Low endocytic pH and capsid protein autocleavage are critical components of Flock House virus cell entry - PubMed (original) (raw)

Low endocytic pH and capsid protein autocleavage are critical components of Flock House virus cell entry

Amy L Odegard et al. J Virol. 2009 Sep.

Abstract

The process by which nonenveloped viruses cross cell membranes during host cell entry remains poorly defined; however, common themes are emerging. Here, we use correlated in vivo and in vitro studies to understand the mechanism of Flock House virus (FHV) entry and membrane penetration. We demonstrate that low endocytic pH is required for FHV infection, that exposure to acidic pH promotes FHV-mediated disruption of model membranes (liposomes), and particles exposed to low pH in vitro exhibit increased hydrophobicity. In addition, FHV particles perturbed by heating displayed a marked increase in liposome disruption, indicating that membrane-active regions of the capsid are exposed or released under these conditions. We also provide evidence that autoproteolytic cleavage, to generate the lipophilic gamma peptide (4.4 kDa), is required for membrane penetration. Mutant, cleavage-defective particles failed to mediate liposome lysis, regardless of pH or heat treatment, suggesting that these particles are not able to expose or release the requisite membrane-active regions of the capsid, namely, the gamma peptides. Based on these results, we propose an updated model for FHV entry in which (i) the virus enters the host cell by endocytosis, (ii) low pH within the endocytic pathway triggers the irreversible exposure or release of gamma peptides from the virus particle, and (iii) the exposed/released gamma peptides disrupt the endosomal membrane, facilitating translocation of viral RNA into the cytoplasm.

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Figures

FIG. 1.

Infectivity of FHV in DL-1 cells in the presence and absence of NH4Cl or bafilomycin A1. DL-1 cells were infected with FHV at an MOI of 1 PFU/cell in the presence or absence of NH4Cl (A) or bafilomycin A1 (BafA1) (B) in the growth medium. Where indicated, FHV was incubated at pH 6.0 for 1 h prior to infection. Samples were harvested at 24 h postinfection, and infectious titers were determined by plaque assay. The amount of FHV progeny produced (log10 growth) was determined by the log10 titer at 24 h minus the log10 titer at 0 h. Each bar represents the average ± standard deviation of three independent infections. Cell viability was determined by incubating uninfected cells in growth medium containing the indicated concentration of NH4Cl or bafilomycin A1. After 24 h of incubation, the proportion of viable cells (percent viability) was determined by trypan blue staining.

FIG. 2.

Electron micrographs of FHV particles. FHV particles were incubated for 1 h at pH 7.0 (A), 6.0 (B), or 5.0 (C). The virus particles were stained with methylamine tungstate and visualized by transmission electron microscopy at a magnification of ×52,000. Bar, 200 nm.

FIG. 3.

Concentration and pH dependence of FHV-mediated dye release from SulfoB-loaded liposomes. FHV particles (0.01 to 0.5 mg/ml) were incubated with liposomes containing the entrapped fluorescent dye SulfoB at pH 7.0 (A) or pH 6.0 (B). The release of SulfoB from liposomes was measured as a function of time over the course of a 20-min incubation.

FIG. 4.

Liposome lysis and bis-ANS fluorescence of wild-type and cleavage-defective FHV particles at various pHs. (A) Reversibility of FHV pH-dependent membrane disruption. FHV particles were incubated at pH 6.0 for 1 h, diluted into pH 7.0 buffer (indicated as 6→7), and incubated for 1 to 20 h. The neutralized particles were added to SulfoB-loaded liposomes at a concentration of 0.1 mg/ml, and the percent SulfoB released was determined following a 20-min incubation. As controls, FHV particles preincubated at pH 6.0 or 7.0 for 1 h were added to SulfoB-loaded liposomes at a concentration of 0.1 mg/ml, and the percent SulfoB released was determined after a 20-min incubation. (B) SulfoB-loaded liposomes were incubated with 0.1 mg/ml of either wt-FHV, FHVD75N, FHVN363T, or FHVD75N/N363T at different pHs (pH 5.0 to 7.0). The release of SulfoB from liposomes was measured after 20 min. (C) FHVwt or FHVD75N particles (1 μg) were incubated in buffer ranging from pH 5.0 to 7.0 in the presence of bis-ANS (50 μM). The amount of bis-ANS fluorescence was measured for each pH condition following a 60-min incubation. Fluorescence intensity is given in arbitrary units (AU). Each error bar represents the average ± standard deviation of three independent experiments.

FIG. 5.

Disrupted FHV particles and fluorescent dye release from liposomes. FHVwt or FHVD75N particles were incubated at 22°C, 45°C, 65°C, or boiled in pH 7.0 buffer as indicated. Following this incubation, large protein aggregates were removed by low-speed centrifugation, and the resulting supernatant was analyzed by the liposome dye release assay. FHVwt (A) or FHVD75N (B) heat-treated virus particles were added to SulfoB-loaded liposomes, and fluorescent dye release was measured as a function of time during a 20-min incubation.

FIG. 6.

Updated model of FHV membrane penetration. Prior to infection, the γ peptides (shown as helices) are noncovalently associated with the interior of the FHV capsid. During uptake into the host cell, FHV is exposed to acidic pH within the endocytic compartment. Acidic endosomal pH irreversibly triggers the virus to undergo partial disassembly and the γ peptides are externalized (step a) or released from the virus particle (step b). Released or particle-associated γ peptides insert into and create a local disruption of the endosomal membrane to facilitate translocation of the RNA or nucleocapsid into the cytoplasm (steps c and d, respectively). Inhibitors that raise endocytic pH, such as NH4Cl and bafilomycin A1, block the low-pH-induced uncoating step (blocked at step a). However, capsid disassembly can be initiated by exposing FHV particles to acidic pH in vitro prior to infection, and these particles no longer require low endosomal pH (step a is bypassed). FHV cleavage-defective mutants also undergo a conformational change upon exposure to acidic pH inside endosomes but cannot fully permeabilize membranes because these particles cannot release the γ peptide (blocked at step b).

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Low endocytic pH and capsid protein autocleavage are critical components of Flock House virus cell entry - PubMed (original) (raw)