Bunyavirus requirement for endosomal K+ reveals new roles of cellular ion channels during infection

In order to multiply and cause disease a virus must transport its genome from outside the cell into the cytosol, most commonly achieved through the endocytic network. Endosomes transport virus particles to specific cellular destinations and viruses exploit the changing environment of maturing endocytic vesicles as triggers to mediate genome release. Previously we demonstrated that several bunyaviruses, which comprise the largest family of negative sense RNA viruses, require the activity of cellular potassium (K+) channels to cause productive infection. Specifically, we demonstrated a surprising role for K+ channels during virus endosomal trafficking. In this study, we have used the prototype bunyavirus, Bunyamwera virus (BUNV), as a tool to understand why K+ channels are required for progression of these viruses through the endocytic network. We report three major findings: First, the production of a dual fluorescently labelled bunyavirus to visualize virus trafficking in live cells. Second, we show that BUNV traffics through endosomes containing high [K+] and that these K+ ions influence the infectivity of virions. Third, we show that K+ channel inhibition can alter the distribution of K+ across the endosomal system and arrest virus trafficking in endosomes. These data suggest high endosomal [K+] is a critical cue that is required for virus infection, and is controlled by cellular K+ channels resident within the endosome network. This highlights cellular K+ channels as druggable targets to impede virus entry, infection and disease

Endosomal ionic balance and its role in bunyavirus entry

The Bunyavirales are the largest group of negative-sense RNA viruses with new members emerging due to changes in virus/host relationships and segment reassortment. The Bunyavirales are of interest as they require host-cell potassium (K+) channels during the early stages of their infectious lifecycle. The mechanism(s) governing this dependence had not been previously defined. In Chapter 1, using the prototype member of the family, Bunyamwera virus (BUNV), the role of K+ channels during virus entry was investigated. It was shown that BUNV encounters high-K+ containing endosomes during virus entry which is controlled by endosome-resident K+ channels. The inhibition of these channels disrupted endosomal K+ uptake and prevented viruses escaping the endosomal system. Mimicking the ionic environment of late endosomes in vitro (pH 6.3/K+) expedited BUNV entry and reduced its susceptibility to K+ channel inhibition. This highlighted endosomal K+ as a biochemical cue for BUNV, explaining its requirement for host-cell K+ channels. In Chapter 2, the effects of pH/K+ priming on virion architecture were investigated using cryo-electron tomography and sub-tomogram averaging (STA). BUNV glycoprotein (GP) spike averages identified key definitions in the GP trimer that became disordered in response to pH/K+ priming. STA indicated uncoupling of the GP trimers in response to K+, likely facilitating a pre-fusion intermediate that exposes the fusion loop. This begins to explain the changes triggered by endocytic pH/K+ to expedite infection. In Chapter 3, through K+ channel silencing it was identified that the two-pore K+ channel TWIK2 was necessary for BUNV infection. TWIK2 localized to endo/lysosomal compartments through which BUNV traversed during infection. This inferred a role for TWIK2 during BUNV entry and revealed this channel as a new anti-BUNV target. The culmination of these findings reveal for the first time, the basis for why inhibiting K+ channels impedes BUNV

Bunyavirus requirement for endosomal K+ reveals new roles of cellular ion channels during infection.

In order to multiply and cause disease a virus must transport its genome from outside the cell into the cytosol, most commonly achieved through the endocytic network. Endosomes transport virus particles to specific cellular destinations and viruses exploit the changing environment of maturing endocytic vesicles as triggers to mediate genome release. Previously we demonstrated that several bunyaviruses, which comprise the largest family of negative sense RNA viruses, require the activity of cellular potassium (K+) channels to cause productive infection. Specifically, we demonstrated a surprising role for K+ channels during virus endosomal trafficking. In this study, we have used the prototype bunyavirus, Bunyamwera virus (BUNV), as a tool to understand why K+ channels are required for progression of these viruses through the endocytic network. We report three major findings: First, the production of a dual fluorescently labelled bunyavirus to visualize virus trafficking in live cells. Second, we show that BUNV traffics through endosomes containing high [K+] and that these K+ ions influence the infectivity of virions. Third, we show that K+ channel inhibition can alter the distribution of K+ across the endosomal system and arrest virus trafficking in endosomes. These data suggest high endosomal [K+] is a critical cue that is required for virus infection, and is controlled by cellular K+ channels resident within the endosome network. This highlights cellular K+ channels as druggable targets to impede virus entry, infection and disease

K⁺ channel modulation arrests BUNV trafficking in endosomes.

<p><b>(A)</b> Cells were treated with TEA (10 mM) for 30 min (or left untreated) and infected with SYTO82/DiD-BUNV for a further 4 hrs in the presence/absence of TEA. EGF-488 was added for the final 15 min of infection and cells were fixed 4 hpi. Confocal images were taken and the EGF-488 fluorescence channel removed in the representative images showing only SYTO82 and DiDvbt (n≥40). Scale bar = 10 μM. <b>(B)(i)</b> Cells treated with TEA (10 mM) or left untreated as in <b><i>A</i></b>, were infected with SYTO82/DiD-BUNV and fixed at 2, 4 or 8 hpi. EGF-488 (2 μg/ml) was added for 15 min prior to fixation as in <b>A</b>, with the representative images showing only SYTO82 and DiDvbt channels (n>65 cells). Scale bar = 10 μM. <b>(ii)</b> As in <b><i>(i)</i></b> but cells were treated with Qd (200 μM) and fixed 8 hpi (n>65 cells). <b>(iii)</b> The number of SYTO82/DiD-BUNV virions per cell were quantified using images from <b>(i)</b> and <b>(ii)</b> for n>65 cells and normalised to the untreated (no-drug) control. <b>(C)</b> A549 cells were infected with SYTO82/DiD-BUNV for 1 hour at 4°C and treated with cytopainter to label lysosomes. Cells were warmed to 37°C for 1 hr, virus/dye removed by washing and cells incubated for up to 8 hpi. Representative live cell images are shown (≥80 cells). Scale bar = 10 μM. <b>(ii)</b> The number of SYTO82/DiD-BUNV virions co-localising with cytopainter positive puncta were calculated and the % of co-localised puncta presented in <b>(ii)</b> (* = p≤0.05).</p

BUNV traffics through endosomes containing K⁺ ions.

<p><b>(A)</b> AG4 (10 μM) was added to A549 cells for 40 min to allow endosomal uptake, alongside <b>(B)(i)</b> Texas Red labelled EGF (2 μg/ml) or Magic Red cathepsin B dye. Non-internalised dyes were subsequently removed and live cells were imaged. Representative images are shown (n≥100 cells). Scale bar = 10 μM. <b>(ii)</b> Total numbers of AG4 positive puncta were counted per cell and % of colocalised AG4 puncta with each marker calculated in ≥100 cells. <b>(C)</b> AG4 (10 μM) was added to A549 cells for 40 min at either 37°C or 4°C and live cells were imaged as in A. Scale bar = 10 μM. <b>(D)</b> Schematic representation of AG4 uptake into endocytic vesicles and increased fluorescence with passage through early endosomes (EE) into late endosomes (LE) identifying K⁺-rich regions, identifiable using Texas Red labelled EGF. AG4 fluorescence decreases with passage into lysosomes (L). <b>(E)</b> A549 cells were infected with labelled-BUNV in the presence of AG4 (10μM) to allow virus penetration into cells and live cells were imaged 2 hrs or 8 hrs post-infection. Images are representative of ≥ 50 cells. <b>(F)(i)</b> A549 cells were infected with SYTO82/DiD-BUNV and EGF-488 (2 μg/ml) for 1 hour at 4°C and cells warmed to 37°C for the indicated timepoints. Images were taken of live cells at the indicated time points post-warming and are representative of ≥60 cells. Scale bar = 10 μM. <b>(ii)</b> Cells were transfected with Rab7-GFP and infected as in <b>F(i)</b> 24 hours post transfection. Images are representative of ≥ 40 cells. Scale bar = 10 μM. <b>G(i)</b> as in <b>F(i)</b> but cells were infected in the presence of 488-labelled Tf (25 μg/mL) or <b>(ii)</b> cells transfected with Rab11-GFP.</p

K⁺ channel modulation can impede normal K⁺ accumulation across the endocytic network.

<p><b>(A)</b> A549 cells were treated for 30 min with 10 mM TEA or left untreated. AG4 (10 μM) was then added in the presence or absence of drug for 40 min. Dye was removed and TEA was re-added onto cells. Fluorescence intensities were quantified using IncuCyte ZOOM imaging and analysis software, and data normalised to untreated (unt) controls over three independent cell populations. NS–no significant difference between no-drug and TEA treated controls (p≥0.05). Scale bar = 10 μM. <b>(B) (i)</b> Cells were treated with 10 mM TEA (or left untreated) and AG4 (10 μm) added as in <b><i>A</i></b>, with the addition of Magic Red during the 40 min incubation with AG4. Representative images are shown (n≥60 cells). Scale bar = 10 μM. <b>(ii)</b> Total number of AG4 positive puncta were counted per cell ± TEA and the % of colocalised puncta presented. n≥60 cells, (* = p≤0.05). Scale bar = 10 μM. <b>(C) (i)</b> Cells were treated with 10 mM TEA or left untreated, and AG4 (10 μM) added as in <b><i>A</i></b>, with the addition of the pH indicator pHrodo red dextran (10 μg/ml) during the 40 min incubation with AG4. Representative images are shown (Scale bar = 10 μM) and the % of co-localised puncta presented in <b>(ii)</b> n≥60 cells (* = p≤0.05). <b>D</b> Fluorescence intensity of Magic Red was quantified using IncuCyte ZOOM imaging and analysis software and data normalised to untreated controls over three independent cell populations. NS–no significant difference between untreated and TEA treated cells (p≥0.05). Representative images are also shown.</p