Thiazolides, a new class of anti-influenza molecules targeting viral hemagglutinin at the post-translational level

Abstract

The emergence of highly contagious influenza A virus strains, such as the new H1N1 swine influenza, represents a serious threat to global human health. Efforts to control emerging influenza strains focus on surveillance and early diagnosis, as well as development of effective vaccines and novel antiviral drugs. Herein we document the anti-influenza activity of the anti-infective drug nitazoxanide and its active circulating-metabolite tizoxanide and describe a class of second generation thiazolides effective against influenza A virus. Thiazolides inhibit the replication of H1N1 and different other strains of influenza A virus by a novel mechanism: they act at post-translational level by selectively blocking the maturation of the viral hemagglutinin at a stage preceding resistance to endoglycosidase H digestion, thus impairing hemagglutinin intracellular trafficking and insertion into the host plasma membrane, a key step for correct assembly and exit of the virus from the host cell. Targeting the maturation of the viral glycoprotein offers the opportunity to disrupt the production of infectious viral particles attacking the pathogen at a level different from the currently available anti-influenza drugs. The results indicate that thiazolides may represent a new class of antiviral drugs effective against influenza A infection.

Figures

FIGURE 1.

Thiazolides inhibit influenza A virus replication acting at a post-entry level. A, structure of NTZ and TIZ. B, NTZ (blue circles) and TIZ (red circles) inhibit the replication of mammalian (PR8 and WSN) and avian (A/Ck) influenza A virus strains in MDCK cells. Virus yield was determined at 24 h p.i. C, antiviral activity of TIZ on influenza A PR8 virus in human monocytic U937 (●) and T-lymphoblastoid Jurkat (▴) cells and WSN virus in human lung epithelial A549 cells (♦). D, MDCK cells were treated with 10 μg/ml TIZ (filled bars) at the indicated times before infection (Pre), immediately after the adsorption period (Post), or only during the adsorption period (Ad, dashed bar). The empty bar represents untreated infected control (C). E, long term antiviral activity of TIZ in PR8-infected MDCK cells treated with 10 μg/ml TIZ (filled circles) or vehicle (empty circles) after virus adsorption. B–E, virus yield, expressed in HAU/ml (B and E) or as a percentage of untreated control (C and D), represents the means ± S.D. of duplicate samples from a representative experiment of three with similar results. *, p < 0.01; **, p < 0.05.

FIGURE 2.

Tizoxanide selectively alters influenza hemagglutinin maturation. A, effect of TIZ on the kinetics of PR8 virus protein synthesis. Autoradiography of [35S]Met/Cys-labeled proteins (1.5-h pulse) at different times p.i. from mock infected (U) or PR8-infected cells treated with 10 μg/ml TIZ after virus adsorption (top panels). The viral proteins are indicated. In the same experiment, protein synthesis was determined by [35S]Met/Cys incorporation into proteins of cells treated with TIZ (●) or vehicle (○) (bottom panels), and phospho-eIF2α protein levels were determined by immunoblot analysis using anti-Ser(P)51-eIF2α (p-eIF2α) or eIF2α panspecific antibodies (middle panels). B, hemagglutinin identification by immunoprecipitation with anti-HA antibodies after [35S]Met/Cys labeling at 5 h p.i. (4-h pulse). Immunoprecipitated proteins (+α-HA, IP) and radiolabeled proteins from the same samples before antibody addition (−α-HA) are shown. Positions of HA uncleaved precursor (HA0) are indicated. C, autoradiography of [35S]Met/Cys-labeled proteins (15-h pulse) from mock infected (U) or PR8-infected cells treated with 10 μg/ml TIZ, 5 μg/ml TM, or vehicle (lane C) after virus adsorption. The white arrowhead and black arrow indicate TM-induced GRP78/BiP and nonglycosylated HA0 (identified by immunoblot; not shown), respectively. D, autoradiography of [35S]Met/Cys-labeled proteins (15-min pulse at 5 h p.i., followed by chase for the indicated times) from PR8-infected cells treated as in A. A–D, the slower- and faster-migrating HA0 forms in untreated or TIZ-treated cells are identified by asterisks and black arrowheads, respectively.

FIGURE 3.

Thiazolides interfere with viral hemagglutinin N-glycosylation. A, mock infected (U) or PR8-infected MDCK cells were treated with 10 μg/ml TIZ, 5 μg/ml TM, or vehicle (lane C) after virus adsorption. At 6 h p.i., the cells were labeled for 4 h with [35S]Met/Cys (top panels), [3H]glucosamine (middle panels), or [3H]mannose (bottom panels). Radiolabeled samples were processed for SDS-PAGE and autoradiography. Sections of fluorograms from SDS-PAGE gels are shown. White arrowheads indicate TM-induced Grp78/BiP. B, mock infected (U) or PR8-infected MDCK cells were treated with 10 μg/ml TIZ, 10 μg/ml swainsonine (SW), 15 μg/ml 1-deoxymannojirimicin (DMJ), or vehicle (lane C) after virus adsorption. At 6 h p.i., the cells were labeled with [35S]Met/Cys (4-h pulse), and radiolabeled samples were processed for SDS-PAGE and autoradiography. C and D, autoradiography of radiolabeled proteins from mock infected (U) or WSN-infected A549 cells (lane C) and mock infected or avian influenza A virus-infected (A/Ck) MDCK cells (D) treated with 5 μg/ml TIZ, 5 μg/ml TM, or vehicle (lane C) after virus adsorption. At 3 h (WSN) or 6 h (A/Ck) p.i., the cells were labeled with [35S]Met/Cys for 15 h (WSN) or 4 h (A/Ck). E and F, autoradiography of radiolabeled proteins from mock infected (U), PR8-infected (E), or avian influenza A virus-infected (A/Ck) (F) MDCK cells treated with 10 μg/ml TIZ, 10 μg/ml NTZ or vehicle (lane C) after virus adsorption. At 6 h p.i., the cells were labeled with [35S]Met/Cys for 4 h. A–F, viral proteins HA0, NP, M1, and NS1 are indicated. The slower- and faster-migrating HA0 forms in untreated or thiazolide-treated cells are identified by asterisks and black arrowheads, respectively.

FIGURE 4.

Tizoxanide blocks HA maturation at an Endo-H-sensitive stage. A, mock infected (U) or PR8-infected MDCK cells treated with 10 μg/ml TIZ (+) or vehicle (−) after virus adsorption were labeled with [35S]Met/Cys (4-h pulse) at 5 h p.i. Radiolabeled proteins were digested (+) or not (−) with PNGase-F or Endo-H and processed for SDS-PAGE and autoradiography. Uncleaved glycosylated (HA0) and nonglycosylated (HAp) hemagglutinin precursor forms are indicated. B, MDCK cells treated as in A were labeled with [35S]Met/Cys (4-h pulse) at 6 h p.i. Radiolabeled proteins were immunoprecipitated (IP) with anti-HA antibodies (α-HA), digested (+) or not (−) with Endo-H, and processed for SDS-PAGE. Sections of fluorograms are shown. C, whole cell extracts from mock infected (U) and PR8-infected MDCK cells treated with TIZ (+) or vehicle (−) were incubated with (+) or without (−) the cross-linking reagent ethylene glycol bis(succinimidylsuccinate) (EGS, 0.2 mm) and processed for Western blot using anti-HA antibodies. HA monomers (mark 1), dimers (mark 2), and trimers (mark 3) are indicated. A–C, slower- and faster-migrating HA0 forms in untreated or TIZ-treated cells are identified by asterisks and black arrowheads, respectively. D, immunofluorescence of mock infected (U) and WSN-infected A549 cells treated with TIZ (5 μg/ml) or vehicle for 24 h, labeled with anti-p230 trans-Golgi (red) and anti-HA (green) antibodies. The nuclei are stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). The overlay of the three fluorochromes is shown (merge). The enlarged areas (insets) highlight the localization of HA in untreated and TIZ-treated cells. The images were captured and deconvolved with a DeltaVision microscope using SoftWoRx-2.50 software. Bar, 5 μm.

FIGURE 5.

Tizoxanide inhibits transport of influenza hemagglutinin to the cell surface. A, levels of total hemagglutinin (green) and α-tubulin (red) were detected in mock infected (U) and untreated or TIZ-treated (10 μg/ml) PR8-infected MDCK cells at 16 h p.i. by indirect immunofluorescence (bar, 10 μm). The nuclei are stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). The overlay of the three fluorochromes is shown (merge). The images were captured and deconvolved with a DeltaVision microscope using the SoftWoRx-2.50 software. B, levels of plasma-membrane hemagglutinin (green) were detected at 16 h p.i. by indirect immunofluorescence (top) in mock infected or PR8-infected cells treated with 10 μg/ml TIZ or 5 μg/ml TM. The nuclei are stained with Hoechst 33342 (blue). The images were processed as in A (bar, 10 μm). The overlay of the two fluorochromes is shown. Erythrocytes hemadsorption on plasma membrane at 5 h p.i. is shown in parallel samples (bottom row) (bar, 35 μm). Hemoglobin levels of bound erythrocytes were quantified spectrophotometrically (λ = 540 nm). The data, expressed in optical density (O.D.), represent the means ± S.D. of duplicate samples from a representative experiment of two with similar results. *, p < 0.05 versus infected-control. C, autoradiography of [35S]Met/Cys-labeled proteins incorporated into viral particles purified at 24 h p.i. from supernatants of mock infected or PR8-infected cells treated as in B. Viral proteins (HA, NP, and M1) are indicated. D, in parallel, virus yield was determined in untreated (empty bars) or TIZ-treated (filled bars) PR8-infected cells at 24 h p.i. by infectivity assay (top) and hemagglutination assay (bottom). The data, expressed in TCID50/ml and HAU/ml respectively, represent the means ± S.D. of duplicate samples from a representative experiment of two with similar results. *, p < 0.05 versus infected control.