Acid-activated structural reorganization of the Rift Valley fever virus Gc fusion protein

Abstract

The entry of the enveloped Rift Valley fever virus (RVFV) into its host cell is mediated by the viral glycoproteins Gn and Gc. We investigated the RVFV entry process and, in particular, its pH-dependent activation mechanism using our recently developed nonspreading-RVFV-particle system. Entry of the virus into the host cell was efficiently inhibited by lysosomotropic agents that prevent endosomal acidification and by compounds that interfere with dynamin- and clathrin-dependent endocytosis. Exposure of plasma membrane-bound virions to an acidic pH (<pH 6) equivalent to the pH of late endolysosomal compartments allowed the virus to bypass the endosomal route of infection. Acid exposure of virions in the absence of target membranes triggered the class II-like Gc fusion protein to form extremely stable oligomers that were resistant to SDS and temperature dissociation and concomitantly compromised virus infectivity. By targeted mutagenesis of conserved histidines in Gn and Gc, we demonstrated that mutation of a single histidine (H857) in Gc completely abrogated virus entry, as well as acid-induced Gc oligomerization. In conclusion, our data suggest that after endocytic uptake, RVFV traffics to the acidic late endolysosomal compartments, where histidine protonation drives the reorganization of the Gc fusion protein that leads to membrane fusion.

Fig 1

Rift Valley fever virus enters the cell via endocytosis. (A) BHK-21 cells were pretreated for 1 h with 2-fold dilutions of the indicated drugs and inoculated with RVFV_ns (MOI of ∼0.1) or VSV_ns (MOI of ∼0.8) for 2 h in the continued presence of the drugs, after which the inoculum was replaced by culture medium containing bafilomycin A1 (10 to 40 nM) to inhibit further RVFV entry (see Fig. 2). Infection was quantified 20 h (RVFV_ns) or 8 h (VSV_ns) postinfection by measuring GFP-positive cells using FACS. The data are the means of three independent experiments done in duplicate. ND, no drugs; SO, solvent (1% DMSO); dyn, dynasore; nys, nystatin; cyt, cytochalasin D; chl, chlorpromazine. (B) BHK-21 cells were incubated with the indicated drugs for 3 h, after which medium containing the drugs was replaced with culture medium containing bafilomycin A1 (20 nM). Twenty hours later, the effect of the chemical compounds on the metabolic activity of the cells was determined using a spectrophotometric assay. Cells were considered viable if the metabolic activity of treated cells remained above 70% (indicated by the dashed line) relative to that of untreated cells (ND). The results shown are representative of two independent experiments performed in triplicate. OD₄₅₀, optical density at 450 nm; dgo, dyngo-4a. (C) BHK-21 cells were infected with RVFV_ns (MOI of ∼0.2) for 2 h, after which infection was continued for 3 h in the presence of 80 or 40 μM dynasore or 20 or 40 μM chlorpromazine. Cells were incubated overnight in culture medium containing bafilomycin A1 (20 nM). At 20 h postinfection, infected (GFP-positive) cells were quantified by FACS. Data shown are representative of two independent experiments performed in duplicate. NI, not infected. (D) Analysis of the effect of dyngo-4a on RVFV_ns and VSV_ns infection (MOI of ∼0.4) of BHK-21 cells. Infection was performed and quantified as described for panel A. (E) Representative fluorescence pictures of BHK-21 cells infected with RVFV_ns or RVFV_rec in the presence of dynasore (80 μM) or chlorpromazine (40 μM). Infection was performed as described for panel A at an MOI of ∼0.2. Nuclei were stained with DAPI. Pictures are representative of two independent experiments performed in duplicate.

Fig 2

Entry of RVFV_ns depends on vacuolar acidification. (A) BHK-21 or A549 cells were pretreated for 1 h with different concentrations of bafilomycin A1 or ammonium chloride (NH₄Cl) and subsequently infected with RVFV_ns (MOI of ∼0.1) or VSV_ns (MOI of ∼0.8). Infection (GFP-positive cells) was quantified by FACS. Results shown are representative of three individual experiments performed in duplicate. NI, not infected; ND, no drugs. (B) RVFV_ns was bound to BHK-21 cells for 1 h in the cold and subsequently warmed to 37°C. NH₄Cl (10 mM) was added at indicated times to instantly raise the endosomal pH, thereby inhibiting further infection. Eighteen hours after warming, infection (GFP-positive cells) was analyzed by fluorescence microscopy (right panel; size bars represent 400 µM) and quantified by FACS (left panel). Untreated cells yielded ∼70% infection. Graphical data shown are representative of two independent experiments performed in duplicate. C, NH₄Cl control (not infected).

Fig 3

Low-pH-activated penetration of cell-bound RVFV_ns and inactivation of unbound RVFV_ns particles. (A) RVFV_ns was allowed to bind in the cold to a confluent monolayer of BHK-21 cells for 2 h before the cell-bound virus was exposed for 3 min at 37°C to the indicated pH. Infection was continued in culture medium containing bafilomycin A1 (baf A1; 20 nM) to inhibit infection via the endocytic route. Infection (GFP-positive cells) was analyzed 20 h after warming by fluorescence microscopy and was quantified by FACS. Untreated cells (pH 7.4) yielded ∼40% infection. The results shown are representative of two individual experiments performed in triplicate. (B) RVFV_ns particles were incubated at the indicated pH for 3 min at 37°C. After neutralization of the medium, infectivity of the virus was assayed on BHK-21 cells. Infection (GFP-positive cells) was analyzed 20 h.p.i. by fluorescence microscopy (left panel; size bars represent 400 µM) and quantified by FACS (right panel). Virus incubated at neutral pH resulted in ∼30% GFP-positive cells. Graphical data shown are representative of four individual experiments performed in duplicate. The controls in the experiments whose results are shown in panels A and B were cells that were not infected (NI).

Fig 4

Acid exposure triggers the formation of an SDS- and temperature-resistant Gc oligomer. (A) RVFV_ns particles were exposed to the indicated pHs for 10 min at 37°C, returned to neutral pH, and subsequently analyzed by Western blotting performed under nonreducing conditions. RVFV glycoproteins were detected with an RVFV antiserum, a monoclonal antibody against Gn, or a polyclonal anti-Gc peptide antiserum. In some cases, Gc migrates as a closely spaced doublet. (B) RVFV_ns particles were exposed for 10 min to pH 5.5 at 37°C, returned to neutral pH, and heated for 20 min at the indicated temperatures. Samples were subsequently analyzed by Western blotting (nonreducing conditions) using an RVFV antiserum or an anti-Gc peptide antiserum. (C) RVFV_ns particles were exposed to pH 5.5 at 37°C for the indicated times and analyzed by Western blotting (nonreducing conditions) using a polyclonal antiserum raised against the purified Gc ectodomain (Gce). OC, oligomeric complex; NC, negative control (medium from mock-transfected replicon cells); RT, room temperature.

Fig 5

Role of histidines in RVFV_ns infectivity. (A) RVFV_ns was pretreated with different concentrations of DEPC for 10 min at 37°C and subsequently assayed for infectivity. Infectivity (GFP-positive cells) was analyzed 20 h postinoculation by fluorescence microscopy and quantified by FACS. BHK-21 cells inoculated with virus treated with 0 μM DEPC yielded ∼20% infection. Graphical data shown are representative of two independent experiments performed in triplicate. SC, solvent control (0.2% ethanol). (B) Membrane topology of the RVFV M segment-encoded polyprotein starting from the fourth methionine (16). The predicted N-linked glycosylation sites (Y symbols) and signal peptidase cleavage sites (scissors) are indicated. The luminal domains of Gn and Gc and the cytoplasmic domains of Gn and Gc are colored in blue, green, gray, and pink, respectively. The orange blocks represent the transmembrane-spanning regions. The positions and amino acid numbering of conserved histidines (H), calculated from the first methionine (GenBank sequence accession number JF784387), are indicated in the linear diagram of the GnGc polyprotein. (C) Wild-type (WT) RVFV_ns and mutants containing histidine-to-alanine substitutions, used in equal amounts as determined by a dot blot assay (2-fold dilutions of viruses are shown), were analyzed for their infectivity on BHK-21 cells. BHK-21 cells inoculated with wild-type RVFV_ns particles resulted in ∼20% infection. Infection (GFP-positive cells) was quantified by FACS. Noninfected (NI) BHK-21 cells were included as a negative control. The data shown represent the combined results for three independently produced batches of wild-type and mutant RVFV_ns particles tested in independent experiments, each performed in triplicate.

Fig 6

Effects of histidine substitutions in RVFV glycoproteins on the formation and stability of the Gc oligomer. (A) Equal amounts of wild-type and mutant RVFV_ns particles were exposed for 10 min to pH 5.5 at 37°C, returned to neutral pH, heated for 20 min at the indicated temperatures, and subsequently analyzed by Western blotting (nonreducing conditions) using a polyclonal antiserum raised against the Gc ectodomain (Gce). In some cases, Gc runs as a closely spaced doublet. The thermostability of the acid-induced Gc oligomer is indicated for the wild-type and Gn- and Gc-His mutant RVFV_ns particles at 20°C and 80°C (left) or for the wild-type and the Gc-His mutant RVFV_ns particles at 40°C, 70°C, and 80°C (right). (B) Equal amounts of wild-type and mutant RVFV_ns particles containing histidine-to-alanine mutations in Gc were exposed to pH 5.5 at 37°C for the indicated times and analyzed by Western blotting (nonreducing conditions) using a polyclonal antiserum raised against the Gc ectodomain. OC, oligomeric complex; WT, wild-type. (C) Equal amounts of wild-type and H857A mutant RVFV_ns particles were exposed for 10 min at 37°C to the indicated pH. The Gc conversion was analyzed by Western blotting as described for panel B.