Zika M-A Potential Viroporin: Mutational Study and Drug Repurposing

Abstract

Genus Flavivirus contains several important human pathogens. Among these, the Zika virus is an emerging etiological agent that merits concern. One of its structural proteins, prM, plays an essential role in viral maturation and assembly, making it an attractive drug and vaccine development target. Herein, we have characterized ZikV-M as a potential viroporin candidate using three different bacteria-based assays. These assays were subsequently employed to screen a library of repurposed drugs from which ten compounds were identified as ZikV-M blockers. Mutational analyses of conserved amino acids in the transmembrane domain of other flaviviruses, including West Nile and Dengue virus, were performed to study their role in ion channel activity. In conclusion, our data show that ZikV-M is a potential ion channel that can be used as a drug target for high throughput screening and drug repurposing.

Keywords: Dengue virus; West Nile virus; Zika virus; drug discovery; drug repurposing; flavivirus; ion channel proteins; membrane glycoprotein; viroporins.

Figure 1

Standardization of ZikV-M as a viroporin in the negative genetic assay. Growth curves (A) and maximal growth rates (B) of DH10B bacteria expressing ZikV-M, grown in LB media with an increasing dose of IPTG, as noted.

Figure 2

Positive genetic assay utilizing K⁺-uptake deficient bacteria, which are unable to grow on LB media without potassium supplement. Growth curves (A) and maximal growth rates (B) of K⁺-uptake deficient bacteria harboring the ZikV-M plasmid grown with different concentrations of IPTG, as noted.

Figure 3

Proton conductivity of flavivirus viroporins and mutants thereof. Wild-type ZikV-M, WNV MgM, DenV MgM, and mutants were transformed into bacteria that harbor a pH-sensitive green fluorescence protein [37,38]. Proton flow for uninduced wild type, induced wild type, and mutants (100 µM IPTG) were recorded after two hours of induction. Panel (A) depicts the H⁺ concentration change as a function of time, while the slopes of the individual curves are listed in panel (B).

Figure 3

Proton conductivity of flavivirus viroporins and mutants thereof. Wild-type ZikV-M, WNV MgM, DenV MgM, and mutants were transformed into bacteria that harbor a pH-sensitive green fluorescence protein [37,38]. Proton flow for uninduced wild type, induced wild type, and mutants (100 µM IPTG) were recorded after two hours of induction. Panel (A) depicts the H⁺ concentration change as a function of time, while the slopes of the individual curves are listed in panel (B).

Figure 4

Effect of mutants of flavivirus membrane proteins in the negative genetic assay. Maximal growth rates of bacteria that express mutants of ZikV-M (A), WNV MgM (B), and DenV MgM (C) as a function of increasing IPTG, as noted.

Figure 5

Effect of mutants of flavivirus membrane proteins in the positive genetic assay. Maximal growth rates of K⁺-uptake deficient bacteria that express mutants of ZikV-M, WNV MgM, and DenV MgM grown with 10 µM IPTG.

Figure 6

Drug screening on wild-type ZikV-M. Maximal growth rate as a function of the different drugs (100 µM) in the negative genetic assay (A) or positive (B) assays. (C) Effect of drugs on proton flow examined on bacteria that express a pH-sensitive GFP [38]. Note, that effective compounds are expected to increase the growth rate in the negative assay (A), decrease growth in the positive assay (B), and decrease H⁺ flow in the pH-assay (C).