Journal: Journal of Virology

Article Title: LY6E Restricts Entry of Human Coronaviruses, Including Currently Pandemic SARS-CoV-2

doi: 10.1128/JVI.00562-20

Figure Lengend Snippet: IFITMs modulate HCoV-OC43 infection of HepG2 and C3A cells to a similar extent and via the same mechanism. HepG2 and C3A cells were stably transduced with a control retroviral vector (pQCXIP) or a retroviral vector expressing an N-terminally FLAG-tagged IFITM1-EX2 or IFITM3-EX2. The resulting cell lines were infected with HCoV-OC43 at 0.5 MOI. (A) Cells were fixed at 24 hpi and virally infected cells were visualized by IF staining of HCoV-OC43 N protein (green). Cell nuclei were visualized by DAPI staining (blue). (B) HCoV-OC43 NP and exogenously expressed N-terminally FLAG-tagged IFITM proteins and total intracellular IFITM2/3 were determined by Western blotting assays with a monoclonal antibody against the FLAG tag and a rabbit polyclonal antibody against IFITM2/3. β-actin served as a loading control. (C) Intracellular viral RNA was quantified by a qRT-PCR assay and presented as copies per 100 ng total RNA. Error bars indicate standard deviations ( n = 4). (D) Viral yields were determined with a plaque assay. Error bars indicate standard deviations ( n = 4). (E) HepG2 and C3A stably transduced with a control retroviral vector (pQCXIP) or a retroviral vector expressing IFITM1, IFITM1-EX2, or IFITM3-EX2 were infected with HCoV-OC43pp. Luciferase activities were determined at 72 hpi. Relative infection represents the luciferase activity normalized to that of HepG2 cells transduced with empty vector (pQCXIP). Error bars indicate standard deviations ( n = 6).

Article Snippet: Rabbit polyclonal antibody against human LY6E was obtained from Proteintech (catalog number 22144-1-AP).

Techniques: Infection, Stable Transfection, Transduction, Control, Retroviral, Plasmid Preparation, Expressing, Staining, Western Blot, FLAG-tag, Quantitative RT-PCR, Plaque Assay, Luciferase, Activity Assay

Journal: Journal of Virology

Article Title: LY6E Restricts Entry of Human Coronaviruses, Including Currently Pandemic SARS-CoV-2

doi: 10.1128/JVI.00562-20

Figure Lengend Snippet: LY6E efficiently suppresses human coronavirus spike-protein-mediated entry. (A) Levels of Ly6E, GILT, and ADAP2 mRNA expression in HepG2 and C3A cells were determined by qRT-PCR assays and normalized to the level of GAPDH. (B) Flp-In T-Rex 293-derived cell lines expressing control protein CAT, GILT, or ADAP2 were cultured in the absence or presence of tet for 24 h. The cells were infected with HCoV-OC43pp and other indicated pseudoviral particles and intracellular luciferase activity were determined at 48 hpi. Relative infection is the ratio of luciferase activity in the same cells cultured in the presence of tet over that in the absence of tet. The error bars refer to standard deviations ( n = 4). (C) Flp-In T-Rex 293-derived cell line expressing a control protein CAT or LY6E were cultured in the absence or presence of tet. Cells were harvested at 24 h after the addition of tet. The cellular expression of LY6E was detected by a Western blot assay. β-actin served as a loading control. (D) Flp-In T-Rex 293-derived cell lines expressing LY6E were cultured in the absence or presence of tet for 24 h. The cells were then infected with lentiviral particles pseudotyped with the envelope protein of the indicated viruses. Luciferase activities were determined at 48 hpi. Relative infection is the ratio of luciferase activity in the same cells cultured in the presence of tet over that in the absence of tet. The error bars refer to standard deviations ( n = 4). **, P < 0.001 compared to the control cells expressing CAT.

Article Snippet: Rabbit polyclonal antibody against human LY6E was obtained from Proteintech (catalog number 22144-1-AP).

Techniques: Expressing, Quantitative RT-PCR, Derivative Assay, Control, Cell Culture, Infection, Luciferase, Activity Assay, Western Blot

Journal: Journal of Virology

Article Title: LY6E Restricts Entry of Human Coronaviruses, Including Currently Pandemic SARS-CoV-2

doi: 10.1128/JVI.00562-20

Figure Lengend Snippet: LY6E inhibits HCoV-OC43 infection in human hepatoma (HepG2 and C3A) and lung cancer (A549) cells. (A) HepG2 cells were stably transduced with scramble shRNA or shRNA targeting LY6E mRNA. The level of cellular LY6E expression was determined by Western blotting using a rabbit polyclonal antibody against LY6E. β-actin served as a loading control. (B) HepG2 cells stably expressing the scramble shRNA or LY6E-specific shRNA were infected with HCoV-OC43 at an MOI of 1.0. Cells were harvested at 24 hpi and intracellular viral RNA was quantified by qRT-PCR assay and presented as copies per 100 ng total RNA. Error bars indicate standard deviations ( n = 4). Differences in viral RNA between scramble or LY6E-specific shRNA-expressing cells were analyzed statistically (**, P < 0.001; Student’s t test). (C to F) C3A or A549 cells were stably transduced with an empty retroviral vector (pQCXIP) or retroviral vector expressing LY6E and infected with HCoV-OC43 at the indicated MOI. The expression of LY6E in the cell lines was confirmed by a Western blot assay. β-actin served as a loading control (C and E). The cells were fixed at 24 hpi. The infected cells were visualized by IF staining of HCoV-OC43 N protein (red); cell nuclei were visualized by DAPI staining (D and F).

Article Snippet: Rabbit polyclonal antibody against human LY6E was obtained from Proteintech (catalog number 22144-1-AP).

Techniques: Infection, Stable Transfection, Transduction, shRNA, Expressing, Western Blot, Control, Quantitative RT-PCR, Retroviral, Plasmid Preparation, Staining

Journal: Journal of Virology

Article Title: LY6E Restricts Entry of Human Coronaviruses, Including Currently Pandemic SARS-CoV-2

doi: 10.1128/JVI.00562-20

Figure Lengend Snippet: Identification of critical structural motifs essential for LY6E to restrict human coronavirus entry. (A) The amino acid sequence alignment of LY6E from multiple vertebrate species is presented, in which the “three finger-fold” structure is highlighted with black boxes. The conserved L36 as well as the GPI anchor and N99 glycosylation sites are indicated. (B) Flp-In T-Rex 293-derived cell lines expressing a control protein CAT or wild-type or mutant LY6E were cultured in the absence or presence of tet for 24 h. LY6E expression was detected by a Western blot assay in which β-actin served as a loading control. (C) Flp-In T-Rex 293-derived cell lines expressing the wild-type or mutant LY6E were cultured in the absence or presence of tet for 24 h. The cells were then infected with the indicated pseudotyped lentivirus. Luciferase activities were measured at 48 hpi. Relative infection is the ratio of luciferase activity in the same cells cultured in the presence of tet over that in the absence of tet. The error bars refer to standard deviations ( n = 4); **, P < 0.001 compared to mutant LY6E. (D) Flp-In T-Rex 293-derived cell lines expressing the wild-type or mutant LY6E were cultured in medium with indicated concentrations of tet for 24 h. LY6E expression was detected by a Western blot assay in which β-actin served as a loading control. (E) Flp-In T-Rex 293-derived cell lines expressing the wild-type or mutant LY6E (L36A) were cultured with or without the indicated concentrations of tet for 24 h. The cells were then infected with the indicated pseudotyped lentivirus. Luciferase activities were measured at 48 hpi. Relative infection is the ratio of luciferase activity in the same cells cultured in the presence of tet over that in the absence of tet. The error bars refer to standard deviations ( n = 4); **, P < 0.001 compared to mutant LY6E.

Article Snippet: Rabbit polyclonal antibody against human LY6E was obtained from Proteintech (catalog number 22144-1-AP).

Techniques: Sequencing, Glycoproteomics, Derivative Assay, Expressing, Control, Mutagenesis, Cell Culture, Western Blot, Infection, Luciferase, Activity Assay

Journal: Journal of Virology

Article Title: LY6E Restricts Entry of Human Coronaviruses, Including Currently Pandemic SARS-CoV-2

doi: 10.1128/JVI.00562-20

Figure Lengend Snippet: LY6E inhibits TMPRSS2-enhanced entry of human coronaviruses. Flp-In T-Rex 293-derived cell lines expressing LY6E were transfected with a control vector (pCAGGS) or a plasmid expressing human TMPRSS2 and cultured in the absence or presence of tet for 24 h. The cells were then infected with the indicated pseudotyped lentivirus. Luciferase activities were measured at 48 hpi. (A) The effect of TMPRSS2 expression on pseudotyped virus infection is normalized to infection efficiency of the cells transfected with control vector plasmid (set as 1) in the cells cultured in the absence of tet. (B) The effect of TMPRSS2 expression on pseudotyped virus infection is normalized to infection efficiency of the cells transfected with control vector plasmid (set as 1) in the cells cultured in the presence of tet. (C) Relative infection refers to the ratio of the luciferase activity in the cells cultured in the presence of tet over that in the cells cultured in the absence of tet. Error bars indicate the standard deviation ( n = 4); **, P < 0.001 compared to cells transfected with the pCAGGS vector.

Article Snippet: Rabbit polyclonal antibody against human LY6E was obtained from Proteintech (catalog number 22144-1-AP).

Techniques: Derivative Assay, Expressing, Transfection, Control, Plasmid Preparation, Cell Culture, Infection, Luciferase, Virus, Activity Assay, Standard Deviation

Journal: Journal of Virology

Article Title: LY6E Restricts Entry of Human Coronaviruses, Including Currently Pandemic SARS-CoV-2

doi: 10.1128/JVI.00562-20

Figure Lengend Snippet: Amphotericin B treatment compromises IFITM3 inhibition of human coronavirus entry, but have no impact on Ly6E inhibition of human coronavirus entry. Flp-In T-Rex 293-derived cell lines expressing IFITM3 (A) or LY6E (B) were cultured in the absence or presence of tet for 24 h. The cells were then infected with the indicated pseudotyped lentivirus in the presence or absence of 1 μM AmphoB. Luciferase activity was measured at 48 h postinfection. Relative infection is the ratio of luciferase activity in the same cells cultured in the presence of tet over that in the absence of tet. The error bars refer to standard deviations ( n = 4); **, P < 0.001 compared to mock treatment.

Article Snippet: Rabbit polyclonal antibody against human LY6E was obtained from Proteintech (catalog number 22144-1-AP).

Techniques: Inhibition, Derivative Assay, Expressing, Cell Culture, Infection, Luciferase, Activity Assay

Journal: Molecular and Cellular Biology

Article Title: Corrected and Republished from: BCL11A Is a Critical Component of a Transcriptional Network That Activates Recombinase Activating Gene Expression and V(D)J Recombination

doi: 10.1128/MCB.00362-17

Figure Lengend Snippet: Modulation of BCL11A expression modulates V(D)J rearrangement. (A) BCL11A-XL overexpression induces Vκ-Jκ recombination in A70-INV pre-B cells. A degenerate Vκ consensus primer (Vcon) and a Jκ2-1 reverse primer (top panel) were used to amplify rearrangements from genomic DNA diluted in 3-fold increments (triangles) to ensure nonsaturating semiquantitative PCR. The strategy predicts products of 190 bp (VJκ2) and 540 bp (VJκ1). Amplified DNAs were resolved in agarose gels, transferred to nylon membranes, and then visualized by autoradiography following hybridization to the Jκ2-2 probe (top panel). (B) Flow cytometric analysis of Bcl11aL/L bone marrow cells following primary infection with the MSCV-Bcl-xL retrovirus and secondary superinfection with a Thy-1.1-expressing MIT or MIT-Cre retrovirus. Cells were stained with antibodies directly conjugated to phycoerythrin (PE-B220) and fluorescein isothiocyanate (FITC–Thy-1.1). (C) Flow cytometric analysis of DNA content/cell cycle progression of Bcl11aL/L bone marrow cells following the retroviral infection protocol detailed for panel B. Detergent-permeabilized cells were stained with propidium iodide and analyzed as described in Materials and Methods, using CellQuest software to calculate percentages of cells in the G1 (M1), G2/M (M2), and S (M1-M2 interval) phases. (D) Cre recombinase-mediated deletion of loxP (L)-modified Bcl11a alleles (Bcl11aL/L) blocks Vκ-Jκ recombination in pre-B/IL-7 bone marrow (BM) cultures. Following retroviral infection as detailed for panel B, semiquantitative and real-time PCRs of genomic DNAs were performed on samples serially diluted 5-fold (triangles). DNAs amplified semiquantitatively were gel fractionated and either directly visualized by ethidium bromide staining (top and bottom panels) or visualized following hybridization as described for panel A (middle panel). PCRs of Cμ served as loading controls. Real-time PCR values, shown as fold reductions (Fold ↓) below corresponding lanes, were determined for each DNA dilution by dividing each MIT control value by the corresponding MIT-Cre value. The analyses are representative of 4 independent experiments performed with 4 separate BM isolates.

Article Snippet: A mouse stem cell virus encoding the antiapoptotic factor Bcl-xL was obtained from Toku-E Pharm (MSCV-Bcl-xLPuro; HCD57).

Techniques: Expressing, Over Expression, Amplification, Autoradiography, Hybridization, Infection, Staining, Software, Modification, Real-time Polymerase Chain Reaction