zikv ns5 protein antibody  (GeneTex)

Journal: Virulence

Article Title: LAMR1 restricts Zika virus infection by attenuating the envelope protein ubiquitination

doi: 10.1080/21505594.2021.1948261

Figure Lengend Snippet: LAMR1 is a host restriction factor against ZIKV infection . (a–d) HeLa cells were transfected with pHA-LAMR1 or empty vector for 16 h and then infected with ZIKV (MOI = 1) for 48 h. The expression levels of ZIKV proteins were detected by immunoblotting (a) and confocal microscopy (c) and the viral RNA content was quantified by qPCR (b) and confocal microscopy (d). (e–g) Hela cells stably expressing LAMR1 or the control gene were generated and analyzed(e). Cells were infected with ZIKV (MOI = 1) for 48 h, following which viral RNA levels were quantified by qPCR (f) and ZIKV E protein levels by high-content analysis (g). (h–m) HeLa cells stably expressing sh-LAMR1 or control sh-RNA were generated and analyzed(h). Cells were infected with ZIKV (MOI = 1) for 48 h, and the cytopathic effects of cells was captured under the microscope (i). The expression levels of viral proteins were assessed by immunoblotting (j), while ZIKV titer in supernatants was calculated through a plaque assay (l, m)

Article Snippet: Antibodies against ZIKV NS5 protein (GTX133312) and E protein (GTX133314) were purchased from GeneTex (Hsinchu, Taiwan, P.R.C).

Techniques: Infection, Transfection, Plasmid Preparation, Expressing, Western Blot, Confocal Microscopy, Stable Transfection, Control, Generated, High Content Screening, Microscopy, Plaque Assay

Journal: Virulence

Article Title: LAMR1 restricts Zika virus infection by attenuating the envelope protein ubiquitination

doi: 10.1080/21505594.2021.1948261

Figure Lengend Snippet: LAMR1 binds to ZIKV E protein through its intracellular domain . (a) HEK293T cells were transfected with plasmids encoding HA-LAMR1 and Flag-E or pFlag-prM. Cell lystes were prepared using lysis buffer and then used for immunoprecipitation (IP) with anti-Flag antibody and analyzed by SDS-PAGE. (b) HEK293T cells were transfected with plasmids encoding HA-LAMR1 and Flag-E. Cell lysates were prepared using lysis buffer and then used for IP with anti-Flag antibody or control IgG and analyzed by SDS-PAGE. (c) Vero cells were infected with ZIKV (MOI = 1) for 48 h and then subjected to IP with anti-LAMR1 antibody or control IgG. (d) A yeast two-hybrid screen was used to identify the interaction between LAMR1 and E protein. (e) HEK293T cells were transfected with pFlag-E or pHA-LAMR1, or co-transfected with pFlag-E and pHA-LAMR1. Immunofluorescence staining showed the sub-cellular localization of, HA-LAMR1 (red) and Flag-E (green); the nucleus is marked with DAPI (blue). (f) HEK293T cells were transfected with plasmids encoding Flag-E and GFP-vector/GFP-LAMR1 (1–85)/GFP-LAMR1 (86–295). Cell lysates were prepared using lysis buffer and then used for IP with anti-GFP antibody and analyzed by SDS-PAGE. (g) The Glutathione Sepharose beads were added to GST-E protein or GST only. The mixtures were then incubated with whole-cell extracts of HEK293T cells transfected with a plasmid encoding Flag-LAMR1 (1–85) (h) HEK293T cells were transfected with pFlag-E and pGFP-vector, pGFP-LAMR1 (1–85), and pGFP-LAMR1 (86–295). The sub-cellular localization of GFP, GFP-LAMR1 (1–85), GFP-LAMR1 (86–295) (green) and Flag-E (red) were analyzed by confocal microscopy; the nucleus is marked with DAPI (blue)

Article Snippet: Antibodies against ZIKV NS5 protein (GTX133312) and E protein (GTX133314) were purchased from GeneTex (Hsinchu, Taiwan, P.R.C).

Techniques: Transfection, Lysis, Immunoprecipitation, SDS Page, Control, Infection, Two Hybrid Screening, Immunofluorescence, Staining, Plasmid Preparation, Incubation, Confocal Microscopy

Journal: Virulence

Article Title: LAMR1 restricts Zika virus infection by attenuating the envelope protein ubiquitination

doi: 10.1080/21505594.2021.1948261

Figure Lengend Snippet: The conserved E protein G282 residue is essential for the binding of LAMR1 . (a) Diagrams of the full-length E protein, truncated forms of E protein (1–505, 52–505, 132–505, 193–505, 280–505, 1–93, 1–296, and 1–406) and deletion forms of E protein (Δ280–283, Δ284–287, Δ288–291, and Δ292–295). (b) HEK293T cells were transfected with plasmids encoding HA-LAMR1 and Flag-vector, Flag-E/Flag-E (52–505), Flag-E (132–505), Flag-E (193–505), and Flag-E (280–505). Cell lysates were prepared using lysis buffer and then analyzed by immunoprecipitation with the indicated antibodies. (c) HEK293T cells were transfected with plasmids encoding HA-LAMR1 and pFlag-vector, Flag-E/Flag-E (1–193), Flag-E (1–296), and pFlag-E (1–406). Cell lysates were prepared using lysis buffer and then analyzed by immunoprecipitation with the indicated antibodies. (d) HEK293T cells were transfected with plasmids encoding HA-LAMR1 and Flag-vector, Flag-E, Flag-E (280–283aa deletion), Flag-E (284–287aa deletion), Flag-E (288–291aa deletion), and Flag-E (292–295aa deletion). Cell lysates were prepared using lysis buffer and then analyzed by immunoprecipitation with the indicated antibodies. (e) HEK293T cells were transfected with plasmids encoding HA-LAMR1 and Flag-vector, Flag-E, Flag-E G282A, and Flag-E DENV II. Cell lysates were prepared with lysis buffer and then analyzed by immunoprecipitation with indicated antibodies. (f) HEK293T cells were transfected with plasmids encoding HA-LAMR1 and Flag-vector, Flag-E, Flag-E A280V, Flag-E K281R, Flag-E G282A, and Flag-E R283A. Cell lysates were prepared with lysis buffer and then analyzed by immunoprecipitation with indicated antibodies. (g) Diagram of the consered G282 site. Viral sequences were downloaded from GenBank, and viewed and aligned using AliView software. (h) The diagrams of amino acid sequences of partial E protein among ZIKV, WNV, JEV and DENV. Sequences were downloaded from GenBank and viewed and aligned using AliView software

Article Snippet: Antibodies against ZIKV NS5 protein (GTX133312) and E protein (GTX133314) were purchased from GeneTex (Hsinchu, Taiwan, P.R.C).

Techniques: Residue, Binding Assay, Transfection, Plasmid Preparation, Lysis, Immunoprecipitation, Software

Journal: Virulence

Article Title: LAMR1 restricts Zika virus infection by attenuating the envelope protein ubiquitination

doi: 10.1080/21505594.2021.1948261

Figure Lengend Snippet: LAMR1 recruits EIF3S5 to deubiquitinate ZIKV E protein . (a) HEK293T cells were co-transfected with pHA-LAMR1 and pFlag-USP13, pFlag-USP15, pFlag-USP26, pFlag-USP30, pFlag-USP38, pFlag-USP49, pFlag-OTUB1, pFlag-EIF3S5, or pFlag-BRCC3. Lysates were prepared and used for IP with an anti-Flag antibody and analyzed by SDS-PAGE. (b) HEK293T cells were co-transfected with pHA-E, pMyc-UB, pFlag-USP13, and pFlag-EIF3S5. Lysates were prepared and used for IP with an anti-HA antibody and then analyzed by SDS-PAGE. (c) EIF3S5-knockdown HEK293T cells and control cells were co-transfected with pFlag-E, pMyc-UB, and pHA-LAMR1. Lysates were prepared and used for IP with an anti-Flag antibody and then analyzed by SDS-PAGE. (d) LAMR1-knockdown HeLa cells and control cells were co-transfected with pFlag-E, pMyc-UB, and pHA-EIF3S5. Lysates were prepared and used for IP with an anti-Flag antibody and then analyzed by SDS-PAGE. (e, f) HEK293T cells were transfected with plasmids encoding Flag-EIF3S5 and HA-LAMR1. Cell lysates were prepared with lysis buffer and then analyzed by IP with the indicated antibodies. (g, h) HEK293T cells were transfected with plasmids encoding Flag-EIF3S5 and HA-E. Cell lysates were prepared with lysis buffer and then analyzed by IP with indicated antibodies and immunoblotting as described above. (i) HEK293T cells were transfected with plasmids encoding Flag-EIF3S5 and GFP-LAMR1, GFP-LAMR1 (1–85aa), and GFP-LAMR1 (86–259aa). Cell lysates were prepared with lysis buffer and then analyzed by IP with the indicated antibodies. (j, k) HeLa cells stably expressing sh-EIF3S5 or control sh-RNA were generated and analyzed. Cells were transfected with pHA-LAMR1 or empty vector for 16 h, and then infected with ZIKV (MOI = 1) for 48 h. The levels of viral protein and RNA were detected by immunoblotting and qPCR, respectively

Article Snippet: Antibodies against ZIKV NS5 protein (GTX133312) and E protein (GTX133314) were purchased from GeneTex (Hsinchu, Taiwan, P.R.C).

Techniques: Transfection, SDS Page, Knockdown, Control, Lysis, Western Blot, Stable Transfection, Expressing, Generated, Plasmid Preparation, Infection

Journal: Emerging Microbes & Infections

Article Title: STAT2-dependent restriction of Zika virus by human macrophages but not dendritic cells

doi: 10.1080/22221751.2021.1929503

Figure Lengend Snippet: MDMs are susceptible to ZIKV infection. Human monocyte-derived macrophages (MDMs) and dendritic cells (moDCs) were uninfected or infected with ZIKV PR or ZIKV U at an MOI of 1.0. At 48 h post-infection (hpi), the cells were fixed with 4% paraformaldehyde and then permeabilized with 0.2% Triton X-100. (A) Viral double-stranded RNA (dsRNA) and (B) envelope (E) protein were identified with J2 and 4G2 antibodies, respectively, and imaged with confocal microscopy. In parallel, MDMs and moDCs were infected with ZIKV PR or ZIKV U at an MOI of 0.1. At 24hpi and 48hpi, ZIKV-infected and mock-infected cells were fixed and stained with J2 or 4G2, and subsequently applied to flow cytometry to determine expression levels of (C) viral dsRNA and (D) E protein. The dot-plot showed data of one representative donor. Statistical analyses in all panels were performed with Student’s t-test and the differences were considered significant when p < 0.05. * p < 0.05, ** p < 0.01. ns, not significant. The histogram showed the mean values and standard deviations from three donors. Bars represented 10 μm.

Article Snippet: After 24hpi, the whole cell lysates were incubated with protein A/G magnetic beads pre-crosslinked with anti-ZIKV NS5 antibody (GeneTex) or anti-STAT2 antibody (SANTA CRUZ BIOTECHNOLOGY).

Techniques: Infection, Derivative Assay, Confocal Microscopy, Staining, Flow Cytometry, Expressing

Journal: Emerging Microbes & Infections

Article Title: STAT2-dependent restriction of Zika virus by human macrophages but not dendritic cells

doi: 10.1080/22221751.2021.1929503

Figure Lengend Snippet: MDMs but not moDCs restrict ZIKV replication. MDMs and moDCs were infected with ZIKV PR or ZIKV U at an MOI of 0.01 or 1.0. Viral genome copy in the ( A ) cell lysates and ( B ) supernatants were determined by qRT-PCR. (C) The live infectious virus titer in the supernatants was measured by plaque assays. (D) MDMs and moDCs were infected with ZIKV PR or ZIKV U at an MOI of 0.1. The supernatants were harvested for determining the infectious virus titer by plaque assays at the indicated days post-infection. The ( E ) cell viability and ( F ) caspase-3/7 activities of uninfected and ZIKV-infected MDMs; and ( G ) cell viability and ( H ) caspase-3/7 activities of uninfected and ZIKV-infected moDCs were determined at the indicated time points by CellTiterGlo and CaspaseGlo 3/7 assays, respectively. Data represented mean and standard deviations from 3–6 donors. Statistical analyses in all panels were performed with two way-ANOVA and the differences were considered significant when p < 0.05. * p < 0.05,** p < 0.01, and *** p < 0.001.

Article Snippet: After 24hpi, the whole cell lysates were incubated with protein A/G magnetic beads pre-crosslinked with anti-ZIKV NS5 antibody (GeneTex) or anti-STAT2 antibody (SANTA CRUZ BIOTECHNOLOGY).

Techniques: Infection, Quantitative RT-PCR, Virus

Journal: Emerging Microbes & Infections

Article Title: STAT2-dependent restriction of Zika virus by human macrophages but not dendritic cells

doi: 10.1080/22221751.2021.1929503

Figure Lengend Snippet: Restricted ZIKV replication in MDMs is not due to inefficient virus entry. ( A ) MDMs were untreated or treated with IL-13 (20 and 50 ng/mL) for 24 h and then subjected to determine the expression levels of ZIKV-related entry factors, including DC-SIGN, AXL, TIM-1, and MERTK by qRT-PCR. moDCs were included as a control. ( B ) MDMs were untreated or treated with IL-13 (50 ng/mL) for 24 h prior to infection with ZIKV PR or ZIKV U at an MOI of 0.01. The viral genome copy in the cell lysates was determined by qRT-PCR at 24hpi and 48hpi. ( C) Viral entry capability in MDMs and moDCs were determined by qRT-PCR and normalized with GAPDH. Data represented mean and standard deviations from 3 donors. Statistical analyses in all panels were performed with one way-ANOVA and the differences were considered significant when p < 0.05. * p < 0.05, ** p < 0.01, and *** p < 0.001. ns, not significant.

Article Snippet: After 24hpi, the whole cell lysates were incubated with protein A/G magnetic beads pre-crosslinked with anti-ZIKV NS5 antibody (GeneTex) or anti-STAT2 antibody (SANTA CRUZ BIOTECHNOLOGY).

Techniques: Virus, Expressing, Quantitative RT-PCR, Control, Infection

Journal: Emerging Microbes & Infections

Article Title: STAT2-dependent restriction of Zika virus by human macrophages but not dendritic cells

doi: 10.1080/22221751.2021.1929503

Figure Lengend Snippet: ZIKV infection does not antagonize type I IFN-mediated responses in MDMs. ( A ) Schematic illustration of human recombinant IFN-α treatment and ZIKV infection. ( B ) MDMs and moDCs were uninfected or infected with ZIKV PR at an MOI of 10.0 for 6 h, followed by mock treatment or treatment with 1000U/mL of recombinant human IFN-α for 6 h. Cell lysates were collected for detection of ISGs induction using qRT-PCR, including IFIT1, ISG15, MX1, and PKR; the fold activations were calculated as compared to mock groups. Data represented mean and standard deviations from 3 donors. Statistical analyses in all panels were performed with one way-ANOVA and the differences were considered significant when p < 0.05. ** p < 0.01, *** p < 0.001, and **** p < 0.0001. ns, not significant.

Article Snippet: After 24hpi, the whole cell lysates were incubated with protein A/G magnetic beads pre-crosslinked with anti-ZIKV NS5 antibody (GeneTex) or anti-STAT2 antibody (SANTA CRUZ BIOTECHNOLOGY).

Techniques: Infection, Recombinant, Quantitative RT-PCR

Journal: Emerging Microbes & Infections

Article Title: STAT2-dependent restriction of Zika virus by human macrophages but not dendritic cells

doi: 10.1080/22221751.2021.1929503

Figure Lengend Snippet: ZIKV infection fails to inhibit the phosphorylation of STAT1 and STAT2 in MDMs. (A) MDMs and moDCs were uninfected or infected with ZIKV PR at an MOI of 1.0. At 48hpi, the cells were untreated or treated with 1000U/mL of recombinant human IFN-α for 40 min. The cell lysates were harvested for detecting expressions of pSTAT1, pSTAT2, STAT1, STAT2, and β-actin by Western blots. Representative blots represented data from three independent experiments. ( B ) Quantitation was calculated as the ratio of pSTAT/STAT protein from three independent experiments. Statistical analyses in all panels were performed with one way-ANOVA and the differences were considered significant when p < 0.05. ** p < 0.01, and *** p < 0.001. ns, not significant.

Article Snippet: After 24hpi, the whole cell lysates were incubated with protein A/G magnetic beads pre-crosslinked with anti-ZIKV NS5 antibody (GeneTex) or anti-STAT2 antibody (SANTA CRUZ BIOTECHNOLOGY).

Techniques: Infection, Phospho-proteomics, Recombinant, Western Blot, Quantitation Assay

Journal: Emerging Microbes & Infections

Article Title: STAT2-dependent restriction of Zika virus by human macrophages but not dendritic cells

doi: 10.1080/22221751.2021.1929503

Figure Lengend Snippet: Depletion of STAT2 but not STAT1 rescues ZIKV and YFV replication in MDMs. ( A ) Schematic illustration of siRNA transfection and ZIKV infection. ( B ) MDMs and moDCs were transfected with scrambled siRNA or siRNA targeting STAT1 or STAT2. The knockdown efficiency of siRNA transfection was determined by Western blots. ( C ) MDMs and moDCs were generated from 2 donors. The cells were transfected with scrambled siRNA or siRNA targeting STAT1 or STAT2, followed by infection with ZIKV PR at an MOI of 1.0. At 2hpi and 24hpi, the cell lysates and supernatants were harvested for detection of viral genome copy and live infectious virus titer by qRT-PCR and plaque assays, respectively. ( D ) MDMs and moDCs were transfected with the same procedure as mentioned above and then infected with YFV at an MOI of 1.0. The cell lysates and supernatants were collected for further detection at 24hpi. Statistical analyses in all panels were performed with one way-ANOVA and the differences were considered significant when p < 0.05. * p < 0.05, and **** p < 0.0001. ns, not significant.

Article Snippet: After 24hpi, the whole cell lysates were incubated with protein A/G magnetic beads pre-crosslinked with anti-ZIKV NS5 antibody (GeneTex) or anti-STAT2 antibody (SANTA CRUZ BIOTECHNOLOGY).

Techniques: Transfection, Infection, Knockdown, Western Blot, Generated, Virus, Quantitative RT-PCR