRNAs upon HTNV infection in HUVECs. Red dots represent up-regulated circRNAs and green dots represent down-regulated circRNAs. (D) Chromosome distribution of DE circRNAs. (E) Heatmap and clustering analysis of DE circRNAs. Each row represents one circRNA and each column represents one sample; −2, −1, 0, 1, and 2 represent fold change. Red indicates high expression and blue represents low expression. CON-1, CON-2 and CON-3 represent three mock-infected samples; HTNV-1, HTNV-2, and HTNV-3 represent three HTNV-infected samples. (F) Verification of dysregulated circRNAs. HUVECs were infected with HTNV 76-118 (MOI = 1) for 3 days. Then the total RNA was extracted and the expression levels of circRNAs were measured by RT-qPCR. Student's t -test, mean ± standard deviation (SD), * P

    Article Snippet: The primary monoclonal anti-HTNV 76-118 strain antibody was purchased from Abnova (MAB5482, Taiwan).

    Techniques: Expressing, Infection, Quantitative RT-PCR, Standard Deviation

    The HTNV Gn/Gc I532K S1094L mutations together enhance plasma membrane localization of Gn/Gc. (A) U2OS cells, co-transfected with plasmids expressing eGFP and wild type, I532K, S1094L or I532K/S1094L forms of HTNV Gn/Gc, were stained for cell surface expression of HTNV Gn or Gc at 48 h post-transfection. (B) Primary human endothelial cells (HUVECs), nucleofected with plasmids encoding eGFP and wild type, I532K, S1094L, or I532K/S1094L versions of HTNV Gn/Gc, were fixed 72 h later, permeabilized, and stained with HTNV Gc-specific antibody. Representative images from a single experiment, illustrating at least 3 independent experiments, are shown for each panel A and B. EV, empty vector. Scale bars, 20 μm. (C) U2OS cells, transfected as described in panel A, were immunostained for cell surface expression of HTNV Gc and analyzed using flow cytometry. Data from 3 independent experiments are shown as mean ± SD. Groups were compared by one-way ANOVA with Tukey’s correction for multiple comparisons. ns, P > 0.05; *, P

    Journal: bioRxiv

    Article Title: Two point mutations in the Hantaan virus glycoproteins afford the generation of a highly infectious recombinant vesicular stomatitis virus vector

    doi: 10.1101/356055

    Figure Lengend Snippet: The HTNV Gn/Gc I532K S1094L mutations together enhance plasma membrane localization of Gn/Gc. (A) U2OS cells, co-transfected with plasmids expressing eGFP and wild type, I532K, S1094L or I532K/S1094L forms of HTNV Gn/Gc, were stained for cell surface expression of HTNV Gn or Gc at 48 h post-transfection. (B) Primary human endothelial cells (HUVECs), nucleofected with plasmids encoding eGFP and wild type, I532K, S1094L, or I532K/S1094L versions of HTNV Gn/Gc, were fixed 72 h later, permeabilized, and stained with HTNV Gc-specific antibody. Representative images from a single experiment, illustrating at least 3 independent experiments, are shown for each panel A and B. EV, empty vector. Scale bars, 20 μm. (C) U2OS cells, transfected as described in panel A, were immunostained for cell surface expression of HTNV Gc and analyzed using flow cytometry. Data from 3 independent experiments are shown as mean ± SD. Groups were compared by one-way ANOVA with Tukey’s correction for multiple comparisons. ns, P > 0.05; *, P

    Article Snippet: Primary human umbilical vein endothelial cells (HUVECs) were nucleofected with 4.75 μg of empty or HTNV Gn/Gc expressing vectors, together with 50 ng eGFP, using the Amaxa Kit (program A-034, Lonza) before staining for total Gn/Gc expression at 72 h post-nucleofection as described above for U2OS cells.

    Techniques: Transfection, Expressing, Staining, Plasmid Preparation, Flow Cytometry

    Two point mutations (I532K S1094L) in the Gn/Gc enhance rVSV-HTNV Gn/Gc spread and replication. (A) Schematic representation of the HTNV Gn/Gc. Location of the point mutations acquired after serial passaging of rVSV expressing HTNV Gn/Gc. (B-C) Growth of WT or mutant rVSV-HTNV Gn/Gc. Supernatants from 293FT cells co-transfected with plasmids encoding rVSV genomes expressing eGFP bearing WT, I532K, S1094L or I532K/S1094L versions of HTNV Gn/Gc with helper plasmids, were used to infect Vero cells. (B) Representative images of eGFP expression in Vero cells at indicated times post infection. (C) Supernatants collected from infected Vero cells at indicated times post-infection were titered on naive Vero cells. Data from two independent experiments (n = 4) are represented as log infectious units (IU) per mL (mean ± SD). “ > ” indicate virus titers that were below the limit of detection (50 IU per mL). Groups were compared by two-way ANOVA with Tukey’s correction for multiple comparisons. ns (not significant), P > 0.05; ****, P

    Journal: bioRxiv

    Article Title: Two point mutations in the Hantaan virus glycoproteins afford the generation of a highly infectious recombinant vesicular stomatitis virus vector

    doi: 10.1101/356055

    Figure Lengend Snippet: Two point mutations (I532K S1094L) in the Gn/Gc enhance rVSV-HTNV Gn/Gc spread and replication. (A) Schematic representation of the HTNV Gn/Gc. Location of the point mutations acquired after serial passaging of rVSV expressing HTNV Gn/Gc. (B-C) Growth of WT or mutant rVSV-HTNV Gn/Gc. Supernatants from 293FT cells co-transfected with plasmids encoding rVSV genomes expressing eGFP bearing WT, I532K, S1094L or I532K/S1094L versions of HTNV Gn/Gc with helper plasmids, were used to infect Vero cells. (B) Representative images of eGFP expression in Vero cells at indicated times post infection. (C) Supernatants collected from infected Vero cells at indicated times post-infection were titered on naive Vero cells. Data from two independent experiments (n = 4) are represented as log infectious units (IU) per mL (mean ± SD). “ > ” indicate virus titers that were below the limit of detection (50 IU per mL). Groups were compared by two-way ANOVA with Tukey’s correction for multiple comparisons. ns (not significant), P > 0.05; ****, P

    Article Snippet: Primary human umbilical vein endothelial cells (HUVECs) were nucleofected with 4.75 μg of empty or HTNV Gn/Gc expressing vectors, together with 50 ng eGFP, using the Amaxa Kit (program A-034, Lonza) before staining for total Gn/Gc expression at 72 h post-nucleofection as described above for U2OS cells.

    Techniques: Passaging, Expressing, Mutagenesis, Transfection, Infection

    High binding affinity of eight Hantaan virus (HTNV) GP nonapeptides to HLA-A*0201 molecules. The T2 cell binding assay was used to quantify the peptide-binding affinity of 34 predicted HTNV GP nonapeptides to HLA-A*0201 molecules. T2 cells incubated with each peptide and β2-microglobulin were then stained with phycoerythrin-labeled HLA-A*02 monoclonal antibody and detected by flow cytometry. The orange curves indicate HLA-A*0201 stabilization with HLA-A*0201-restricted HTNV NP FA9 (aa129–aa137, FVVPILLKA) peptides serving as positive controls. The red curves indicate T2 cells incubated without peptide serving as negative controls. The blue curves indicate T2 cells incubated with each HTNV GP nonapeptide. The overlay of the three conditions in histograms clearly show that the curve of T2 cells incubated with each of the eight HTNV GP nonapeptides was shifted more to the right with a higher fluorescence intensity of HLA-A*0201 molecules than those incubated without peptide, indicating that these nonapeptides have a high binding affinity to the HLA-A*0201 molecule. GP, glycoprotein; NP, nucleoprotein.

    Journal: Frontiers in Immunology

    Article Title: Novel Identified HLA-A*0201-Restricted Hantaan Virus Glycoprotein Cytotoxic T-Cell Epitopes Could Effectively Induce Protective Responses in HLA-A2.1/Kb Transgenic Mice May Associate with the Severity of Hemorrhagic Fever with Renal Syndrome

    doi: 10.3389/fimmu.2017.01797

    Figure Lengend Snippet: High binding affinity of eight Hantaan virus (HTNV) GP nonapeptides to HLA-A*0201 molecules. The T2 cell binding assay was used to quantify the peptide-binding affinity of 34 predicted HTNV GP nonapeptides to HLA-A*0201 molecules. T2 cells incubated with each peptide and β2-microglobulin were then stained with phycoerythrin-labeled HLA-A*02 monoclonal antibody and detected by flow cytometry. The orange curves indicate HLA-A*0201 stabilization with HLA-A*0201-restricted HTNV NP FA9 (aa129–aa137, FVVPILLKA) peptides serving as positive controls. The red curves indicate T2 cells incubated without peptide serving as negative controls. The blue curves indicate T2 cells incubated with each HTNV GP nonapeptide. The overlay of the three conditions in histograms clearly show that the curve of T2 cells incubated with each of the eight HTNV GP nonapeptides was shifted more to the right with a higher fluorescence intensity of HLA-A*0201 molecules than those incubated without peptide, indicating that these nonapeptides have a high binding affinity to the HLA-A*0201 molecule. GP, glycoprotein; NP, nucleoprotein.

    Article Snippet: Peptide/HLA-A*0201 Tetramer Staining PE-labeled HLA-A*0201 tetramers refolded separately with the HTNV GP nonapeptides were customized by QuantoBio (Beijing, China).

    Techniques: Binding Assay, Cell Binding Assay, Incubation, Staining, Labeling, Flow Cytometry, Cytometry, Fluorescence

    Puumala virus (PUUV) L protein possesses a functional endonuclease domain. ( A ) Sequence alignment of the N-terminal domains of HTNV and PUUV L protein. ( B ) BSR T7/5 cells were transfected with wt or D97A PUUV and HTNV L protein N-terminal constructs along with an NLuc reporter control plasmid. Cells were lyzed 48 hours post-transfection. Proteins were separated by SDS-PAGE and transferred on membranes for immunoblotting. ( C ) NLuc activity was measured using the Nano Glow ® Luciferase Assay System (Promega). Data are expressed as mean ± SD ( n = 3) and were analyzed by one-way ANOVA with p-values indicated.

    Journal: Viruses

    Article Title: Conserved Endonuclease Function of Hantavirus L Polymerase

    doi: 10.3390/v8050108

    Figure Lengend Snippet: Puumala virus (PUUV) L protein possesses a functional endonuclease domain. ( A ) Sequence alignment of the N-terminal domains of HTNV and PUUV L protein. ( B ) BSR T7/5 cells were transfected with wt or D97A PUUV and HTNV L protein N-terminal constructs along with an NLuc reporter control plasmid. Cells were lyzed 48 hours post-transfection. Proteins were separated by SDS-PAGE and transferred on membranes for immunoblotting. ( C ) NLuc activity was measured using the Nano Glow ® Luciferase Assay System (Promega). Data are expressed as mean ± SD ( n = 3) and were analyzed by one-way ANOVA with p-values indicated.

    Article Snippet: Protein Expression and Purification The coding region of the N-terminal 220 residues of HTNV L protein (Accession number X55901) was optimized for expression in E. coli and synthetized (DNA2.0).

    Techniques: Functional Assay, Sequencing, Transfection, Construct, Plasmid Preparation, SDS Page, Activity Assay, Luciferase

    In vitro analysis of HTNV endonuclease. ( A ) The N-terminal 220 residues of HTNV L protein were expressed in E. coli and purified using TALON ® metal affinity resin as described in the methods section. A fraction of the purified material was separated by SDS-PAGE and stained with Coomassie blue. ( B ) Divalent cation-dependent nuclease activity. Single-stranded M13mp18 DNA (25 ng/μL) was incubated at 37 °C during 60 min in presence of 0, 5 or 10 μM of purified HTNV N-terminal domain and 2 mM of the indicated divalent cations, or 10 mM ethylenediaminetetraacetic acid (EDTA).

    Journal: Viruses

    Article Title: Conserved Endonuclease Function of Hantavirus L Polymerase

    doi: 10.3390/v8050108

    Figure Lengend Snippet: In vitro analysis of HTNV endonuclease. ( A ) The N-terminal 220 residues of HTNV L protein were expressed in E. coli and purified using TALON ® metal affinity resin as described in the methods section. A fraction of the purified material was separated by SDS-PAGE and stained with Coomassie blue. ( B ) Divalent cation-dependent nuclease activity. Single-stranded M13mp18 DNA (25 ng/μL) was incubated at 37 °C during 60 min in presence of 0, 5 or 10 μM of purified HTNV N-terminal domain and 2 mM of the indicated divalent cations, or 10 mM ethylenediaminetetraacetic acid (EDTA).

    Article Snippet: Protein Expression and Purification The coding region of the N-terminal 220 residues of HTNV L protein (Accession number X55901) was optimized for expression in E. coli and synthetized (DNA2.0).

    Techniques: In Vitro, Purification, SDS Page, Staining, Activity Assay, Incubation

    Model of the putative active sites of prototypic Hantaan virus (HTNV) and Andes (ANDV) superposed on the active site of orthobunyavirus La Crosse (LACV). ( A ) Schematic representation of HTNV L protein. The L segment of the prototypic HTNV strain 76/118 has 6533 nucleotides and encodes the L protein of 2151 amino acids (aa). HTNV L contains a polymerase (P) domain (aa 956–1142) and a putative endonuclease (E) domain (aa 1–216). ( B ) Models of the putative active sites of HTNV and ANDV superposed on the active site of LACV. Ribbon diagrams of LACV (PDB entry 2XI5), HTNV and ANDV after structural superposition of key active site residues. Secondary structure classifications was calculated using definition of secondary structure proteins (DSSP) [ 33 ] for LACV or predicted using the method/program PSI-PRED [ 34 ]. Helix residues have been colored yellow and β-strand residues have been colored blue in the ribbon diagrams as well as in the alignment, according to DSSP or PSI-PRED results, respectively. Annotations in ribbon diagrams follow the HTNV residue numbering in the alignment. Key residues are colored red in the alignment and shown with side chains in the ribbon diagrams. The ribbon diagrams to the right have been rotated 90 degrees around the vertical coordinate axis. ( C ) Amino acid residues of the active site of HTNV L endonuclease derived from the model in ( A ).

    Journal: Viruses

    Article Title: Conserved Endonuclease Function of Hantavirus L Polymerase

    doi: 10.3390/v8050108

    Figure Lengend Snippet: Model of the putative active sites of prototypic Hantaan virus (HTNV) and Andes (ANDV) superposed on the active site of orthobunyavirus La Crosse (LACV). ( A ) Schematic representation of HTNV L protein. The L segment of the prototypic HTNV strain 76/118 has 6533 nucleotides and encodes the L protein of 2151 amino acids (aa). HTNV L contains a polymerase (P) domain (aa 956–1142) and a putative endonuclease (E) domain (aa 1–216). ( B ) Models of the putative active sites of HTNV and ANDV superposed on the active site of LACV. Ribbon diagrams of LACV (PDB entry 2XI5), HTNV and ANDV after structural superposition of key active site residues. Secondary structure classifications was calculated using definition of secondary structure proteins (DSSP) [ 33 ] for LACV or predicted using the method/program PSI-PRED [ 34 ]. Helix residues have been colored yellow and β-strand residues have been colored blue in the ribbon diagrams as well as in the alignment, according to DSSP or PSI-PRED results, respectively. Annotations in ribbon diagrams follow the HTNV residue numbering in the alignment. Key residues are colored red in the alignment and shown with side chains in the ribbon diagrams. The ribbon diagrams to the right have been rotated 90 degrees around the vertical coordinate axis. ( C ) Amino acid residues of the active site of HTNV L endonuclease derived from the model in ( A ).

    Article Snippet: Protein Expression and Purification The coding region of the N-terminal 220 residues of HTNV L protein (Accession number X55901) was optimized for expression in E. coli and synthetized (DNA2.0).

    Techniques: Derivative Assay

    Mutational analysis of HTNV endonuclease domain. ( A ) Schematic representation of the constructs. ( B ) BSR T7/5 cells were transfected with wild-type (wt) or D97A HTNV L protein N-terminal constructs along with an nanoluciferase (NLuc) reporter control plasmid. Cells were lyzed 48 h post-transfection. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred on membranes for immunoblotting. ( C ) NLuc activity was measured using the Nano Glow ® Luciferase Assay System (Promega). Data represent mean ± SD ( n = 3). One representative experiment out of three is shown. Data were analyzed using one-way ANOVA with p-values indicated. ( D ) BSR T7/5 cells were co-transfected with HA-tagged WT L protein N-terminal constructs and the indicated mutants together with an NLuc reporter plasmid. Cells were lyzed 48 h post-transfection and HA-tagged endonuclease constructs detected in Western blot. ( E ) Nanoluciferase activity was measured using the Nano Glow ® Luciferase Assay System (Promega). Data represent mean ± SD ( n = 3). One representative experiment out of three is shown. Data were analyzed using one-way ANOVA with p -values *** p

    Journal: Viruses

    Article Title: Conserved Endonuclease Function of Hantavirus L Polymerase

    doi: 10.3390/v8050108

    Figure Lengend Snippet: Mutational analysis of HTNV endonuclease domain. ( A ) Schematic representation of the constructs. ( B ) BSR T7/5 cells were transfected with wild-type (wt) or D97A HTNV L protein N-terminal constructs along with an nanoluciferase (NLuc) reporter control plasmid. Cells were lyzed 48 h post-transfection. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred on membranes for immunoblotting. ( C ) NLuc activity was measured using the Nano Glow ® Luciferase Assay System (Promega). Data represent mean ± SD ( n = 3). One representative experiment out of three is shown. Data were analyzed using one-way ANOVA with p-values indicated. ( D ) BSR T7/5 cells were co-transfected with HA-tagged WT L protein N-terminal constructs and the indicated mutants together with an NLuc reporter plasmid. Cells were lyzed 48 h post-transfection and HA-tagged endonuclease constructs detected in Western blot. ( E ) Nanoluciferase activity was measured using the Nano Glow ® Luciferase Assay System (Promega). Data represent mean ± SD ( n = 3). One representative experiment out of three is shown. Data were analyzed using one-way ANOVA with p -values *** p

    Article Snippet: Protein Expression and Purification The coding region of the N-terminal 220 residues of HTNV L protein (Accession number X55901) was optimized for expression in E. coli and synthetized (DNA2.0).

    Techniques: Construct, Transfection, Plasmid Preparation, Polyacrylamide Gel Electrophoresis, SDS Page, Activity Assay, Luciferase, Western Blot

    Colocalization of HTNV N with ERGIC-53 and redistribution of N with BFA. Vero E6 cells were infected with HTNV at an MOI of 0.1, and after 3 days slides were acetone fixed (except for Mann II staining, in which case paraformaldehyde was used). Prior to

    Journal: Journal of Virology

    Article Title: Dynein-Dependent Transport of the Hantaan Virus Nucleocapsid Protein to the Endoplasmic Reticulum-Golgi Intermediate Compartment ▿

    doi: 10.1128/JVI.00418-07

    Figure Lengend Snippet: Colocalization of HTNV N with ERGIC-53 and redistribution of N with BFA. Vero E6 cells were infected with HTNV at an MOI of 0.1, and after 3 days slides were acetone fixed (except for Mann II staining, in which case paraformaldehyde was used). Prior to

    Article Snippet: A murine MAb (E-314) was raised to the same HTNV N antigen by Cell Essentials, Inc. (Boston, MA).

    Techniques: Infection, Staining

    Colocalization studies of HTNV N in Vero E6 cells expressing N alone, as well as redistribution of N with BFA, with various subcellular markers. Vero E6 cells were transfected with pcHTNVN, which expresses N alone, and after 18 h, the cells were fixed

    Journal: Journal of Virology

    Article Title: Dynein-Dependent Transport of the Hantaan Virus Nucleocapsid Protein to the Endoplasmic Reticulum-Golgi Intermediate Compartment ▿

    doi: 10.1128/JVI.00418-07

    Figure Lengend Snippet: Colocalization studies of HTNV N in Vero E6 cells expressing N alone, as well as redistribution of N with BFA, with various subcellular markers. Vero E6 cells were transfected with pcHTNVN, which expresses N alone, and after 18 h, the cells were fixed

    Article Snippet: A murine MAb (E-314) was raised to the same HTNV N antigen by Cell Essentials, Inc. (Boston, MA).

    Techniques: Expressing, Transfection

    Association of HTNV N with membrane fractions. Vero E6 cells were transfected with pcHTNVN, and after 18 h they were subjected to membrane floatation (A) or subcellular fractionation (B). Fractions were subjected to Western blotting and were probed with

    Journal: Journal of Virology

    Article Title: Dynein-Dependent Transport of the Hantaan Virus Nucleocapsid Protein to the Endoplasmic Reticulum-Golgi Intermediate Compartment ▿

    doi: 10.1128/JVI.00418-07

    Figure Lengend Snippet: Association of HTNV N with membrane fractions. Vero E6 cells were transfected with pcHTNVN, and after 18 h they were subjected to membrane floatation (A) or subcellular fractionation (B). Fractions were subjected to Western blotting and were probed with

    Article Snippet: A murine MAb (E-314) was raised to the same HTNV N antigen by Cell Essentials, Inc. (Boston, MA).

    Techniques: Transfection, Fractionation, Western Blot

    Examination of TNF-α-induced NF-κB p65 nuclear translocation and degradation of IκBα in HTNV-infected cells. A549 cells were mock infected (M) or infected with HTNV (V) at an MOI of 5. On day 5, mock- and HTNV-infected cells were left untreated (−) or treated (+) with 50 ng/ml of TNF-α for 15 min. (A) After fixation, cells were stained with antibodies against NF-κB p65 (green) and HTNV N protein (red) and stained with DAPI to highlight nuclei (blue). (B) Cell lysates were separated into cytoplasmic and nuclear fractions. (C) Total cell lysates were prepared for immunoblotting, and proteins were transferred onto PVDF membranes. Blots were probed with antibodies against IκBα, p50, p65, HTNV N protein, GAPDH, or histone H2B.

    Journal: Journal of Virology

    Article Title: Hantaan Virus Nucleocapsid Protein Binds to Importin ? Proteins and Inhibits Tumor Necrosis Factor Alpha-Induced Activation of Nuclear Factor Kappa B ▿

    doi: 10.1128/JVI.00986-08

    Figure Lengend Snippet: Examination of TNF-α-induced NF-κB p65 nuclear translocation and degradation of IκBα in HTNV-infected cells. A549 cells were mock infected (M) or infected with HTNV (V) at an MOI of 5. On day 5, mock- and HTNV-infected cells were left untreated (−) or treated (+) with 50 ng/ml of TNF-α for 15 min. (A) After fixation, cells were stained with antibodies against NF-κB p65 (green) and HTNV N protein (red) and stained with DAPI to highlight nuclei (blue). (B) Cell lysates were separated into cytoplasmic and nuclear fractions. (C) Total cell lysates were prepared for immunoblotting, and proteins were transferred onto PVDF membranes. Blots were probed with antibodies against IκBα, p50, p65, HTNV N protein, GAPDH, or histone H2B.

    Article Snippet: After being blocked with 10% goat serum in PBS, cells were sequentially stained, first with antibodies to p65 (Santa Cruz Biotechnologies) and then with antibodies to HTNV N (Abcam) and HTNV Gc (mouse immune sera).

    Techniques: Translocation Assay, Infection, Staining

    Analysis of HTNV N protein interaction with importin α proteins. A549 cells were cotransfected with 0 or 1.25 μg of a FLAG-importin α1 (Iα1), FLAG-importin α2 (Iα2), FLAG-importin α3 (Iα3), or FLAG-importin α4 (Iα4) construct and 1.25 μg of pWRG7077-Empty or pWRG7077-HTNV-S plasmid for 24 h and left untreated (−TNF-α) or treated with 50 ng/ml TNF-α (+TNF-α) for 15 min. Immunoprecipitations (IP) were performed with anti-HTNV N protein antibody bound to Sepharose beads. The single asterisks indicate the location of HTNV N, whereas double asterisks identify the heavy chain. Immunoprecipitates and whole-cell lysates (WCL) were analyzed by Western blotting for the expression of importin α proteins and HTNV N protein. −, no importin α.

    Journal: Journal of Virology

    Article Title: Hantaan Virus Nucleocapsid Protein Binds to Importin ? Proteins and Inhibits Tumor Necrosis Factor Alpha-Induced Activation of Nuclear Factor Kappa B ▿

    doi: 10.1128/JVI.00986-08

    Figure Lengend Snippet: Analysis of HTNV N protein interaction with importin α proteins. A549 cells were cotransfected with 0 or 1.25 μg of a FLAG-importin α1 (Iα1), FLAG-importin α2 (Iα2), FLAG-importin α3 (Iα3), or FLAG-importin α4 (Iα4) construct and 1.25 μg of pWRG7077-Empty or pWRG7077-HTNV-S plasmid for 24 h and left untreated (−TNF-α) or treated with 50 ng/ml TNF-α (+TNF-α) for 15 min. Immunoprecipitations (IP) were performed with anti-HTNV N protein antibody bound to Sepharose beads. The single asterisks indicate the location of HTNV N, whereas double asterisks identify the heavy chain. Immunoprecipitates and whole-cell lysates (WCL) were analyzed by Western blotting for the expression of importin α proteins and HTNV N protein. −, no importin α.

    Article Snippet: After being blocked with 10% goat serum in PBS, cells were sequentially stained, first with antibodies to p65 (Santa Cruz Biotechnologies) and then with antibodies to HTNV N (Abcam) and HTNV Gc (mouse immune sera).

    Techniques: Construct, Plasmid Preparation, Western Blot, Expressing

    Endogenous NF-κB transcription activation in cells expressing HTNV N protein. A549 cells were transfected with 500 ng of pWRG7077-Empty, pWRG7077-HTNV-S, or pWRG7077-HTNV-M for 24 h. After treatment of the cells with 50 ng/ml of TNF-α for 15 min, cytoplasmic and nuclear extracts from lysates were prepared. (A) Nuclear extracts were allowed to bind to NF-κB consensus sequence oligonucleotides on 96-well plates and then probed with antibodies specific for NF-κB p65 or NF-κB p50. The absorbance reading for each sample was determined using a spectrophotometer. OD, optical density; MUT oligo, mutated consensus oligonucleotide; WT oligo, wild-type consensus oligonucleotide. (B) Cytoplasmic extracts were used for immunoblotting to detect HTNV N protein and GAPDH. Each point represents an average ± standard deviation of results for six samples. The statistical significance of results for HTNV S with TNF and MG132 with TNF was determined by comparing the results to those for the empty vector with TNF. Asterisks indicate significant differences ( P

    Journal: Journal of Virology

    Article Title: Hantaan Virus Nucleocapsid Protein Binds to Importin ? Proteins and Inhibits Tumor Necrosis Factor Alpha-Induced Activation of Nuclear Factor Kappa B ▿

    doi: 10.1128/JVI.00986-08

    Figure Lengend Snippet: Endogenous NF-κB transcription activation in cells expressing HTNV N protein. A549 cells were transfected with 500 ng of pWRG7077-Empty, pWRG7077-HTNV-S, or pWRG7077-HTNV-M for 24 h. After treatment of the cells with 50 ng/ml of TNF-α for 15 min, cytoplasmic and nuclear extracts from lysates were prepared. (A) Nuclear extracts were allowed to bind to NF-κB consensus sequence oligonucleotides on 96-well plates and then probed with antibodies specific for NF-κB p65 or NF-κB p50. The absorbance reading for each sample was determined using a spectrophotometer. OD, optical density; MUT oligo, mutated consensus oligonucleotide; WT oligo, wild-type consensus oligonucleotide. (B) Cytoplasmic extracts were used for immunoblotting to detect HTNV N protein and GAPDH. Each point represents an average ± standard deviation of results for six samples. The statistical significance of results for HTNV S with TNF and MG132 with TNF was determined by comparing the results to those for the empty vector with TNF. Asterisks indicate significant differences ( P

    Article Snippet: After being blocked with 10% goat serum in PBS, cells were sequentially stained, first with antibodies to p65 (Santa Cruz Biotechnologies) and then with antibodies to HTNV N (Abcam) and HTNV Gc (mouse immune sera).

    Techniques: Activation Assay, Expressing, Transfection, Sequencing, Spectrophotometry, Standard Deviation, Plasmid Preparation

    Effect of HTNV N protein on NF-κB gene expression. (A) 293T cells were cotransfected with 500 ng of pNF-κB-hrGFP and 500 ng of pWRG7077-Empty, pWRG7077-HTNV-S, or pWRG7077-HTNV-M. After 24 h, cells were incubated in medium with or without TNF-α (0 to 100 ng/ml) for 4 h. As a positive control for inhibition in our assay, cells transfected with only 500 ng of pNF-κB-hrGFP were pretreated with 50 μM MG132 for 2 h before the addition of TNF-α and throughout the experiment. (B) Cells were cotransfected with 500 ng of pNF-κB-hrGFP and 5 to 500 ng of pWRG7077-Empty, pWRG7077-HTNV-S, or pWRG7077-HTNV-M or transfected with 500 ng of pNF-κB-hrGFP and treated with 0 to 50 μM MG132. After 24 h, cells were incubated in medium with or without TNF-α (10 ng/ml) for 4 h. Following a 4-h treatment, medium was removed from all wells and replaced with medium lacking TNF-α for an additional 24 h. (C) Cells were cotransfected with 500 ng of pCAGGS-GFP and 500 ng of pWRG7077-Empty, pWRG7077-HTNV-S, or pWRG7077-HTNV-M. After 24 h, cells were incubated in medium with TNF-α (10 ng/ml) for an additional 24 h. GFP expression was examined by fluorescence microscopy.

    Journal: Journal of Virology

    Article Title: Hantaan Virus Nucleocapsid Protein Binds to Importin ? Proteins and Inhibits Tumor Necrosis Factor Alpha-Induced Activation of Nuclear Factor Kappa B ▿

    doi: 10.1128/JVI.00986-08

    Figure Lengend Snippet: Effect of HTNV N protein on NF-κB gene expression. (A) 293T cells were cotransfected with 500 ng of pNF-κB-hrGFP and 500 ng of pWRG7077-Empty, pWRG7077-HTNV-S, or pWRG7077-HTNV-M. After 24 h, cells were incubated in medium with or without TNF-α (0 to 100 ng/ml) for 4 h. As a positive control for inhibition in our assay, cells transfected with only 500 ng of pNF-κB-hrGFP were pretreated with 50 μM MG132 for 2 h before the addition of TNF-α and throughout the experiment. (B) Cells were cotransfected with 500 ng of pNF-κB-hrGFP and 5 to 500 ng of pWRG7077-Empty, pWRG7077-HTNV-S, or pWRG7077-HTNV-M or transfected with 500 ng of pNF-κB-hrGFP and treated with 0 to 50 μM MG132. After 24 h, cells were incubated in medium with or without TNF-α (10 ng/ml) for 4 h. Following a 4-h treatment, medium was removed from all wells and replaced with medium lacking TNF-α for an additional 24 h. (C) Cells were cotransfected with 500 ng of pCAGGS-GFP and 500 ng of pWRG7077-Empty, pWRG7077-HTNV-S, or pWRG7077-HTNV-M. After 24 h, cells were incubated in medium with TNF-α (10 ng/ml) for an additional 24 h. GFP expression was examined by fluorescence microscopy.

    Article Snippet: After being blocked with 10% goat serum in PBS, cells were sequentially stained, first with antibodies to p65 (Santa Cruz Biotechnologies) and then with antibodies to HTNV N (Abcam) and HTNV Gc (mouse immune sera).

    Techniques: Expressing, Incubation, Positive Control, Inhibition, Transfection, Fluorescence, Microscopy

    Examination of NF-κB p50 and p65 levels and TNF-α-induced IκBα degradation in HTNV N-expressing cells. A549 cells were transfected with 500 ng of pWRG7077-Empty, pWRG7077-HTNV-S, or pWRG7077-HTNV-M for 24 h. To induce the degradation of IκBα, cells were treated with 50 ng/ml of TNF-α for 15 min, and lysates were prepared for immunoblotting. For uninduced samples, cells were left untreated. Proteins were transferred onto PVDF membranes and probed with antibodies against IκBα, p50, p65, HTNV N protein, or GAPDH. +, with; −, without.

    Journal: Journal of Virology

    Article Title: Hantaan Virus Nucleocapsid Protein Binds to Importin ? Proteins and Inhibits Tumor Necrosis Factor Alpha-Induced Activation of Nuclear Factor Kappa B ▿

    doi: 10.1128/JVI.00986-08

    Figure Lengend Snippet: Examination of NF-κB p50 and p65 levels and TNF-α-induced IκBα degradation in HTNV N-expressing cells. A549 cells were transfected with 500 ng of pWRG7077-Empty, pWRG7077-HTNV-S, or pWRG7077-HTNV-M for 24 h. To induce the degradation of IκBα, cells were treated with 50 ng/ml of TNF-α for 15 min, and lysates were prepared for immunoblotting. For uninduced samples, cells were left untreated. Proteins were transferred onto PVDF membranes and probed with antibodies against IκBα, p50, p65, HTNV N protein, or GAPDH. +, with; −, without.

    Article Snippet: After being blocked with 10% goat serum in PBS, cells were sequentially stained, first with antibodies to p65 (Santa Cruz Biotechnologies) and then with antibodies to HTNV N (Abcam) and HTNV Gc (mouse immune sera).

    Techniques: Expressing, Transfection

    Translocation of NF-κB p65 in HTNV N-expressing cells. 293T cells were cotransfected with 500 ng of pCAGGS-GFP-p65 and 500 ng of pWRG7077-Empty, pWRG7077-HTNV-S, or pWRG7077-HTNV-M for 24 h. After incubation, cells were fixed and stained with antibodies against HTNV N or Gc protein (red) and with DAPI to highlight nuclei (blue). Arrows indicate areas of protein accumulation.

    Journal: Journal of Virology

    Article Title: Hantaan Virus Nucleocapsid Protein Binds to Importin ? Proteins and Inhibits Tumor Necrosis Factor Alpha-Induced Activation of Nuclear Factor Kappa B ▿

    doi: 10.1128/JVI.00986-08

    Figure Lengend Snippet: Translocation of NF-κB p65 in HTNV N-expressing cells. 293T cells were cotransfected with 500 ng of pCAGGS-GFP-p65 and 500 ng of pWRG7077-Empty, pWRG7077-HTNV-S, or pWRG7077-HTNV-M for 24 h. After incubation, cells were fixed and stained with antibodies against HTNV N or Gc protein (red) and with DAPI to highlight nuclei (blue). Arrows indicate areas of protein accumulation.

    Article Snippet: After being blocked with 10% goat serum in PBS, cells were sequentially stained, first with antibodies to p65 (Santa Cruz Biotechnologies) and then with antibodies to HTNV N (Abcam) and HTNV Gc (mouse immune sera).

    Techniques: Translocation Assay, Expressing, Incubation, Staining