Potential Viroporin Candidates From Pathogenic Viruses Using Bacteria-Based Bioassays

Abstract

Viroporins are a family of small hydrophobic proteins found in many enveloped viruses that are capable of ion transport. Building upon the ability to inhibit influenza by blocking its archetypical M2 H+ channel, as a family, viroporins may represent a viable target to curb viral infectivity. To this end, using three bacterial assays we analyzed six small hydrophobic proteins from biomedically important viruses as potential viroporin candidates. Our results indicate that Eastern equine encephalitis virus 6k, West Nile virus MgM, Dengue virus 2k, Dengue virus P1, Variola virus gp170, and Variola virus gp151 proteins all exhibit channel activity in the bacterial assays, and as such may be considered viroporin candidates. It is clear that more studies, such as patch clamping, will be needed to characterize the ionic conductivities of these proteins. However, our approach presents a rapid procedure to analyze open reading frames in other viruses, yielding new viroporin candidates for future detailed investigation. Finally, if conductivity is proven vital to their cognate viruses, the bio-assays presented herein afford a simple approach to screen for new channel blockers.

Blockers of the SARS-CoV-2 3a Channel Identified by Targeted Drug Repurposing

“…This system ensures targeting of the protein to the inner bacterial membrane. While this membrane may not be identical to the eukaryotic Golgi-ER compartment where 3a is thought to reside [ 55 , 56 ], it was used to express and examine many other viral ion channels successfully [ 35 , 53 , 55 , 57 , 58 ].…”

“…Future electrophysiological studies will be needed to assess the detailed inhibitory mechanisms of each chemical. In addition, in vitro and in vivo studies will be required to establish the anti-viral activity of the different blockers considering the intracellular localization of the protein and the ability of individual hits to access it [ 55 , 56 ].…”

“…Three bacteria-based assays were performed as described previously when analyzing the E protein from SARS-CoV-2 [ 35 , 55 ].…”

Targeted Drug Repurposing Against the SARS-CoV-2 E channel Identifies Blockers Within vitroAntiviral Activity

“…To ensure proper membrane reconstitution, we used the MBP Fusion and Purification System (New England BioLabs, Ipswich, MA) in which the E protein was fused to the carboxy terminus of the maltose-binding protein. This system has been used to express and study numerous other viral ion channels successfully (23–26). Finally, we have recently shown that SARS-CoV-2 E protein is also expressed in functional form utilizing this construct (16).…”

“…These bacteria are incapable of growth unless the media is supplemented by K + (27). However, when a channel capable of K + transport is heterologously expressed, the bacteria can thrive even in low K + media (25, 26). Hence, in this instance, the viral channel positively impacts the bacteria, and channel blockers result in growth retardation.…”