Journal: PLoS Neglected Tropical Diseases

Article Title: Inhibition of Lassa Virus Glycoprotein Cleavage and Multicycle Replication by Site 1 Protease-Adapted α 1 -Antitrypsin Variants

doi: 10.1371/journal.pntd.0000446

Figure Lengend Snippet: A. CHO-K1, Vero E6, and SRD-12B cells were infected either with the recombinant virus VSVΔG/LASVGP or wild-type VSV (VSVwt) at an MOI of 0.02. Supernatants were collected at different times and titrated by plaque assay. The growth curves shown are the mean result±standard deviation of three independent experiments. B. CHO-K1 Tet-On cells were infected with VSVΔG/LASVGP in the presence (+) or absence (−) of doxycycline (Dox, 1 µg/ml) at an MOI of 0.02. Supernatants were sampled 24 h and 48 h post-infection and analyzed by plaque assay.

Article Snippet: To generate stably expressing cell lines, Chinese hamster ovary (CHO)-K1 Tet-On cells (Clontech) were transfected with pTRE2hyg containing the α 1 -antitrypsin constructs using Lipofectamine 2000 (Invitrogen) according to manufacturer's instructions.

Techniques: Infection, Recombinant, Plaque Assay, Standard Deviation

Journal: PLoS Neglected Tropical Diseases

Article Title: Inhibition of Lassa Virus Glycoprotein Cleavage and Multicycle Replication by Site 1 Protease-Adapted α 1 -Antitrypsin Variants

doi: 10.1371/journal.pntd.0000446

Figure Lengend Snippet: A. Schematic representation of constructed α 1 -antitrypsin variants used for generation of stable cell lines. The amino acid sequences of the different motifs introduced into the RCL by recombinant PCR technology are shown in one-letter code. To facilitate their detection, a flag epitope was introduced at the C-terminus as indicated. B. Cellular expression of α 1 -antitrypsin variants. CHO-K1 Tet-On α 1 -ATs cells were induced with doxycycline (Dox, 1 µg/ml) or left untreated. At 24 h post-induction, cell lysates were analyzed by immunoblot analysis using a mouse anti-flag antibody. β-tubulin served as a loading control using a rabbit anti-ß-tubulin antibody. C. Immunofluorescence analysis of α 1 -antitrypsin expression. CHO-K1 Tet-On and α 1 -antitrypsin expressing cells were fixed with 4% PFA and permeabilized using 0.1% Triton X-100. α 1 -antitrypsin variants were visualized with a primary anti-flag mouse antibody and a secondary anti-mouse antibody coupled to rhodamine. Cell nuclei were stained with DAPI.

Article Snippet: To generate stably expressing cell lines, Chinese hamster ovary (CHO)-K1 Tet-On cells (Clontech) were transfected with pTRE2hyg containing the α 1 -antitrypsin constructs using Lipofectamine 2000 (Invitrogen) according to manufacturer's instructions.

Techniques: Construct, Stable Transfection, Recombinant, FLAG-tag, Expressing, Western Blot, Immunofluorescence, Staining

Journal: PLoS Neglected Tropical Diseases

Article Title: Inhibition of Lassa Virus Glycoprotein Cleavage and Multicycle Replication by Site 1 Protease-Adapted α 1 -Antitrypsin Variants

doi: 10.1371/journal.pntd.0000446

Figure Lengend Snippet: A. CHO-K1 Tet-On cells (lanes 1 and 2), S1P-deficient SRD-12B cells (lanes 3 and 4) and CHO-K1 Tet-On cell lines stably transfected with plasmids encoding α 1 -antitrypsin variants (lanes 5–14) were infected with VSVΔG/LASVGP at an MOI of 0.2 in the presence (+) or absence (−) of doxycycline (Dox, 1 µg/ml) as indicated. At 10 h post-infection cell lysates were analyzed by Western blot analysis using specific antibodies as described in the . B. Efficiency of GP-C cleavage was quantified by infrared fluorescent imaging using the Odyssey infrared imaging system. The amount of GP-2 was quantified compared to total amount of GP-C. Detected GP-2 in induced cell lines was calculated in relation to the corresponding non-induced cells, which were adjusted to 100%. The negative control (SRD-12B cells) was calculated with reference to the positive control (CHO-K1 Tet-On cells). Quantified GP-2 values are means±standard deviations of three independent experiments. Asterisks indicate differences that are statistically significant (**, p<0.002; ***, p<0.0001).

Article Snippet: To generate stably expressing cell lines, Chinese hamster ovary (CHO)-K1 Tet-On cells (Clontech) were transfected with pTRE2hyg containing the α 1 -antitrypsin constructs using Lipofectamine 2000 (Invitrogen) according to manufacturer's instructions.

Techniques: Stable Transfection, Transfection, Infection, Western Blot, Imaging, Negative Control, Positive Control

Journal: PLoS Neglected Tropical Diseases

Article Title: Inhibition of Lassa Virus Glycoprotein Cleavage and Multicycle Replication by Site 1 Protease-Adapted α 1 -Antitrypsin Variants

doi: 10.1371/journal.pntd.0000446

Figure Lengend Snippet: CHO-K1 Tet-On cells and α 1 -AT variant RRIL CHO cell line were infected with (A) VSVΔG/LASVGP or (B) VSVwt at an MOI of 0.2 in the presence (+) or absence (−) of doxycycline (Dox, 1 µg/ml). At 24 h post-infection, virions in cell culture supernatants were pelleted through a 20% sucrose cushion by ultracentrifugation. Cell lysates and purified virions were subjected to SDS-PAGE followed by immunoblotting using antisera specific for LASV GP and VSV proteins, respectively. Intracellular α 1 -AT expression was analyzed using an anti-flag antibody. β-tubulin was used as a loading control.

Article Snippet: To generate stably expressing cell lines, Chinese hamster ovary (CHO)-K1 Tet-On cells (Clontech) were transfected with pTRE2hyg containing the α 1 -antitrypsin constructs using Lipofectamine 2000 (Invitrogen) according to manufacturer's instructions.

Techniques: Variant Assay, Infection, Cell Culture, Purification, SDS Page, Western Blot, Expressing

Journal: PLoS Neglected Tropical Diseases

Article Title: Inhibition of Lassa Virus Glycoprotein Cleavage and Multicycle Replication by Site 1 Protease-Adapted α 1 -Antitrypsin Variants

doi: 10.1371/journal.pntd.0000446

Figure Lengend Snippet: A. CHO-K1 Tet-On cells, SRD-12B cells and S1P-adapted α 1 -antitrypsin expressing CHO cell lines were infected with VSVΔG/LASVGP at an MOI of 0.02 in the presence (+) or absence (−) of doxycycline (Dox, 1 µg/ml). Infected cells were immunostained 24 h post-infection using an antiserum against VSV and HRP-linked secondary antibody. Individual cells and virus spread were visualized by subsequent application of True Blue peroxidase substrate. B. CHO-K1 Tet-On cells, S1P-adapted α 1 -antitrypsin variant RRIL cell line, as well as the furin-specific α 1 -antitrypsin variant RVKR cell line, were infected with Fowl Plague virus (FPV) at an MOI of 0.001 in the presence (+) or absence (−) of doxycycline. At 24 h post-infection, immunostaining was performed as described above using a polyclonal FPV antiserum.

Article Snippet: To generate stably expressing cell lines, Chinese hamster ovary (CHO)-K1 Tet-On cells (Clontech) were transfected with pTRE2hyg containing the α 1 -antitrypsin constructs using Lipofectamine 2000 (Invitrogen) according to manufacturer's instructions.

Techniques: Expressing, Infection, Variant Assay, Immunostaining

Journal: PLoS Neglected Tropical Diseases

Article Title: Inhibition of Lassa Virus Glycoprotein Cleavage and Multicycle Replication by Site 1 Protease-Adapted α 1 -Antitrypsin Variants

doi: 10.1371/journal.pntd.0000446

Figure Lengend Snippet: CHO-K1 Tet-On, SRD-12B and α 1 -AT variant RRIL CHO cell lines were infected with LASV (strain Josiah) at an MOI of 0.1 in the presence (+) or absence (−) of doxycycline (Dox, 1 µg/ml). At various times post-infection as indicated, cell culture supernatants were collected and virus yields were determined by TCID 50 . Values shown are mean results±standard deviation of three independent experiments performed in duplicate. Asterisks indicate differences that are statistically significant between induced α 1 -AT variant RRIL cells in comparison to non-induced cells (*, p<0.05).

Article Snippet: To generate stably expressing cell lines, Chinese hamster ovary (CHO)-K1 Tet-On cells (Clontech) were transfected with pTRE2hyg containing the α 1 -antitrypsin constructs using Lipofectamine 2000 (Invitrogen) according to manufacturer's instructions.

Techniques: Variant Assay, Infection, Cell Culture, Standard Deviation