Heat shock protein 90 chaperone activity is required for hepatitis A virus replication

Abstract

HSP90 heat shock chaperones are essential for maintaining cellular proteostasis, as well as the ATP-dependent folding and functional maturation of many viral proteins. As a result, inhibitors of HSP90 have broad antiviral activity, disrupting replication of many viruses at concentrations below those causing cytotoxicity. Among the Picornaviridae, HSP90 inhibitors block replication of multiple Enterovirus, Aphthovirus, and Cardiovirus species, in some cases, by preventing post-translational processing and assembly of P1 capsid proteins. Hepatitis A virus (HAV), classified within the genus Hepatovirus, has been suggested to be an exception among picornaviruses and to replicate independently of HSP90, possibly because its slow translational kinetics could facilitate co-translational folding and assembly of its capsid proteins. However, we show here that HAV replication is highly dependent upon HSP90, both in human hepatocyte-derived cell lines, in which the 50% inhibitory concentration of geldanamycin was 8.7-11.8 nM, and in vivo in Ifnar1^-/- mice. Label-free proteomics experiments suggested that HSP90 interacts with capsid proteins or their precursors and may thus facilitate the folding and assembly of capsid proteins, as it does for enteroviruses and aphthoviruses. By contrast, there was no evidence for HSP90 interacting with any nonstructural protein, and HSP90 inhibitors did not impair 3C^pro proteolytic activity. Despite this, and in contrast to previous studies of enteroviruses and aphthoviruses, geldanamycin potently inhibited replication of a subgenomic HAV replicon. We conclude that HAV is no exception from the HSP90-dependent nature of other picornaviruses and indeed is more dependent on HSP90 than other picornaviruses for amplification of its genome.IMPORTANCEHepatitis A virus (HAV), a common cause of acute infectious hepatitis, has been reported to differ from other picornaviruses in not requiring heat shock protein HSP90 for efficient replication. However, we show here that productive HAV infection is highly dependent on HSP90 and that HAV replication is potently blocked both in cell culture and in vivo in the murine liver by chemical inhibitors of HSP90. Such inhibitors also disrupt the replication of a subgenomic HAV RNA replicon, indicating that HSP90 is required for the assembly of functional replication organelles. This highlights a key difference from other picornaviruses for which HSP90 is required primarily, if not exclusively, for the maturation of the P1 capsid proteins.

Keywords: ACC1; HD-PTP; HSP70; HSP90 inhibitor; antiviral; chaperone; hepatovirus; mouse model; picornavirus; quasi-enveloped virus; replicon.

Fig 1

The heat shock pathway is required for HAV replication. (A) Screen for inhibitors of infectious virus production using the 18f-NLuc reporter virus and a commercial 326 chemical compound library. Huh-7.5.1 cells were treated with compounds (10 µM) from 6 to 72 hours after infection, with cell viability assessed at 72 hours. Supernatant fluids collected at 72 hours were assayed for virus by measuring nanoluciferase expressed in a second round infection. Results were normalized for cytotoxicity. (B) Results of the chemical screen. The top six inhibitory compounds are highlighted: GA, geldanamycin; CVi, crystal violet; EtB, ethidium bromide; DPV, dapivirine; 4270, 4270-0405; DhS, dehydroandrographolide succinate. See also Table S1. (C) Geldanamycin inhibition of infectious virus release. Huh-7.5.1 cells were treated, and cell viability was assessed as in panel A. HAV was assayed in supernatant fluids by quantifying viral RNA 48 hours after infection of naive cells. (D) Antiviral activity of HSP90 inhibitors against HM175/18f virus. Huh7.5.1 cells were infected at a multiplicity of 100 GE/cell and treated with increasing concentrations of inhibitors from 6 to 72 hours post-infection. HAV RNA was quantified at 72 hours by RT-qPCR. Dashed lines indicate cell viability with the DMSO-treated control set at 1.0. Geldanamycin (open red symbols) IC₅₀ = 11.8 nM, CC₅₀ 333 nM; 17-AAG (shaded black symbols) IC₅₀ = 14.2 nM, CC₅₀ 418 nM. Data are means ± SD, n = 3. (E) Antiviral activity of geldanamycin against the 18f-NLuc reporter virus in Huh-7.5 (open red symbols, IC₅₀ = 8.7 nM) or FRhK4 (shaded black symbols, IC₅₀ = 804 nM) cells. NLuc activity was measured 48 hours post-infection. Dashed lines indicate cell viability with the DMSO-treated control set at 1.0. Data are means ± SD, n = 3. (F) Geldanamycin inhibition of NLuc activity expressed by a PV1 reporter virus 16 hours after infection of Huh-7.5 and FRhK4 cells. Huh-7.5 IC₅₀ = 183 nM; FRhK4 IC₅₀ = >10 µM. (G) Client protein flow through the heat shock pathway. DNAJA1 is a co-chaperone for HSP70. STIP1 facilitates the transfer of client proteins from HSP70 to HSP90 and interacts with both heat shock proteins. (H) Fold increase in individual sgRNAs targeting heat shock proteins in Huh-7.5 cells following selection against lethal HAV infection in a previously described genome-wide CRISPR screen (18). Dashed line indicates a twofold change in sgRNA counts; red symbols, false discovery rate < 0.0005 by ANOVA. (I) Bubble plot showing heat shock proteins identified by mass spectrometry in two independent stable isotope labeling by amino acids (SILAC) analyses of gradient-purified quasi-enveloped eHAV (19). Mean fold enrichment of mass-tagged heat shock proteins identified in virus samples from cells grown in each experiment using media with “heavy” versus “light” isotopes from the first and second SILAC experiments (#1 and #2) are plotted on X and Y axes, respectively, while the size of bubbles represents the relative mean intensity of peptides.

Fig 2

Antiviral activity of the HSP90 inhibitor 17-AAG in infected Ifnar1^-/- mice. (A) Infection scheme: mice infected by i.v. inoculation of 10⁶ GE mouse-passaged HAV were treated with 1 mg 17-AAG or vehicle (DMSO), only administered by i.p. injection daily for 6 days. The experiment was terminated, and mice were necropsied 7 days post-infection (dpi). (B) Serum alanine aminotransferase activities at 7 dpi. (C–E) HAV RNA quantified by RTqPCR in (C) serum, (D) feces, and (E) liver at 7 dpi. n = 5–6; P-values were calculated by two-way t-test. (F) Hematoxylin and eosin-stained sections of liver from (left to right) an uninfected mouse (mock) and infected Ifnar1^-/- mice treated with 17-AAG or vehicle only at 7 dpi. (G) Immunohistochemical staining for macrophages with antibody to ionized calcium-binding adapter molecule 1 in serial sections of mouse livers. (H) Digital image scoring of IBA1-positive cells (cells/mm²) and areas of inflammation (percent area) in liver sections from untreated and 17-AAG-treated HAV-infected Ifnar1^-/- mice. Three regions of interest were scanned in each of three different sections from each mouse liver. Liver sections from uninfected mice are included for comparison. n = 5–6; P-values were calculated by two-way ANOVA. Scale bars = 50 µm.

Fig 3

HSP90 activity is required for HAV replication. (A) NLuc expressed by cells treated with the HSP90 inhibitors geldanamycin (GA, 50 nM) or 17-AAG (50 nM) for 48 hours prior to or 6 hours after infection with the 18f-NLuc reporter virus. (B) Inhibition of luciferase expressed by cells transfected with a subgenomic HAV replicon RNA, HAV-FLuc, or similarly constructed human rhinovirus (RV-B14-FLuc) and human parechovirus A1 (PeV-A1-NLuc) replicons, in the presence of increasing geldanamycin concentrations. HAV-FLuc IC₅₀ = 29.1 nM (95% CI 12.9–66.1 nM). (C) Immunoblot of GFP expressed by cells transfected with a circular RNA HAV IRES reporter, circRNA-HAV (22), in the presence of geldanamycin (100 nM), 17-AAG (100 nM), or no inhibitor (∅). (D) Polyprotein processing leading to mature VP1 and 3C^pro proteins. Primary polyprotein cleavage occurs at the P1/P2 junction between the structural (pink) and nonstructural (blue) protein precursors. All cleavages are catalyzed by 3C^pro. (E) HA immunoblot of lysates from geldanamycin-treated cells transfected with a vector expressing HA-3ABCD. Only one protein band, HA-3ABC, is apparent. (F) Immunoblots of lysates from cells transfected with vectors expressing GFP-MAVS and HA-3ABC or HAV-3ABC-C172 (catalytically inactive 3C mutant). GFP-MAVS*, 3C^pro GFP-MAVS cleavage product. (G) Anti-VP1 and anti-3C immunoblots of proteins present in lysates of cells infected for 72 hours with 18f virus, then treated with the indicated concentration of geldanamycin for 24 hours. *Nonspecific protein band.

Fig 4

Label-free proteomics analysis of HSP90-interacting proteins. (A) Immunoblots of proteins immunoprecipitated (IP) from 18f-infected Huh-7.5 cell lysates with anti-HSP90 antibody (α-HSP90) or isotype control antibody (IgG). The lysate is in lane 1. (B) Summed MS intensities of peptides derived from VP0, VP2, and VP1pX in proteins pulled down from infected Huh-7.5 cell lysates with anti-HSP90 versus isotype control IgG antibody. Data are means ± SD, n = 3 independent precipitates; IP versus lysate P = 0.0037 by two-way ANOVA. (C) Distribution of HAV-related peptide intensities identified in anti-HSP90 pulldown products versus infected whole cell lysate. ∅, none identified. Peptide intensities were summed as in panel B, then computed as a percentage of the total. (D) Number of unique peptides derived from the structural (P1) versus nonstructural (P2P3) segments of the HAV polyprotein identified in the anti-HSP90 precipitate. P-value was calculated by Fisher’s exact test. (E) HSP90 immunoblot of anti-HA immunoprecipitates (HA IP) from cells ectopically expressing HA-3C^pro and HA-3D^pol. ∅, mock transfection. (F) Volcano plot of 691 host cell proteins co-immunoprecipitating with HSP90 from HAV p16 virus-infected Huh-7.5 cells. The ratio of peptide MS intensities of proteins pulled down by anti-HSP90 versus isotype control IgG is plotted against P-value by t-test. ACC1, acetyl-CoA carboxylase 1. Shaded area, P > 0.05; n = 3. (G) Immunoblot of ACC1 in lysates of Huh-7.5 cells with or without 24 hours treatment with 200 nM geldanamycin (GA).

Fig 5

Airyscan super-resolution confocal immunofluorescence microscopy of Huh-7.5 cells infected with HM175/p16 virus for 7 days. (A) At the top are low magnification images of 3D volume reconstructions of cells labeled with antibodies to (left) HSP90 (green) and dsRNA (red, J2 antibody), or (right) HSP90 (green), dsRNA (red), and VP1 aa7-143 (white). Sections (0.55 µ) of the region highlighted in yellow are shown below labeled as indicated. (B) Sections from cells labeled with antibodies to HSP90 (green) and the assembled HAV capsid (K3-2F2, red). (C) Percent volume in 3D image reconstructions of infected cells containing HSP90 signal above an arbitrary threshold that also contains signal above thresholds for dsRNA, VP1 aa7-143, capsid (K3-2F2), and HD-PTP signals. (D) Percent volume containing dsRNA, VP1 aa7-143, capsid (K3-2F2), or HD-PTP signals above threshold that also contain HSP90 signal above threshold. (E) Section of a cell labeled with antibody to HSP90 (green), the ESCRT-associated protein HD-PTP (red), and VP1 (white). On the right is a detailed image of the region highlighted in yellow. (F) Percent shared volume colocalization of HSP90 with HD-PTP and vice versa in uninfected (mock) and p16 virus-infected (HAV) cells. P-values by unpaired two-sided t-test. Scale bars as labeled in each panel.