Antiviral activity of interferon-α/β against the cytopathology of KFDV and VSV-GFP.

<p>Antiviral activity of interferon-α/β against the cytopathology of KFDV and VSV-GFP.</p

IFN-α2a does not clear KFDV infection in BHK-21 cells.

<p>BHK-21 cells were infected with a 11 TCID₅₀ units (MOI of 0.00001) of the indicated virus, and either treated or mock-treated with 2, 000 U/mL of IFN-α2a (designated as P0). Monolayers were passaged when untreated controls reached CPE of 90%, 96 and 48 hpi for KFDV and VSV-GFP, respectively and 2, 000 U/mL of IFN-α2a was either added or omitted (P1) and after 72 hpi for KFDV and 48 hpi for VSV-GFP. This procedure was repeated again for passage 2 (P2). (A) Before each passage, supernatants were harvested for titration by TCID₅₀ assay determination on BHK-21 cells. The averages and standard deviation from three biological replicates are shown graphically and expressed in log₁₀ scale TCID₅₀/mL. Statistical significance is denoted as * P < 0.1, ** P < 0.05, *** P < 0.01. (B) Cell monolayers were visualized with light microscopy prior to passaging Cell monolayers were photographed prior to passaging. Mock, non-IFN treated/infected controls. UI, Un-infected controls.</p

IFN-α2a does not clear KFDV infection in A549 cells.

<p>A549 cells were infected with a 11 TCID₅₀ units (MOI of 0.00001) of the indicated virus, and either treated or mock-treated with 2, 000 U/mL of IFN-α2a (designated as P0). Monolayers were passaged when untreated controls reached CPE of 90%, 96 and 48 hpi for KFDV and VSV-GFP, respectively and 2, 000 U/mL of IFN-α2a was either added or omitted (P1) and after 72 hpi for KFDV and 48 hpi for VSV-GFP. This procedure was repeated again for passage 2 (P2). (A) Before each passage, supernatants were harvested for titration by TCID₅₀ assay determination on BHK-21 cells. The averages and standard deviation from three biological replicates are shown graphically and expressed in log₁₀ scale TCID₅₀/mL. Statistical significance is denoted as * P < 0.1, ** P < 0.05, *** P < 0.01. (B) Cell monolayers were visualized with light microscopy prior to passaging. Mock, non-IFN treated/infected controls. UI, Un-infected controls.</p

Antiviral activity of interferon-α/β against KFDV and VSV-GFP virion production.

<p>Antiviral activity of interferon-α/β against KFDV and VSV-GFP virion production.</p

KFDV NS5 impedes the cellular antiviral state.

<p><b>(A)</b> VeroE6 (ATCC) cells were transfected with plasmid encoding KFDV NS proteins and Ebola virus VP24 and treated with 1, 000 U/mL of Universal IFN, 24 hpi. After a 24-hour incubation period, cells were infected with VSV-GFP (MOI of 2) and, pictures were taken with fluorescent (top panel) and light (bottom panel) microscopy 24 hours later. <b>(B)</b> VeroE6 (ATCC) cells were transfected with KFDV NS5-pCAGGS and treated with 1, 000 U/mL of commercially available type I IFNs, 24 hpi. After a 24-hour incubation period, cells were infected with VSV-GFP (MOI of 2) and, 24 hours later, the virus-containing supernatants were harvested for virus quantification. Dark grey bars indicate experiments in which cells were un-transfected. Light grey bars indicate NS5-expressing cells. Mock, no IFN treatment of cells lacking NS5 expression (dark gray bar) and with NS5 expression (light gray bar); UI represents uninfected/un-treated cells. Universal IFN controls included VP24-pCAGGS as anti-IFN control. The graph represents the log₁₀ scale TCID₅₀/mL averages and standard deviations from three biological repetitions. ***, Significant difference of NS5-expressing cells compared to VP24-expressing cells (P < 0.01).</p

Screening interferon-α/β subtypes against KFDV.

<p>Cultures of A549 cells were pre-treated (grey bars) 24 hours prior to infection or post-treated (black bars) 1 hour after infection with 1, 000 U/mL of IFN-α (B2, C, D, F, G, H2, I, J1, K, WA, 2a, 2b or 4b), IFN-β (beta-1) and a recombinant IFN-α (Universal) subtypes and infected with KFDV at a MOI of 1. Supernatants were harvested for each treatment after 72 hours of incubation and quantified (expressed in log₁₀ scale TCID₅₀/mL) on BHK-21 (ATCC) when the mock-treated control cells displayed CPE near 100%. Pre-infection treatment experiments were assayed in two biological replicates and post-infection treatment experiments were assayed in three biological replicates; the resulting averages and standard deviations are presented. Mock, Mock-treated with IFN. UI, Un-infected control. IFN-α2b was excluded from 24-hour pre-infection treatment. * Significant compared to mock-treated samples (P < 0.1). *** Significant compared to mock-treated samples (P < 0.01).</p

Binding characteristics of FluA and FluB PB1-derived peptide chimera.

<p>Inhibitory concentrations of FluA/FluB-derived peptides determined by competitive ELISA. Competitor peptides (0.048 to 3000 nM) were mixed with cell extracts containing HA-tagged PA from either FluA or FluB. Letters in red indicate FluB, letters in blue FluA specific aa. S.D. is indicated in parenthesis. Asterisks indicate highest concentrations of peptides used without reaching 50% inhibition.</p

Virus type-specific conservation of the PA-binding domain and interaction of PA with PB1.

<p>(A) Upper panel: Alignment of the N-terminal 25 aa of FluA and FluB PB1. The dotted box indicates the 3₁₀-helix comprising the core PA-binding domain of PB1. FluA-specific (blue) and FluB-specific (red) aa are highlighted. Middle and lower panels: Alignment of the N-terminal 25 aa of all available FluA and FluB sequences available in the NCBI influenza virus database. Figures on the right hand side indicate the number of sequences present in the database. Grey bars highlight aa which reconstitute the 3₁₀-helix of FluA PA and possibly of FluB PA. (B) A/SC35M- and B/Yamagata/73-derived PB1 chimeras used in (b). Note that all PB1 proteins were expressed with C-terminal HA-tags. (C) Human 293T cells were transfected with expression plasmids coding for the indicated PB1 proteins and the C-terminally hexahistidine-tagged PA of FluA (FluA-PA_His). Cell lysates were prepared 24 hours post transfection and subjected to immunoprecipitation (IP) using anti-HA (αHA) agarose. Precipitated material was separated by SDS-PAGE and analyzed by Western blot for the presence of either His- or HA-tagged polymerase subunits using appropriate antibodies. Protein expression was controlled by analyzing equal amounts of cell lysate. Molecular weights are shown in kilodaltons.</p

Peptides used for the plaque-reduction assay.

<p>The underlined Y highlights the tyrosine residue derived from the PB1 FluB sequence. Bold letters in italics represent the HIV-Tat derived sequence that allow the peptides to cross the plasma membrane.</p

Quantification of the interaction between PB1_1–25 and PA.

<p>(A) Determination of the 50% inhibitory concentration (IC₅₀) of PB1_1–25A by competitive ELISA using the indicated increasing concentrations of peptides and cell extract containing HA-tagged PA of FluA. Error bars represent standard deviations from triplicate experiments. (B) IC50 of PB1_1–25A-derived peptides. S.D. is shown in parenthesis. Asterisks indicate highest concentrations of peptides (3000 nM) used without detectable inhibitory effect. Grey boxes highlight amino acids that are part of the 3₁₀-helix, which was postulated to comprise the core PA-binding region of PB1. Amino acids known to form hydrogen bonds with PA residues are represented in bold.</p