Priming and Acid-Bypass Fusion of BUNV Virions

Pre-infection priming of the BUNV virions was carried out using the pH 6.3 (-K+) CTL buffer and the pH 6.3, high K+ buffer. Primed virions were added to A549 cells for 1.5 hrs on ice, followed by a wash step with cold DMEM to remove unbound virions. Acid-bypass to induce virion fusion at the plasma membrane was then carried out following the procedure outlined by Stauffer et al 2014. Warm pH 5.0 fusion buffer (DMEM containing 50 mM sodium citrate) was added to cells for a 2 min pulse at 37°C, alongside control samples incubated with either an acid-bypass control buffer (DMEM, 50 mM HEPES, 20 mM NH4Cl, at pH 7.4) or DMEM alone. Cells were subsequently washed twice with cold DMEM, then warm acid-bypass control buffer was added to all except the DMEM alone control wells, where DMEM was re-added (allows endocytic entry of virus, to confirm successful virus priming). Infected cells were incubated for 17 hrs and lysed.

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Hover S., Foster B., Fontana J., Kohl A., Goldstein S.A., Barr J.N, & Mankouri J. (2018). Bunyavirus requirement for endosomal K+ reveals new roles of cellular ion channels during infection. PLoS Pathogens, 14(1), e1006845.

Publication 2018

A549 cells Acid Buffer Cells Cold Hepes Infection Membrane fusion Plasma Pulse Sodium citrate Virion Virus Virus entry

Corresponding Organization : University of Leeds

Other organizations : MRC University of Glasgow Centre for Virus Research, Loyola University Chicago

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Protocol cited in 4 other protocols

Variable analysis

independent variables

PH of the buffer used for priming the BUNV virions (pH 6.3 (-K+) CTL buffer and pH 6.3, high K+ buffer)
Type of buffer used for acid-bypass to induce virion fusion (warm pH 5.0 fusion buffer (DMEM containing 50 mM sodium citrate) and acid-bypass control buffer (DMEM, 50 mM HEPES, 20 mM NH4Cl, at pH 7.4))

dependent variables

Infection of A549 cells by the primed BUNV virions (as measured by cell lysis after 17 hrs of incubation)

control variables

Temperature (all steps were carried out on ice or at 37°C)
Cell line (A549 cells were used)

controls

Positive control: Acid-bypass control buffer (DMEM, 50 mM HEPES, 20 mM NH4Cl, at pH 7.4) - allows endocytic entry of virus to confirm successful virus priming
Negative control: DMEM alone (no acid-bypass treatment)

Annotations

Based on most similar protocols

Optimize BUNV virion priming by testing a range of pH buffers (pH 7.3, 6.3, 5.3) and ionic concentrations (with or without 140 mM K+) as done in Protocol 1. This helps determine the optimal conditions for virus entry.

Validate BUNV priming and cell binding by assessing BUNV-N expression levels at multiple time points (18 and 24 hpi) using western blot as done in Protocol 1. This confirms successful virus priming and entry.

Use a high-titer BUNV preparation (~ 1 × 10^9 PFU/mL) for the immunofluorescence binding assay, as described in Protocol 1, to improve the detection of bound virions.

Perform a wash step with 0.1x PBS or 0.1% trypsin after virus binding to remove unbound virions, as described in the initial wash test assay in Supplementary Fig. 8a of Protocol 1.

Neutralize primed BUNV virions with a BUNV-Gc specific monoclonal antibody (mAb-742) to confirm that the observed effects are due to specific virus entry, as done in the neutralization assays of Protocol 1.

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