Potential Viroporin Candidates From Pathogenic Viruses Using Bacteria-Based Bioassays

Figures

• 

  Figure 1. Sequence and topology of candidate viroporins. Sequences of the proteins analyzed in the current study alongside their predicted topologies according to Phobius [39,40]. The shaded region indicates the presumed position of the lipid bilayer. The lengths of the proteins are stated in the parentheses and the accession numbers in gray. The figure was prepared using TEXtopo version 1.5 [56].

• 

  Figure 2. Membrane incorporation assay. Test for proper insertion and orientation of viral chimera based on the malE- complementation assay [62]. Bacteria lacking any maltose binding protein (MBP) were transformed with various viroporins which were fused to MBP and cultivated on M9 agar containing 0.4% maltose and 10 μM isopropyl β-d-1-thiogalactopyranoside (IPTG).

• 

  Figure 3. Positive genetic test for viroporin activity. Impact of viral protein expression on the growth of K+-uptake-deficient bacteria [43]. The relative growth enhancements in comparison to bacteria that harbor an empty vector (MBP on its own) are indicated by the gray arrows. Generic TM indicates a chimera in which MBP is fused at its carboxy terminus to the transmembrane domain of glycophorin A containing two monomerizing mutations [48,49]. M2 + Rim is a sample in which rimantadine, a specific blocker of the influenza M2 channel [10], was added at a concentration of 50 μM. Note that rimantadine did not affect other proteins (data not shown). The results are an average of three independent experiments, with standard deviations depicted as error bars.

• 

  Figure 4. Negative genetic test for viroporin activity. The impact of viral protein expression on bacterial growth as a function of the concentration of the inducer IPTG varied between 0 to 100 μM, as indicated. The relative growth inhibitions in comparison to bacteria that harbor an empty vector (MBP on its own) are indicated by the gray arrows. Generic TM indicates a chimera in which MBP is fused at its carboxy terminus to the transmembrane domain of glycophorin A containing two monomerizing mutations [48,49]. M2 + Rim is a sample in which rimantadine, a specific blocker of the influenza M2 channel [10], was added at a concentration of 50 μM. Note that rimantadine did not affect other proteins (data not shown). The results are an average of three independent experiments, with standard deviations depicted as error bars.

• 

  Figure 5. Fluorescence-based H+ conductivity assay. The fluorescence of bacteria that harbor pHluorin, a pH-sensitive green fluorescence protein (GFP) [44], was examined as a function of the expressed viral chimera [46,47]. Protein levels were governed by the level of the inducer (50 μM IPTG), as indicated. At time 0, a concentrated acid solution was injected into the media. Consequently, a drop of the fluorescence ratio between excitation at 390 nm relative to excitation at 466 nm (shown on the left axis) is indicative of a pH decrease, as shown on the right axis. The ΔpH values indicated in the parentheses correspond to the difference in the pH after 30 s in bacteria that had undergone protein induction. Generic TM indicates a chimera in which MBP is fused at its carboxy terminus to the transmembrane domain of glycophorin A containing two monomerizing mutations [48,49]. + Rim is a sample in which rimantadine, a specific blocker of the influenza M2 channel [10], was added at a concentration of 50 μM. Note that rimantadine did not affect other proteins (data not shown). The results are an average of three independent experiments, with standard deviations depicted as error bars.