Allosteric heat shock protein 70 inhibitors block hepatitis C virus assembly

Abstract

The human molecular chaperones heat shock protein 70 (Hsp70) and heat shock cognate protein 70 (Hsc70) bind to the hepatitis C viral nonstructural protein 5A (NS5A) and regulate its activity. Specifically, Hsp70 is involved in NS5A-augmented internal ribosomal entry site (IRES)-mediated translation of the viral genome, whilst Hsc70 appears to be primarily important for intracellular infectious virion assembly. To better understand the importance of these two chaperones in the viral life cycle, infected human cells were treated with allosteric Hsp70/Hsc70 inhibitors (AHIs). Treatment with AHIs significantly reduced the production of intracellular virus at concentrations that were non-toxic to human hepatoma Huh7.5 cells. The supernatant of treated cultures was then used to infect naïve cells, revealing that AHIs also lowered levels of secreted virus. In contrast to their effects on virion assembly, AHIs did not impact the stability of NS5A or viral protein translation in IRES assays. These results suggest that Hsc70 plays a particularly important and sensitive role in virion assembly. Indeed, it was found that combination of AHIs with a peptide-based viral translation inhibitor exhibited additive antiviral activity. Together these results suggest that the host Hsc70 is a new antiviral target and that its inhibitors utilise a new mechanism of action.

Keywords: Allosteric heat shock protein inhibitors; Hepatitis C virus; Hsc70; Hsp70; Viral assembly; Viral translation.

Fig. 1

Cytotoxicity profile of allosteric Hsp70/Hsc70 inhibitors (AHIs). (A) Cellular toxicity of the AHIs in the concentration range of 100 pM to 10 μM. Huh7.5 cells were treated with the indicated concentrations of the AHIs and MTT assays were then performed every 24 h up to 72 h. The 72-h data points are shown. (B) Cellular toxicity of the AHIs in the concentration range of 1μM to 10 μM. Huh7.5 cells were treated with the indicated concentrations of the AHIs and MTT assays were then performed as above. The 72-hour data points are shown. (C) Cellular toxicity of the AHIs at 1 μM concentration over a time course of 72 h. Huh7.5 cells were treated with 1 μM of the AHIs and MTT assays were then performed as above. DMSO, dimethyl sulphoxide.

Fig. 2

The allosteric Hsp70/Hsc70 inhibitors (AHIs) YM-01, JG-40 and JG-98 significantly attenuate virus production. (A) Intracellular virus production assay indicates that YM-01, JG-40 and JG-98 significantly block virus production. (B) Viral secretion is significantly attenuated by AHIs as demonstrated by a long-term infectious virion secretion assay. (C) AHIs significantly block virus production. The intracellular virus production assay was performed as in (A) using wild-type virus and Western analyses were performed with antibodies against the viral core and cellular actin proteins. *** P < 0.0005. DMSO, dimethyl sulphoxide.

Fig. 3

Allosteric Hsp70/Hsc70 inhibitors (AHIs) block virus production in a dose-dependent manner. Dose response of the AHIs in the concentration range of 100 pM to 1 μM as determined by intracellular virus production assays. IC₅₀, half-maximal inhibition concentration.

Fig. 4

Allosteric Hsp70/Hsc70 inhibitors (AHIs) do not affect viral entry. (A) Schematic of the entry assay. (B) Huh7.5 cells were treated with the indicated compounds and were immediately infected for 3 h, at which point, the medium was replaced and the infection was allowed to proceed for 18 h. The cells were then lysed and luciferase activity was measured. *** P < 0.0005. DMSO, dimethyl sulphoxide.

Fig. 5

Allosteric Hsp70/Hsc70 inhibitors (AHIs) do not affect viral protein translation and internal ribosomal entry site (IRES) activity. (A) Schematic of the intracellular viral protein production assay. (B) Intracellular viral protein production assay demonstrates that the AHIs do not affect viral protein translation. (C) Schematic of the bicistronic construct utilised for determining the levels of NS5A-augmented IRES-mediated translation. (D) NS5A-augmented IRES-mediated translation is not affected by the AHIs as determined by IRES reporter assays. *** P < 0.0005.

Fig. 6

Allosteric Hsp70/Hsc70 inhibitors (AHIs) block intracellular infectious virion assembly. (A) Schematic of the assembly assay. BFA, brefeldin A. (B) Intracellular infectious virion assembly assays performed with the Renilla reporter virus indicate that YM-01, JG-40 and JG-98 significantly block infectious virion assembly. (C) AHIs block intracellular assembly of infectious virus. Intracellular infectious virion assembly assays was performed as in (A) and (B) using wild-type virus and Western analyses were performed on the cells infected with intracellular virus with antibodies against the viral core, NS3 and NS5B as well as cellular actin proteins. (D) AHIs do not affect viral protein levels. Intracellular infectious virion assembly assay was performed as in (A) and (B) using wild-type virus and Western analyses were performed on the cells treated with AHIs for 8 h. DMSO, dimethyl sulphoxide.

Fig. 7

Allosteric Hsp70/Hsc70 inhibitors (AHIs) significantly affect infectious virion secretion. (A) Schematic of the secretion assay. (B) Short-term infectious virion secretion assays demonstrate the significant effect of YM-01, JG-40 and JG-98 on the levels of infectious virion secretion. (C) The combination of the AHIs and the HCV4 peptide displays significantly improved antiviral activity as demonstrated by the intracellular virus production assays. *** P < 0.0005. DMSO, dimethyl sulphoxide.