Journal: The Journal of General Virology

Article Title: Ubiquitin-like modifier-activating enzyme 1 interacts with Zika virus NS5 and promotes viral replication in the infected cell

doi: 10.1099/jgv.0.002063

Figure Lengend Snippet: UBA1 interacts with orthoflaviviral NS5 proteins. ( a ) UBA1 is co-immunoprecipitated with ZIKV, USUV and WNV NS5 proteins. 293 T cells were transfected with constructs expressing HA-tagged versions of each NS5 (ZIKV, USUV and WNV), EYFP (pEYFP-N1, shown as pEYFP) or an empty vector (pcDNA3.1, shown as pcDNA). NS5 proteins were immunoprecipitated with anti-HA agarose beads. ( b ) UBA1 coprecipitated with recombinant His-tagged ZIKV NS5 in assays using Ni-NTA agarose resin (Invitrogen). For the assay, 3 mg of whole-cell protein, extracted from 293 T cells, was incubated in the presence of either 90 µg of recombinant BSA or His-tagged ZIKV NS5.

Article Snippet: The UBA1 transcript was then amplified using the Q5 High-Fidelity DNA Polymerase (New England Biolabs) and specific primers.

Techniques: Immunoprecipitation, Transfection, Construct, Expressing, Plasmid Preparation, Recombinant, Incubation

Journal: The Journal of General Virology

Article Title: Ubiquitin-like modifier-activating enzyme 1 interacts with Zika virus NS5 and promotes viral replication in the infected cell

doi: 10.1099/jgv.0.002063

Figure Lengend Snippet: Increased UBA1 protein levels in ZIKV- and USUV-infected cells. ( a ) Immunofluorescence assay (IFA) to the detection of UBA1 in mock or ZIKV-infected A549 cells. (b–e) UBA1 protein levels detected in A549 cell lysates collected at different time points in mock and infected cultures. ( b, c ) Representative blot of UBA1 kinetics in cells infected with either ZIKV (b) or USUV (c). ( d, e ) UBA1 protein level kinetics in cells infected with either ZIKV ( d ) or USUV ( e ). Each value in the graph is the average of three independent biological replicas ( n =3). Bars represent the sem for each data point. Significant differences between mock and infected samples are indicated (two-way ANOVA, Sidak’s multiple comparison test; * P <0.05; ** P <0.01). ( f ) Virus titre kinetics in the supernatant of A549 cells infected with either ZIKV (dark grey) or USUV (light grey).

Article Snippet: The UBA1 transcript was then amplified using the Q5 High-Fidelity DNA Polymerase (New England Biolabs) and specific primers.

Techniques: Infection, Immunofluorescence, Comparison, Virus

Journal: The Journal of General Virology

Article Title: Ubiquitin-like modifier-activating enzyme 1 interacts with Zika virus NS5 and promotes viral replication in the infected cell

doi: 10.1099/jgv.0.002063

Figure Lengend Snippet: ZIKV NS5 colocalizes with UBA1 in the infected cell. Fluorescence signals emitted by UBA1 and NS5 overlap in nuclear and cytoplasmic regions of the infected cells at 36 h post-infection (merge). To detect NS5 (green) and UBA1 (red), specific rabbit anti-NS5 and mouse anti-UBA1 antibodies were used. Nuclei were stained with DAPI (blue). All signals combined are represented (merge). ( a, c ) UBA1 and NS5 are detected in the nuclei of ZIKV-infected cells. ( b, d ) Close-up view of the areas where colocalization between UBA1 and NS5 was observed. An amplification of the image selected in the dotted square area in ( a ) and (c) is provided in (b) and ( d ), respectively. ( e ) Colocalization of NS5 and UBA1 in the cytoplasmic region of A549 cells. ( f ) An amplification of the image selected in the dotted square area in ( e ) is provided. Arrows indicate those regions where colocalization is detected.

Article Snippet: The UBA1 transcript was then amplified using the Q5 High-Fidelity DNA Polymerase (New England Biolabs) and specific primers.

Techniques: Infection, Fluorescence, Staining, Amplification

Journal: The Journal of General Virology

Article Title: Ubiquitin-like modifier-activating enzyme 1 interacts with Zika virus NS5 and promotes viral replication in the infected cell

doi: 10.1099/jgv.0.002063

Figure Lengend Snippet: UBA1 colocalizes with dsRNA in ZIKV-infected cells. Fluorescence emitted by UBA1 and dsRNA overlap in the cytoplasm of A549 cells at 48 h post-infection. To detect dsRNA (green) and UBA1 (red), specific mouse anti-dsRNA and rabbit anti-UBA1 antibodies were used. Nuclei are stained with DAPI (blue). All signals combined are represented (merge). ( a, c ) UBA1 and dsRNA are detected in the cytoplasm of ZIKV-infected cells. ( b, d ) Close-up view of the areas where colocalization between UBA1 and dsRNA was observed. An amplification of the image selected in the dotted square area in ( a ) and ( b ) is provided in ( c ) and ( d ), respectively. Arrows indicate those regions where colocalization is detected.

Article Snippet: The UBA1 transcript was then amplified using the Q5 High-Fidelity DNA Polymerase (New England Biolabs) and specific primers.

Techniques: Infection, Fluorescence, Staining, Amplification

Journal: The Journal of General Virology

Article Title: Ubiquitin-like modifier-activating enzyme 1 interacts with Zika virus NS5 and promotes viral replication in the infected cell

doi: 10.1099/jgv.0.002063

Figure Lengend Snippet: ZIKV and USUV replication is restricted in UBA1-depleted cells. ( a ) RT-qPCR analysis of UBA1 expression in A549 cells transfected with either a UBA1-targeting or a non-human target DsiRNA (NegC). UBA1 RNA expression was normalized to GAPDH mRNA levels. Values obtained are the average of four independent biological replicas ( n =4), and relative to untransfected cells carried out in parallel. Significant differences observed between samples are indicated (two-way ANOVA, Sidak’s multiple comparisons test; ** P <0.01; *** P <0.001). ( b, c ) UBA1 protein levels detected by WB in A549 cells previously transfected with either a specific (UBA1) or a non-human target (NegC) DsiRNA. Protein levels were normalized to the detection of tubulin. ( b ) Values are the average of three independent experiments ( n =3) and represented as a percentage of those observed in untransfected cells. A significant difference between samples was observed ( t -test; *** P <0.001). ( c ) Representative WB of UBA1 protein levels in cells transfected with DsiRNAs. ( d ) UBA1 detection by IFA in cells previously transfected with either a specific (DsiUBA1) or a non-human target (DsiNegC) DsiRNA. To detect UBA1 (red), a specific mouse anti-UBA1 antibody was used. Nuclei were stained with DAPI (blue). (e–h) Virus infectivity was examined by TCID 50 assays of samples collected at different time points from cells previously transfected with 3 ( e, g ) or 10 pmol ( f, h ) of each DsiRNA, as indicated in ( a ). Twenty-four hours after the second transfection with DsiRNAs, cells were infected with ZIKV ( e, f ) or USUV ( g, h ) at an m.o.i. of 0.1 TCID 50 /cell. Each value in the graph is the average of at least three independent biological replicas ( n ≥3). Significant differences observed between unspecific and UBA1-targeting DsiRNA-transfected cells are indicated (two-way ANOVA, Sidak’s multiple comparisons test; * P <0.05; ** P <0.01; *** P <0.001). Bars represent the sem for each data point.

Article Snippet: The UBA1 transcript was then amplified using the Q5 High-Fidelity DNA Polymerase (New England Biolabs) and specific primers.

Techniques: Quantitative RT-PCR, Expressing, Transfection, RNA Expression, Staining, Virus, Infection

Journal: The Journal of General Virology

Article Title: Ubiquitin-like modifier-activating enzyme 1 interacts with Zika virus NS5 and promotes viral replication in the infected cell

doi: 10.1099/jgv.0.002063

Figure Lengend Snippet: Enhanced ZIKV replication in cells overexpressing UBA1. ( a, b ) UBA1 is overexpressed in 293 T cells transfected with either pUBA1 ( a ) or pFLAG-UBA1 ( b ) when compared to untransfected cultures (mock) or cells transfected with an empty construct (pcDNA3.1, namely, pcDNA). ( a ) Larger levels of UBA1 detected in cells transfected with pUBA1; tubulin was used as a reference loading control. ( b ) Larger amounts of UBA1 detected in cells transfected with pFLAG-UBA1 when using a specific anti-UBA1 antibody. A positive band of the expected size for UBA1 was detected with an anti-FLAG antibody. Tubulin was used as a reference loading control. Representative images were included, which were obtained from two independent biological samples (two different WBs for each sample). ( c ) Virus titres obtained in cells previously transfected with pUBA1, pFLAG-UBA1 or an empty vector (pcDNA). Cells were infected with ZIKV at an m.o.i. of 1 TCID 50 /cell, and samples collected at different time points were titrated (four independent biological replicas). Significant differences with respect to mock-transfected cells (pcDNA) are indicated (two-way ANOVA, Dunnett’s multiple comparisons test; * P <0.05). ( d ) Representative WB of ZIKV NS5 and UBA1 protein levels in cells previously transfected (36 h earlier) with pUBA1, pFLAG-UBA1 or an empty vector (pcDNA); as a loading reference control, an anti-tubulin antibody was used (four independent biological replicas). ( e ) Ratio of NS5 and UBA1 protein levels relative to mock-transfected cells (empty vector) at 36 h post-infection. Tubulin was used as a reference control (four biological replicas).

Article Snippet: The UBA1 transcript was then amplified using the Q5 High-Fidelity DNA Polymerase (New England Biolabs) and specific primers.

Techniques: Transfection, Construct, Control, Virus, Plasmid Preparation, Infection

Journal: The Journal of General Virology

Article Title: Ubiquitin-like modifier-activating enzyme 1 interacts with Zika virus NS5 and promotes viral replication in the infected cell

doi: 10.1099/jgv.0.002063

Figure Lengend Snippet: UBA1 drugs PYR-41 and TAK-243 inhibit orthoflaviviral replication in cell culture. (a–f) UBA1 drugs PYR-41 and TAK-243 inhibit ZIKV and USUV replication in Vero cells. ( a, d ) Relative number of metabolically live Vero cells after treatment with increasing concentrations of PYR-41 ( a ) and TAK-243 ( d ) during 24 h. The relative amount of metabolically active cells in treated samples is calculated by using the CellTiter-Blue Cell Viability Assay kit as suggested by the manufacturer (Promega). Values are represented as a percentage relative to mock-treated cells. ( b, c, e, f ) The antiviral activity was examined by TCID 50 assays of samples collected from infected cells treated with increasing concentrations of drugs. To this, Vero cells were inoculated with either ZIKV at an m.o.i. of 0.2 TCID 50 /cell or USUV at 0.02 TCID 50 /cell. At 24 h post-infection, the cellular supernatants were collected and the virus titres were quantified. ( b, c ) Virus titres at 24 h post-infection in PYR-41-treated cells, previously infected with ZIKV ( b ) or USUV ( c ). ( e, f ) Virus titres at 24 h post-infection in TAK-243-treated cells, previously infected with ZIKV ( e ) or USUV ( f ). (g–l) UBA1 drug TAK-243 inhibits ZIKV and USUV replication in human A549 cells. ( g, j ) Relative number of metabolically active A549 cells after treatment with increasing concentrations of TAK-243 at 24 ( a ) and 48 ( d ) h. The relative number of viable cells is calculated as mentioned above. ( h, i, k, l ) The antiviral activity was examined by TCID 50 assays of samples collected at 24 or 48 h post-infection from cells treated with increasing concentrations of TAK-243. The treated cultures were previously inoculated with either ZIKV or USUV at an m.o.i. of 0.1 TCID 50 /cell. ( b, e ) ZIKV titres at 24 ( h ) and 48 ( k ) h post-infection. ( i, l ) USUV titres at 24 ( i ) and 48 ( l ) h post-infection. Each value in the graph is the average of at least three independent biological replicas ( n ≥3). Bars represent the sem for each data point. Significant differences between untreated controls and treated samples are indicated (one-way ANOVA test; * P <0.05; ** P <0.01; *** P <0.001).

Article Snippet: The UBA1 transcript was then amplified using the Q5 High-Fidelity DNA Polymerase (New England Biolabs) and specific primers.

Techniques: Cell Culture, Metabolic Labelling, Viability Assay, Activity Assay, Infection, Virus

Journal: Nature Communications

Article Title: HIV-1 promotes ubiquitination of the amyloidogenic C-terminal fragment of APP to support viral replication

doi: 10.1038/s41467-023-40000-x

Figure Lengend Snippet: a Transfection of HIV-1 JR-CSF reduces Flag-C99, but not Flag-C83 or GAPDH-HA in HEK293A cells. b Densitometry analysis of effects in ( a ) from 3 independent replicates. c Ubiquitination inhibitor TAK-243 stabilizes exogenous Flag-C99 but not C83-V5 in HEK293A cells transfected with either empty vector control (pcDNA3.1) or JR-CSF. d Densitometry analysis of effects in c from 3 independent replicates. e MG132 and TAK-243, but not DMSO control, stabilizes exogenous Flag-C99 in HEK293A cells transfected with JR-CSF. f Densitometry analysis of effects in ( e ) from 3 independent replicates. g Ubiquitin-activating enzyme E1 (UBE1) specific siRNA, but not negative control siRNA (NC2), suppresses downregulation of exogenous Flag-C99 in JR-CSF transfected HEK293A cells. h Densitometry analysis of effects in ( g ) from 3 independent replicates. i Schematic of N-terminally Flag-tagged WT or mutant C99 constructs used in ( j ). K: Lysine; A. Alanine. Each mutation site at the corresponding amino acid is represented by X. WB ( j ) and densitometry analysis ( k ) of effects of single or combined lysine-alanine (K-A) mutations on Flag-C99 stability in control pcDNA3.1 or JR-CSF transfected HEK293A cells. Complete stabilization mirroring TAK-243 treatment occurs in the C99-7KA mutant. l Representative ( n = 3) anti-Flag IP showing ubiquitination of Flag-C99 but not Flag-C99-7KA mutant in lysates from HEK293A transfected with JR-CSF. Red arrows highlight ubiquitinated bands. Quantification of the protein levels from 3 independent replicates is presented in b , d , f , h , k . Data is presented as mean, SEM . b one-way ANOVA with Tukey’s multiple comparisons test; d unpaired two-tailed t test with Welch’s correction; f unpaired two-tailed t test with Welch’s correction; h and k : two-way ANOVA with Sidak’s multiple comparisons test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns: not significant. Source data are provided as a Source Data file.

Article Snippet: Antibodies against APP/CTFs Y188 (ab32136, Abcam, western blotting (WB) 1:1000), APP (LN27, Invitrogen, 130200, immunofluorescene (IF) assay 1:150), ACTIN (A2103, Sigma, WB 1:1000), PARP (9542, Cell Signaling Technology, WB 1:1000), V5 (D3H8Q, 13202, Cell Signaling Technology, WB 1:1000), HIV-1 Pr55/p24/p17(ab63917, Abcam, WB 1:1000, IF assay 1:200) (labeled as Pr55 Gag in the Figures), HIV-1 p24 (ab9071, Abcam, IF assay 1:200), Ubiquitin (P4D1, 3936, Cell Signaling Technology, WB 1:1000), Flag (F7425, Sigma, WB 1:1000), Flag (L5, NBP1-06712, Novus, IF assay 1:100), Flag (M2, Sigma, see immunoprecipitation assay section), HA (H3663, Sigma, WB 1:1000), Rab7 (D95F2, 9367, Cell Signaling Technology, IF assay 1:200), EEA1 (C45B10, 3288, Cell Signaling Technology, IF assay 1:200), CD63 (H5C6, DSHB, IF assay 1:100), LAMP1 (1D4B, sc-19992, Santa Cruz, IF assay 1:100) and UBE1 (15912-1-AP, Proteintech, WB 1:1000) were used according to manufacturer’s instructions.

Techniques: Transfection, Ubiquitin Proteomics, Plasmid Preparation, Control, Negative Control, Mutagenesis, Construct, Two Tailed Test