Zika Virus Dependence on Host Hsp70 Provides a Protective Strategy against Infection and Disease

Abstract

The spread of mosquito-borne Zika virus (ZIKV), which causes neurological disorders and microcephaly, highlights the need for countermeasures against sudden viral epidemics. Here, we tested the concept that drugs targeting host proteostasis provide effective antivirals. We show that different cytosolic Hsp70 isoforms are recruited to ZIKV-induced compartments and are required for virus replication at pre- and post-entry steps. Drugs targeting Hsp70 significantly reduce replication of different ZIKV strains in human and mosquito cells, including human neural stem cells and a placental trophoblast cell line, at doses without appreciable toxicity to the host cell. By targeting several ZIKV functions, including entry, establishment of active replication complexes, and capsid assembly, Hsp70 inhibitors are refractory to the emergence of drug-resistant virus. Importantly, these drugs protected mouse models from ZIKV infection, reducing viremia, mortality, and disease symptoms. Hsp70 inhibitors are thus attractive candidates for ZIKV therapeutics with the added benefit of a broad spectrum of action.

Keywords: Hsp70; Hsp70 inhibitor; Zika virus; antiviral; chaperone; viral assembly; viral capsid; viral entry; viral replication complex.

Figure 1.. Cytosolic Hsp70 Isoforms Are Involved in the ZIKV Life Cycle

(A and B) Depletion of cytosolic Hsp70 isoforms reduces Zika virus (ZIKV) replication. Indicated knockdown (KD) cells were infected with ZIKV MR766 at MOI 0.5; 48 h post-infection (hpi), supernatants were harvested and extracellular virus titrated by focus-forming assay (FFA) as described in STAR Methods (A). Intra- cellular RNA was harvested, and viral RNA and host GAPDH mRNA was measured by qRT-PCR (B). Data represent means ± SD of three independent exper- iments, each carried out in triplicate. *p < 0.01; **p < 0.005. (C–F) Cytosolic Hsc70s and Hsp70s colocalize with ZIKV replication compartments. Huh7 cells infected with ZIKV MR766 were stained at 36 hpi with a pan- cytosolic HSC70 and HSP70 antibody (green); dsRNA, which marks viral replication sites (blue); and E or NS3 proteins (red) (C). Huh7 expressing GFP-HSPA8 (D), GFP-HSPA1 (E), or GFP-HSPA1L (F) cells were infected as in (C). Scale bars, 10 μm.

Figure 2.. Hsp70 Functions Are Universally Required for ZIKV Propagation in Two Distinct Hosts: Human and Mosquito Cells

(A) Chemical structures of small compounds: JG40, JG345, and JG132, specific Hsp70 inhibitors; negative control, JG258; and 2⁰CMA, a viral NS5 polymerase inhibitor. (B and C) Hsp70 inhibitors suppress propagation of three ZIKV strains in human and mosquito cells in a dose-dependent manner. Huh7 (B) or C6/36 (C) cells were infected with (i) MR766, (ii) P6–740, and (iii) PRVABC59 at MOI 0.5 for 1 h, and inhibitors were added for 36 h (Huh7 cells) or 48 h (C6/36 cells). Extracellular virus infectivity was determined by FFA. All data are expressed as means ± SD of three independent experiments, each carried out in triplicate, and analyzed by one-way ANOVA with post hoc Tukey honestly significant difference (HSD) test. *p < 0.05; **p < 0.01; ***p < 0.005.

Figure 3.. Hsp70s Facilitate Two Distinct Steps in the ZIKV Life Cycle

(A) Hsp70 inhibition blocks viral entry and post-entry steps. Time-of-drug addition experiment with an Hsp70 inhibitor (JG40), entry inhibitor (heparin), and viral NS5 polymerase inhibitor (2⁰CMA). Huh7 cells infected with ZIKV MR766 at MOI 0.5 were treated with inhibitors as indicated. Intracellular ZIKV RNA and extracellular virus infectivity was measured by qRT-PCR and FFA, respectively. (B) Hsp70 is required for RNA replication during early infection. Huh7 cells were infected with ZIKV MR766 at MOI 0.5. At 6 hpi, cells were treated with 3 mM 2⁰CMA or JG40, and intracellular viral RNA levels were monitored every 3 h. (C–F) Hsp70 is required for infectious viral particle production of ZIKV. Drug-chase experiment comparing the effects of 2⁰CMA and JG40 on viral RNA (vRNA) replication and virion production (C). Huh7 cells infected with ZIKV MR766 at MOI 0.5 were treated at 24 hpi with 3 mM 2⁰CMA or JG40, and levels of intracellular ZIKV RNA (D) and intra- and extracellular infectious virion levels (E and F) were measured by qRT-PCR and FFA, respectively. All data are expressed as mean ± SD of three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.005; ****p < 0.001.

Figure 4.. Hsp70s Maintain Conformational Stabilization of Capsid and Its Localization on Lipid Droplets

(A) Processed and unprocessed capsid variants physically associate with Hsc70. FLAG-tagged capsids were co-expressed together with GFP-tagged HSPA8 or control GFP in 293T cells, and their interactions were examined by co-immunoprecipitation. (B) Hsp70 inhibition reduces expressions of processed and unprocessed capsids. FLAG-tagged processed and unprocessed capsid variants were expressed in Huh7 cells, and cells were treated with JG40 for 42 h. Cell lysates were subjected to immunoblot (left) and quantified using infrared (IR) fluorescence on a Li-Cor Odyssey System (right). Data are mean ± SD of three independent experiments. *p < 0.05; **p < 0.01. (C) Capsid localization to lipid droplets is decreased by Hsp70 inhibition. Huh7 cells stably expressing FLAG-tagged capsid 1–104 were treated with JG40 for 12 h, fixed by PFA, and permeabilized with saponin. FLAG-capsid (red) and ADRP (blue) were stained with specific primary antibodies, and the lipid droplets (green) were stained with BODIPY. (D–G) Hsp70 inhibition reduces capsid levels in the light fraction of density gradients. (D) Scheme of experiments. At 24 hpi, Huh7 cells infected with ZIKV MR766 were treated with the indicated drugs for 12 h. Cell lysates were fractionated by iodixanol-gradient centrifugation. (E) Each fraction was subjected to SDS-PAGE followed by immunoblot. (F) Multimerization of the capsid is impaired under Hsp70 inhibition. Fractionated lysates as in (D) were subjected to blue-native PAGE and analyzed by immunoblot. (G) Effect of Hsp70 inhibition on capsid stability in the whole-cell lysate or fraction 2. Samples were subjected to SDS-PAGE and immunoblot as in (E).

Figure 5.. Involvement of Hsp70 Function in Infectious ZIKV Particle Production

(A) Blocking degradation does not abrogate JG40 inhibition of ZIKV propagation. Huh7 cells infected with ZIKV at MOI 0.5 were treated at 24 hpi with 3 mM CM and MG132 or JG40, and extracellular virion levels were measured by FFA. (B) Hsp70 inhibition results in a reduced ratio of infectivity per particle. (i) At 24 hpi (Huh7) or (ii) 36 hpi (C6/36), ZIKV-infected cells were treated with the indicated Hsp70 inhibitors and incubated for 12 h. The ratio of infectivity to vRNA was calculated based on the amount of vRNA (Figure S5A) and infectivity (Figure S5B) in the supernatant. (C) Post-translationally modified capsids incorporate into extracellular particles upon Hsp70 inhibition. ZIKV-infected Huh7 cells were treated with JG40 as in Figure 5B. The cells and supernatant were lysed and subjected to immunoblot analysis with anti-capsid (left) and anti-prM antibodies (right). (D) Influence of Hsp70/proteasome inhibition on the incorporation of viral proteins into particles. ZIKV-infected Huh7 cells were treated with JG40/MG132 as in Figure 5B. The viral proteins in the supernatant were detected by immunoblot analysis. (E) Serial passages of ZIKV MR766 in inhibitor-treated conditions to test the development of drug resistance. At each passage, Huh7 cells were infected with ZIKV at an MOI 0.5 in the presence or absence of JG40 or 2⁰CMA. The sensitivity of inhibitors was examined for the indicated virus passages obtained as above using an MOI 0.5 to infect Huh7 cells. Extracellular infectivity was quantified by FFU assay. The passage experiments were performed three times independently (replica I is shown here, and replicas II and III are shown in Figure S5G). All data are expressed as mean ± SD of three independent experiments. *p < 0.05.

Figure 6.. Hsp70 Inhibitors Suppress ZIKV Propagations in Clinically Relevant Cells and Protect Mice from Lethal ZIKV Infection

(A and B) Hsp70 inhibitors potently inhibit different ZIKV strains in clinically relevant cell lines. JEG3 cells (A) or H9-derived human neural stem cells (B) were infected with the ZIKV strains at MOI 0.5 and treated with JG compounds at the indicated concentrations for 48 h, and then infectivity was determined by FFA in Huh7 cells. Data are mean ± SD of three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.005. (C) Schedule of virus challenge in mice model. (D) Survival rates of ZIKV-infected mice with solvent or JG345 at the indicated concentrations, monitored for 14 days. n = 18 in each cohort. (E) Administration of Hsp70 inhibitor alleviates the clinical manifestation of ZIKV infection. Clinical score determination is described in STAR Methods. n = 18 in each cohort. (F) At day 5, peripheral blood was harvested from ZIKV-infected mice, and infectivity in the serum was determined by FFA using Vero cells.