Evolutionary constraints on chaperone-mediated folding provide an antiviral approach refractory to development of drug resistance

Abstract

The genome diversity of RNA viruses allows for rapid adaptation to a wide variety of adverse conditions. Accordingly, viruses can escape inhibition by most antiviral compounds targeting either viral or host factors. Here we exploited the capacity of RNA viruses for rapid adaptation to explore the evolutionary constraints of chaperone-mediated protein folding. We hypothesized that inhibiting a host molecular chaperone required for folding of a viral protein would force the virus to evolve an alternate folding strategy. We identified the chaperone Hsp90 as an essential factor for folding and maturation of picornavirus capsid proteins. Pharmacological inhibition of Hsp90 impaired the replication of poliovirus, rhinovirus, and coxsackievirus in cell culture. Strikingly, anti-Hsp90 treatment did not yield drug-resistant viruses, suggesting that the complexity of capsid folding precludes the emergence of alternate folding pathways. These results reveal tight evolutionary constraints on chaperone-mediated protein folding, which may be exploited for viral inhibition in vivo. Indeed, Hsp90 inhibitors drastically reduced poliovirus replication in infected animals without the emergence of drug-resistant escape mutants. We propose that targeting folding of viral proteins may provide a general antiviral strategy that is refractory to development of drug resistance.

Figure 1.

The Hsp90 inhibitor GA reduces picornavirus replication in cultured cells. (A) Outline of the experiment. (B–E) Effect of GA on poliovirus (B,E), rhinovirus (C), and coxsackievirus (D) production in HeLa S3 cells (B–D) or primary human foreskin fibroblasts (E). Data are represented as the number of plaque-forming units (PFU) or 50% tissue culture infective dose (TCID₅₀) produced per cell and, for comparison reasons, standardized between experiments so as to yield the same number of PFU or TCID₅₀ per cell for DMSO-treated conditions. Results indicate mean and SEM of three independent experiments. (*) p < 0.05, (**) p < 0.001 relative to DMSO-treated condition by t-test.

Figure 2.

Inhibition of Hsp90 specifically affects production of mature capsid proteins. (A) Schematic representation of picornavirus life cycle. (B) GA inhibits poliovirus replication from a transfected infectious genomic RNA. Data represent the mean and SEM of three independent experiments. (C) GA does not inhibit translation and replication of a poliovirus luciferase replicon (PLuc), in which the capsid-coding sequence is replaced with luciferase (Herold and Andino 2000). The time course of luciferase activity reports on viral translation and replication. Data show mean and SEM of three independent experiments. (D) Poliovirus-encoded polyprotein, highlighting the processing events for the capsid precursors. (E,F) GA decreases capsid protein production. (E) Steady-state ³⁵S-labeling of poliovirus proteins from infected cells grown in the presence or absence of GA. Total cytoplasmic extracts (lanes 3,4) and immunoprecipitated capsid proteins (lanes 1,2) separated by SDS-PAGE were visualized by autoradiography. P1-derived (labeled arrows) and P2- and P3-derived (arrowheads) proteins are indicated. (F) Relative band intensity of P1 and P1-derived capsid proteins in control and GA-treated cells. Data show means and SEM of four independent experiments performed as in E. (*) p < 0.05, (**) p < 0.001 relative to control-treated cells by t-test.

Figure 3.

Hsp90 associates with the capsid precursor P1 and is required for its processing to mature capsid proteins. (A) Association of ³⁵S-labeled P1 with Hsp90 and its cochaperone p23 in the presence or absence of GA, measured by immunoprecipitation. (NI) Nonimmune control. (B) Pulse-chase analysis of poliovirus proteins from infected cells grown in the presence or absence of GA. Total cytoplasmic extracts separated by SDS-PAGE were visualized by autoradiography. P1-derived (labeled arrows) and P2- and P3-derived (arrowheads) proteins are indicated. (C) Relative band intensity of P1 and P1-derived capsid proteins in control and GA-treated cells, calculated from B as percent of P1 at 15-min chase time point. (D) GA treatment promotes P1 degradation by the proteasome. The effect of GA on degradation of ³⁵S-labeled P1, expressed in cells by infection with a recombinant vaccinia virus (VV-P1) (Ansardi et al. 1991), was examined in the presence or absence of the proteasome inhibitors LC and ALLN, and the lysosomal protease inhibitor E64. (E) Processing of in vitro translated P1 into capsid proteins by purified 3C^pro is blocked by GA even in the absence of proteasomal function. (CHX) Cycloheximide. (F) Role of Hsp90 in picornavirus capsid maturation. Hsp90 binds newly translated P1, probably in cooperation with Hsp70 (see Discussion; Macejak and Sarnow 1992). Together with ATP and its cofactors, such as p23, Hsp90 folds P1 to a processing-competent conformation (P1*) and protects it from proteasomal degradation. Upon cleavage by 3C^Pro, the mature capsid proteins no longer interact with Hsp90.

Figure 4.

Poliovirus cannot bypass the Hsp90 requirement. (A) Poliovirus can gain resistance to BFA but not GA within 10 passages. For each passage, 10⁶ viruses (MOI of <0.2) were used to inoculate a new dish in the presence of BFA, GA, or no drug. After 10 passages, the sensitivity of each virus to BFA or GA was tested as in Figure 1B. Data represent the mean number of PFU per cell and SEM. (B) Poliovirus remains GA-sensitive following extensive serial passage in the presence of GA. For each passage, an MOI of 0.1–0.002 was used to inoculate a new dish of cells in the presence GA. Data are represented as the number of PFUs produced per cell. To facilitate comparison between experiments, data were standardized to yield the same number of PFUs for DMSO-treated conditions. (**) p < 0.01 relative to the virus passaged untreated by t-test.

Figure 5.

GA inhibits viral replication in poliovirus-infected animals without eliciting drug resistance. (A) Outline of the experiment. (B) Viral load in the brains of poliovirus-infected cPVR transgenic mice treated with vehicle or GA is expressed as number of PFUs per gram of brain (n = 10 per group, p < 0.01 by Wilcoxon two-sample test). (C) Viral populations recovered from GA-treated animals remain GA sensitive. Poliovirus isolated from the brains of infected animals from B was used to infect HeLa S3 cells at a low MOI (10⁻⁴) in the presence or absence of 1 μM GA. Virus production was measured after 48 h by standard plaque assay. Data represent the average number of PFUs produced per cell from all 10 GA-treated animals and four control animals.

Figure 6.

17AAG inhibits viral replication in poliovirus-infected animals. Viral load in the brains of poliovirus-infected cPVR transgenic mice treated with vehicle or 17AAG expressed as in Figure 5B (n = 8 per group, p < 0.001 for 2.5 mg/kg group and p < 0.005 for 25 mg/kg group by Wilcoxon two-sample test). Animals with no detectable virus (four of eight mice treated with 2.5 mg/kg 17AAG and five of eight mice treated with 25 mg/kg 17AAG) are plotted below the hatched line indicating the detection limit.