Journal: Journal of Virology

Article Title: pH Optimum of Hemagglutinin-Mediated Membrane Fusion Determines Sensitivity of Influenza A Viruses to the Interferon-Induced Antiviral State and IFITMs

doi: 10.1128/JVI.00246-17

Figure Lengend Snippet: Inhibition of single-cycle infection and plaque formation in MDCK cells stably expressing IFITM2 and IFITM3 proteins. (a, b) Virus titers in MDCK, MDCK-IFITM2, and MDCK-IFITM3 cells were determined with a single-cycle focus formation assay. Titers are expressed in log10 FFU per milliliter, and the graphs depict the calculated reduction of the titers in IFITM-expressing cells with respect to control MDCK cells. Three (a) and four (b) independent experiments were performed on different days with the same results. The panels show results of representative experiments with three biological replicates for each condition. (c, d) The diameters of 40 to 100 plaques formed by the viruses under semisolid overlay medium were measured for each cell type, and percentages with respect to the mean plaque diameter in MDCK cells (control) were calculated. Asterisks indicate the statistical significance of differences with respect to Mem-H1N1 (a, c) and HK-H3N2 (b, d).

Article Snippet: The expression of IFITM2 and IFITM3 was verified by immunoblot analysis employing a polyclonal IFITM2 antiserum, which is cross-reactive with IFITM3 (12769-1-AP; Proteintech, Chicago, IL, USA).

Techniques: Inhibition, Infection, Stable Transfection, Expressing, Tube Formation Assay

Journal: Virology

Article Title: Primate lentiviruses are differentially inhibited by interferon-induced transmembrane proteins

doi: 10.1016/j.virol.2014.10.015

Figure Lengend Snippet: Knockdown of endogenous IFITM2 and 3 increases the entry of SIV AGM-sab . (A) Knockdown of endogenous IFITM2 and 3 in TZM-bl cells. Three shRNA clones targeting both IFITM2 and 3 were transduced into TZM-bl cells with or without IFNα-2b treatment (1000 U/ml, 16 h). Levels of endogenous IFITM2 and 3 were assessed by Western blotting. (B) Effect of endogenous IFITM2 and 3 on the infection of SIV AGM-sab and SIV AGM-tan . IFITM2/3-knockdown TZM-bl cells were first treated with IFNα-2b and then infected with SIV AGM-sab or SIV AGM-tan . Viral infection was determined by measuring luciferase activities in the infected TZM-bl cells. Results of three independent experiments are summarized in the bar graph with the infection of control TZM-bl cells arbitrarily set as 100%. (C) Effect of endogenous IFITM2 and 3 on SIV AGM-sab entry in TZM-bl cells. BlaM-Vpr-containing SIV AGM-sab was used to infect the IFITM2/3-knockdown TZM-bl cells. Levels of virus entry were assessed by measuring the cleavage of CCF2 using flow cytometry. (D) The results of three independent virus entry experiments are summarized in the bar graph. (E) Knockdown of endogenous IFITM2 and IFITM3 in C8166 cells. The knockdown efficiency was examined by western blotting. (F and G) Effect of endogenous IFITM2 and 3 in C8166 cells on SIV AGM-sab entry. Details refer to the legend to (C) and (D). The p Values were calculated and are presented as ⁎ ( p <0.05), ⁎⁎ ( p <0.01).

Article Snippet: Anti-Flag antibody was purchased from Sigma, anti-IFITM2 and anti-IFITM3 antibodies from ProteinTech.

Techniques: shRNA, Clone Assay, Western Blot, Infection, Luciferase, Flow Cytometry

Journal: Virology

Article Title: Primate lentiviruses are differentially inhibited by interferon-induced transmembrane proteins

doi: 10.1016/j.virol.2014.10.015

Figure Lengend Snippet: Effects of different agents on SIV infection. (A and B) Different doses of amphotericin B were used to treat TZM-bl cell lines that stably express IFITM2 and 3. The control cell line was transduced with the empty pQCXIP retroviral vector. After one-hour pretreatment, cells were infected with SIV AGM-sab (A) or SIV AGM-tan (B). Virus infection was monitored by measuring luciferase activity in TZM-bl cell lysates. (C and D) TZM-bl cells were treated with different doses of chloroquine or bafilomycin A1 to neutralize the low pH in late endosomes and lysosomes. Cells were infected with SIV AGM-sab , SIV AGM-tan or VSV-G pseudotyped HIV-1 NL4-3 . Virus infection was determined by measuring luciferase activity in TZM-bl cell lysates. (E) TZM-bl cells were treated with or without dynasore for 1 h followed by infection with SIV AGM-sab , SIV AGM-tan or VSV-G pseudotyped HIV-1 NL4-3 . Levels of virus infection were monitored by measuring luciferase activity in TZM-bl cell lysates. Results shown are the averages of three independent experiments. The p -Values were calculated and are presented as ⁎⁎ ( p <0.001).

Article Snippet: Anti-Flag antibody was purchased from Sigma, anti-IFITM2 and anti-IFITM3 antibodies from ProteinTech.

Techniques: Infection, Stable Transfection, Transduction, Plasmid Preparation, Luciferase, Activity Assay

Journal: Virology

Article Title: Primate lentiviruses are differentially inhibited by interferon-induced transmembrane proteins

doi: 10.1016/j.virol.2014.10.015

Figure Lengend Snippet: Inhibition of HIV and SIV by IFITM1 and 3 of African green monkeys. (A) Sequence alignment of agmIFITM1 and 3. IFITM1 and IFITM3 cDNAs were cloned from African green monkey kidney cell lines COS-7 and Vero. Their protein sequences are aligned with orthologs from macaques (mac), humans and chimpanzees (chimp). No distinct IFITM2 was identified. Four agmIFITM3 sequences were found that vary at amino acid positions 22 and 38. (B) TZM-bl cells were stably transduced with agmIFITM1 and four agmIFITM3 variants, then challenged with HIV-1 NL4-3 , HIV-1 YU-2 , HIV-1 A/G , HIV-2 ROD , SIV AGM-tan , SIV AGM-sab and SIV MAC . Results shown are the averages of three independent experiments, with the infectivity in control cells arbitrarily set as 100% for each virus. The p Values were calculated and are presented as ⁎ ( p <0.05), ⁎⁎⁎ ( p <0.01).

Article Snippet: Anti-Flag antibody was purchased from Sigma, anti-IFITM2 and anti-IFITM3 antibodies from ProteinTech.

Techniques: Inhibition, Sequencing, Clone Assay, Stable Transfection, Transduction, Infection