pH regulation in early endosomes and interferon-inducible transmembrane proteins control avian retrovirus fusion

Abstract

Enveloped viruses infect host cells by fusing their membranes with those of the host cell, a process mediated by viral glycoproteins upon binding to cognate host receptors or entering into acidic intracellular compartments. Whereas the effect of receptor density on viral infection has been well studied, the role of cell type-specific factors/processes, such as pH regulation, has not been characterized in sufficient detail. Here, we examined the effects of cell-extrinsic factors (buffer environment) and cell-intrinsic factors (interferon-inducible transmembrane proteins, IFITMs), on the pH regulation in early endosomes and on the efficiency of acid-dependent fusion of the avian sarcoma and leukosis virus (ASLV), with endosomes. First, we found that a modest elevation of external pH can raise the pH in early endosomes in a cell type-dependent manner and thereby delay the acid-induced fusion of endocytosed ASLV. Second, we observed a cell type-dependent delay between the low pH-dependent and temperature-dependent steps of viral fusion, consistent with the delayed enlargement of the fusion pore. Third, ectopic expression of IFITMs, known to potently block influenza virus fusion with late compartments, was found to only partially inhibit ASLV fusion with early endosomes. Interestingly, IFITM expression promoted virus uptake and the acidification of endosomal compartments, resulting in an accelerated fusion rate when driven by the glycosylphosphatidylinositol-anchored, but not by the transmembrane isoform of the ASLV receptor. Collectively, these results highlight the role of cell-extrinsic and cell-intrinsic factors in regulating the efficiency and kinetics of virus entry and fusion with target cells.

Keywords: endocytosis; fusion protein; pH regulation; viral protein; virus entry.

Figure 1.

Progression of the ASLV and VSV pseudovirus entry and fusion with CV-1- and A549-derived cells. A and B, ASLV pseudovirus fusion with CV-1/TVA950 (A) and CV-1/TVA800 (B) cells was measured using the BlaM assay, as described under “Experimental procedures.” Fusion inhibitors (R99, NH₄Cl, or low temperature (TB)) were applied at the indicated times after virus entry/fusion was initiated by raising the temperature to 37 °C. C, VSV pseudovirus fusion with CV-1/TVA950 cells experiments were carried out, as described above. D and E, ASLV pseudovirus fusion with A549 cells expressing either TVA950 (D) or TVA800 (E). Data are mean ± S.E. from 3 independent triplicate experiments.

Figure 2.

Kinetics of ASLV fusion and measurement of endosomal pH. A, illustration of the virus labeling strategy with Gag-mCherry (red) and EcpH-ICAM (green) to assess the pH drop in virus-carrying endosomes (top) and images of CV-1 cells before (0 h) and after (1 h) internalization of labeled viruses (bottom). Virus entry into acidic endosomes is manifested in disappearance of the EcpH signal and accumulation of Gag-mCherry in the perinuclear areas. B, kinetics of ASLV fusion with A549/TVA950 cells in DMEM measured by the BlaM assay and EcpH quenching measured in parallel imaging experiments. C, images of Gag-mCherry/EcpH-ICAM co-labeled ASLV particles internalized by A549/TVA950 cells at different pH. Viruses were pre-bound to cells in the cold and allowed to enter by incubation at 37 °C for 15 min. Cells were then placed in buffers of the indicated acidity supplemented with monensin and nigericin to equilibrate the external and endosomal pH (see “Experimental procedures” for details). The EcpH signal is virtually lost in the background cell fluorescence at pH ≤ 6.2. A triangle shows the expected fluorescence ratio in DMEM equilibrated with air (pH ∼ 7.9). D, calibration of the mean ratio of EcpH and mCherry signals from intracellular compartments as a function of endosomal pH (as illustrated in panel C). Data are mean ratios ± S.E. from at least 4 image fields acquired for each pH value. The light pink and blue colored regions represent the pH range conducive for ASLV fusion and the background EcpH/mCherry ratio, respectively. E and F, kinetics of ASLV fusion with A549/TVA950 cells in LIB (E) or DMEM buffered with HEPES at pH 7.4 (F), as measured by the BlaM assay. EcpH quenching in panel E was measured in parallel imaging experiments. Data are mean ± S.E. from 3 (panels B and E) and 2 (panel F) independent triplicate experiments.

Figure 3.

Effect of extracellular buffers on endosomal pH and on ASLV fusion. A, calibration of the pH in early endosomes by measuring the ratio of fluorescence signals from a mixture of internalized transferrin labeled with FITC (pH sensitive) or with AF594, as described under “Experimental procedures.” Briefly, cells were incubated with transferrin mixture for 15 min at 37 °C and exposed to buffers of different acidity supplemented with monensin and nigericin. The resulting changes in transferrin fluorescence are illustrated in supplemental Fig. S2. Data are mean ratios ± S.E. from at least 4 image fields acquired for each pH value. B, the dynamics of endosomal pH in A549/TVA950 and CV-1/TVA950 cells bathed in DMEM versus LIB measured by FITC/AF594 transferrin fluorescence ratio, as also illustrated in supplemental Fig. S1. Data are mean ratios ± S.E. from 4 or more image fields acquired for each pH value. C, the efficiency of ASLV fusion with A549 and CV-1 cells expressing TVA950 in different extracellular buffers (DMEM and LIB). The total number of co-labeled particles analyzed for each condition (2–3 independent experiments) is shown in the graph. D, the kinetics of single ASLV (co-labeled with YFP-Vpr and Gag-imCherry) fusion with TVA950-expressing A549 and CV-1 cells bathed in DMEM or LIB. Note that, despite different fusion efficiencies (panel C), a comparable number of events were annotated and plotted to ensure an appropriate sample size for all conditions. **, p < 0.01; ***, p < 0.001.

Figure 4.

Ectopic expression and virus restriction activity of IFITM3. A, immunofluorescence staining for IFITM3 expression in A549/TVA800 and A549/TVA950 cells transduced with the IFITM3-encoding vector or an empty vector. B, effect of IFITM3 expression on ASLV, VSV, and influenza A virus (IAV) pseudovirus fusion with A549/TVA950 and A549/TVA800 cells was measured by the BlaM assay. Data are mean ± S.E. from 3 independent triplicate experiments. C, immunostaining of CV-1/TVA800 cells ectopically expressing or lacking IFITM3 with anti-IFITM3 serum. D, effect of IFITM3 expression on ASLV and VSV pseudovirus fusion with CV-1/TVA950 and CV-1/TVA800 cells. Data are mean ± S.E. from 3 independent triplicate experiments. **, p < 0.01; ***, p < 0.001.

Figure 5.

The effect of IFITM3 on extent and kinetics of single ASLV uptake and fusion with A549 cells expressing alternative TVA receptor isoforms. A, percent of double-labeled ASLV pseudoviruses that fused with the indicated cell lines expressing or lacking IFITM3 bathed in LIB. Data are mean ± S.E. from 4 to 8 independent experiments. B, kinetics of ASLV uptake and entry into acidic endosomes in A549/TVA800 and A549/TVA950 cells expressing or lacking IFITM3, as measured by EcpH quenching (see Fig. 2 and “Experimental procedures”). Data are mean ± S.E. from 3 independent experiments. Solid lines are curve fits using a single- or double-exponential raise model. C, the kinetics of single ASLV (co-labeled with YFP-Vpr and Gag-imCherry) fusion with A549/TVA950 and A549/TVA800 cells expressing or lacking IFITM3. Measurements were done in LIB. Despite different fusion efficiencies, a comparable number of events were annotated and plotted to ensure an appropriate sample size for all conditions. Note that a roughly equal number of data points was analyzed for each condition. ***, p < 0.001; NS, not significant.