The SH protein of mumps virus is a druggable pentameric viroporin

Abstract

Viral infections are on the rise and drugs targeting viral proteins are needed. Viroporins constitute a growing group of virus-encoded transmembrane oligomeric proteins that allow passage of small molecules across the membrane. Despite sparsity in viroporin structures, recent work has revealed diversity in both the number of transmembrane helices and oligomeric states. Here, we provide evidence that the small hydrophobic protein (SH) from mumps virus is a pentameric viroporin. From extensive biophysical data, a HADDOCK model of full-length SH shows its intracellular C-terminal region to form an extended structure crucial to stabilization of the pentamer. Heterologous expression of wild-type SH and variants in Xenopus laevis oocytes reveals the viroporin as a chloride channel, with transport facilitated by conserved hydroxyl-carrying residues lining the pore. The channel function of SH is inhibited by the small-molecule BIT225, highlighting the potential for antiviral targeting through SH.

Fig. 1.. Oligomerization and structural characterization of SH_FL.

(A) Consensus amino acid sequence of SH from genotype G. (B) Membrane topology of SH. (C) Left: SDS-PAGE of SH_FL in 100 mM SDS. Right: SDS-PAGE of SH_FL in 1:120 POPC. (D) Native MS of SH_FL in DPC. (E) Far-UV CD spectrum of SH_FL in 1:500 DHPC. (F) ¹H-¹⁵N HSQC spectra of SH_FL in different membrane mimetics and detergents as indicated. (G) SCS analysis of SH_FL in 66% TFE:33% H₂O (v/v). (H) ¹H-¹⁵N HSQC spectra of the Trp⁵¹ indole peak from (F). Peaks colored by lipid or detergent head group charge. Anionic, red; Zwitterionic, blue.

Fig. 2.. SH is helical only in its TMD.

(A) ¹H-¹⁵N HSQC spectrum of SH_1–34 in 1:500 DHPC, with assignment. (B) SCS analysis of SH_1–34 in (A). R₁, R₂, and HetNOE values for SH_1–34 in 1:500 DHPC. (C) Far-UV CD spectra of SH_1–34 in 1:500 DHPC. (D) ¹H-¹⁵N HSQC spectrum of SH_2–12, with assignment. (E) SCS analysis of SH_2–12 in (D). (F) Far-UV CD spectra of SH_2–12. (G) ¹H-¹⁵N HSQC spectrum of SH_39–57, with assignment. (H) SCS analysis of SH_39–57 in (G). (I) Far-UV CD spectra of SH_35–57.

Fig. 3.. The SH pentamer.

(A) Five helix bundle of the transmembrane region SH_7-34. (B) Simplified two helix view of pore-lining residues and HOLE-calculated pore diameter. (C) Pore radius of SH_7–34 along with pore-lining residues. (D) Examples of helix stabilizing residues in the helix-helix interface. (E) Clusters from MD simulation of SH_35–57. (F) Two pentameric models of SH_FL with extended C-termini. Insert: Zoom of Trp⁵¹ proximity to “membrane.”

Fig. 4.. SH is a viroporin and conducts Cl⁻ currents in Xenopus l. oocytes.

(A) Illustration of the two-electrode voltage clamp setup employed. One microelectrode is used for current injection, and one is used for voltage sensing, measuring the membrane potential (V_m) compared to a command voltage, which is leveled by an amplifier. (B) Summarized and averaged current-voltage (I/V) relations in SH_FL-expressing oocytes compared to control (uninjected) oocytes. n = 11. (C) Current activity at −85 mV in SH_FL-expressing oocytes compared to uninjected oocytes from (B). Inset: Representative current traces. (D) Summarized I/V curves of uninjected or with 4 or 50 ng of SH_FL RNA/oocyte microinjected. n = 9. (E) Current activity at −85 mV in SH_FL-expressing oocytes compared to uninjected oocytes from (D). (F) Summarized I/V curves of SH_FL-induced currents in the presence or absence of Cl⁻. n = 10. (G) Normalized data at V_m = −85 mV from (F). (H) Summarized I/V curves of SH_FL-mediated currents in the presence or absence of Ca²⁺. n = 9. (I) Normalized data at V_m = −85 mV from (H). (J) Summarized I/V curves of SH_FL-induced currents in the presence or absence of Na⁺. n = 7. (K) Normalized data at V_m = −85 mV from (J). All summarized current traces represent means ± SEM. Each point in the bar charts indicates an individual oocyte. The magnitude of SH_FL-mediated currents (at V_m = −85 mV) was compared to those of uninjected oocytes using an unpaired t test (C), one-way analysis of variance (ANOVA) (E), or between the presence or absence of indicated ion with paired t test [(G), (I), and (K)]. *P < 0.033, **P < 0.002, and ***P < 0.001.

Fig. 5.. SH channel function is dependent on oligomerization and polar residues and inhibited by BIT225.

(A) Left: Schematic representation of SH variants. Right: Sequences of SH WT and variants. All electrophysiology experiments included a C-terminal His6-tag. SH_LW: L24A, W27A. SH_Y: Y19A. (B) Summarized I/V curves of SH variants. n = 9,22. (C) Data at V_m = −85 mV from (B). (D) Surface side view of SH_FL and SH_LW. (E) Surface N-terminal view of SH_FL and SH_Y. (F) Summarized I/V curves of SH_FL-expressing oocytes with 45-min preincubation with 10 μM BIT225 or matched negative controls. n = 9. (G) Data at V_m = −85 mV from (F). (H) Concentration dependent inhibition of SH_FL by BIT225. All summarized current traces represent means ± SEM. Each point in the bar charts indicates an individual oocyte. Statistical significance was determined with unpaired t test. *P < 0.033, **P < 0.002, and ***P < 0.001.