Journal: Biomedical Journal

Article Title: Interferon-alpha and MxA inhibit BK polyomavirus replication by interaction with polyomavirus large T antigen

doi: 10.1016/j.bj.2023.100682

Figure Lengend Snippet: Inhibition of BKPyV replication by IFNα HRPTECs were infected with BKPyV (1 × 10 6 copies/mL) for 2 h and then incubated in the presence or absence of human IFNα (0−20 ng/mL) for an additional 72 h. At the end of experiments, cell lysates were collected for Western blot analysis (A) to determine the protein expression of MxA and VP1, and the BKPyV viral titer was measured by qPCR (B) from the collected supernatant. (C) Eight-to ten-week Balb/c female mice received 30 min-ischemic reperfusion injury (IRI) of a unilateral kidney. Mice were then intraperitoneally inoculated with MuPyV wild-type strain (2 × 10 6 plaque-forming units). Two doses of IFNα (1 MIU/Kg) or PBS were subcutaneously injected on day 1 and day 4. After seven days of inoculation, the mice were sacrificed, and fold change of MuPyV VP1 mRNA relative expression using 18S rRNA (18S) as reference in the IRI and non-IRI kidneys was determined by RT-qPCR. The results of cell culture experiments represent three independent experiments. For animal study, each experimental group comprised 4 to 6 animals. The results are presented as mean ± SEM. Statistical significance: ns, not significant; ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.005; ∗∗∗∗, p < 0.001.

Article Snippet: Human primary renal proximal tubular epithelial cells (HRPTECs) were purchased from the American Type Culture Collection (ATCC).

Techniques: Inhibition, Infection, Incubation, Western Blot, Expressing, Injection, Quantitative RT-PCR, Cell Culture

Journal: Biomedical Journal

Article Title: Interferon-alpha and MxA inhibit BK polyomavirus replication by interaction with polyomavirus large T antigen

doi: 10.1016/j.bj.2023.100682

Figure Lengend Snippet: Inhibition of BKPyV replication by MxA HRPTECs were transfected with varying doses (0.1a2 μg) of FLAG-tagged MxA-expressing plasmids or an empty plasmid for 12 h. Subsequently, they were infected with BKPyV (1 × 10 6 copies/mL) for 2 h. After replacing the medium, the cells were cultured for an additional 72 h. BKPyV-infected cells transfected with the empty vector served as the negative control, while BKPyV-infected cells treated with 10 ng/mL of IFNα served as the positive control. At the end of the experiments, viral titer analysis (A) was performed on the collected supernatant, and Western blot analysis (B) was conducted on cell lysates to determine the expressions of TAg, VP1, and MxA proteins. Bar graphs depict the normalized levels of BKPyV TAg (C) and VP1 (D) relative to GAPDH. The data represent three independent experiments. The results are presented as mean ± SEM. Statistical significance: ns, not significant; ∗∗, p < 0.01; ∗∗∗, p < 0.005; ∗∗∗∗, p < 0.001.

Article Snippet: Human primary renal proximal tubular epithelial cells (HRPTECs) were purchased from the American Type Culture Collection (ATCC).

Techniques: Inhibition, Transfection, Expressing, Plasmid Preparation, Infection, Cell Culture, Negative Control, Positive Control, Western Blot

Journal: Biomedical Journal

Article Title: Interferon-alpha and MxA inhibit BK polyomavirus replication by interaction with polyomavirus large T antigen

doi: 10.1016/j.bj.2023.100682

Figure Lengend Snippet: MxA silencing diminished IFNα′s anti-BKPyV effect HRPTECs were transfected with varying doses of MxA siRNA or scrambled RNA for 12 h. Afterward, they were treated with IFNα (10 ng/mL) and infected with BKPyV (1 × 10 6 copies/mL) for an additional 72 h. Western blot analysis was performed to assess the expression levels of TAg, VP1, and MxA proteins (A). Bar graphs depict the normalized levels of BKPyV MxA (B) and VP1 (C) relative to GAPDH. The data represent three independent experiments. The results are presented as mean ± SEM. Statistical significance: ns, not significant; ∗, p < 0.05; ∗∗∗∗, p < 0.001.

Article Snippet: Human primary renal proximal tubular epithelial cells (HRPTECs) were purchased from the American Type Culture Collection (ATCC).

Techniques: Transfection, Infection, Western Blot, Expressing

Journal: Biomedical Journal

Article Title: Interferon-alpha and MxA inhibit BK polyomavirus replication by interaction with polyomavirus large T antigen

doi: 10.1016/j.bj.2023.100682

Figure Lengend Snippet: Mutation of MxA residue 103 decreases antiviral activity against BK polyomavirus . HRPTECs were transfected with plasmids encoding FLAG-tagged wild-type MxA, GTP binding-defective MxA mutants (K83A and D250 N), or the GTPase-deficient MxA mutant (T103A) for 12 h. Cells transfected with the empty vector served as the negative control. Subsequently, the cells were infected with BKPyV (1 × 10 6 copies/mL) for an additional 72 h. Western blot analysis was conducted to determine the expression levels of wild-type MxA, MxA mutants, BKPyV TAg, and VP1 proteins. Bar graphs represent the normalized levels of BKPyV TAg (B) and VP1 (C) relative to GAPDH. The results are presented as mean ± SEM. Statistical significance: ns, not significant; ∗, p < 0.05; ∗∗, p < 0.01.

Article Snippet: Human primary renal proximal tubular epithelial cells (HRPTECs) were purchased from the American Type Culture Collection (ATCC).

Techniques: Mutagenesis, Residue, Activity Assay, Transfection, Binding Assay, Plasmid Preparation, Negative Control, Infection, Western Blot, Expressing

Journal: Biomedical Journal

Article Title: Interferon-alpha and MxA inhibit BK polyomavirus replication by interaction with polyomavirus large T antigen

doi: 10.1016/j.bj.2023.100682

Figure Lengend Snippet: BKPyV TAg is co-immunoprecipitated with wild-type MxA but not with the MxA T103A mutant . A. HRPTECs were co-transfected with BKPyV TAg-expressing plasmid and FLAG-tagged wild-type MxA-expressing plasmid. Control transfections were the TAg-expressing or wild-type MxA-expressing plasmid alone. Immunoprecipitation assay was performed by immunoprecipitation with anti-SV40 TAg antibody. The TAg-binding MxA in the TAg-associated immunocomplexes was assessed by subsequent immunoblotting with an anti-FLAG antibody. The protein levels of inputted MxA, TAg, and GAPDH were determined through immunoblotting using their respective specific antibodies. B. HRPTECs were co-transfected with either FLAG-tagged MxA or FLAG-tagged MxA mutants (K83A and T103A)-expressing plasmids, along with the TAg-expressing plasmid. The immunoprecipitation assay was conducted using an anti-SV40 TAg antibody, followed by immunoblotting with an anti-FLAG antibody.

Article Snippet: Human primary renal proximal tubular epithelial cells (HRPTECs) were purchased from the American Type Culture Collection (ATCC).

Techniques: Immunoprecipitation, Mutagenesis, Transfection, Expressing, Plasmid Preparation, Control, Binding Assay, Western Blot

Journal: Biomedical Journal

Article Title: Interferon-alpha and MxA inhibit BK polyomavirus replication by interaction with polyomavirus large T antigen

doi: 10.1016/j.bj.2023.100682

Figure Lengend Snippet: MxA and BKPyV TAg are colocalized in the cytoplasm . A. HRPTECs were infected with BKPyV (1 × 10 6 copies/mL). After 72 h, cells were fixed and stained for MxA and TAg. B&C. HRPTECs were initially infected with BKPyV (1 × 10 6 copies/mL) for 2 h, followed by incubation in the absence (B) or presence (C) of human IFNα (10 ng/mL) for an additional 72 h. D. HRPTECs were transfected with wild-type MxA-expressing plasmids for 12 h and infected with BKPyV (1 × 10 6 copies/mL). After 72 h, cells were fixed and stained for MxA and TAg. MxA was labeled with anti-MxA and a green fluorescent (Alexa Fluor 488) secondary antibody, and BKV TAg was labeled with anti-SV40 TAg and a red fluorescent (Alexa Fluor 647) secondary antibody. In MxA-expressing cells, MxA exhibited colocalization with BKPyV TAg in the cytoplasm, displaying a diffuse granulate pattern (indicated by white arrowheads) (C&D). In BKPyV infected non-MxA expressing cells, TAg could be seen exclusively in the nucleus (indicated by grey arrows) (A, C&D).

Article Snippet: Human primary renal proximal tubular epithelial cells (HRPTECs) were purchased from the American Type Culture Collection (ATCC).

Techniques: Infection, Staining, Incubation, Transfection, Expressing, Labeling