Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2007 Oct;81(19):10758-68.
doi: 10.1128/JVI.00725-07. Epub 2007 Jul 11.

Role of endocytosis and low pH in murine hepatitis virus strain A59 cell entry

Patricia Eifart  1 , Kai Ludwig, Christoph Böttcher, Cornelis A M de Haan, Peter J M Rottier, Thomas Korte, Andreas Herrmann
Affiliations

Role of endocytosis and low pH in murine hepatitis virus strain A59 cell entry

Patricia Eifart et al. J Virol. 2007 Oct.

Abstract

Infection by the coronavirus mouse hepatitis virus strain A59 (MHV-A59) requires the release of the viral genome by fusion with the respective target membrane of the host cell. Fusion is mediated by the viral S protein. Here, the entry pathway of MHV-A59 into murine fibroblast cells was studied by independent approaches. Infection of cells assessed by plaque reduction assay was strongly inhibited by lysosomotropic compounds and substances that interfere with clathrin-dependent endocytosis, suggesting that MHV-A59 is taken up via endocytosis and delivered to acidic endosomal compartments. Infection was only slightly reduced in the presence of substances inhibiting proteases of endosomal compartments, precluding that the endocytic uptake is required to activate the fusion potential of the S protein by its cleavage. Fluorescence confocal microscopy of labeled MHV-A59 confirmed that virus is taken up via endocytosis. Bright labeling of intracellular compartments suggests their fusion with the viral envelope. No fusion with the plasma membrane was observed at neutral pH conditions. However, when virus was bound to cells and the pH was lowered to 5.0, we observed a strong labeling of the plasma membrane. Electron microscopy revealed low pH triggered conformational alterations of the S ectodomain. Very likely, these alterations are irreversible because low-pH treatment of viruses in the absence of target membranes caused an irreversible loss of the fusion activity. The results imply that endocytosis plays a major role in MHV-A59 infection and the acidic pH of the endosomal compartment triggers a conformational change of the S protein mediating fusion.

PubMed Disclaimer

Figures

FIG. 1.
FIG. 1.
Inhibition of MHV-A59 infection of different murine cells by substances interfering with the endocytic pathway and endosomal acidification. Infection by MHV-A59 was assessed by plaque assay. (A) Infection of 17Cl-1, LR-7, and DBT cells by MHV-A59 in the presence of chlorpromazine-HCl (Chlorpr; 15 μM, gray bar), bafilomycin A1 (Baf A1; 100 nM, dark gray bar), monensin (Mon; 10 μM, black bar), concanamycin A (Con; 10 nM, striped gray bar), and ammonium chloride (NH4Cl; 20 mM, gray hatched bar). (B) Cells were treated with 20 mM ammonium chloride (NH4Cl) or 100 nM bafilomycin A1 (Baf A1) and infected with either MHV-A59 (black bar) or MHV-S4 (gray bar) in the presence of the drug. (C) Influence of cholesterol depletion of 17Cl-1 cells on infection by MHV-A59. Bars: light gray, cholesterol depletion by MβCD treatment; dark gray, preincubation of cells with Filipin III. For treatment of the cells with substances, see Materials and Methods. As a control, VSV, which is known to enter cells independently of cholesterol and/or rafts, was used (black bar). (D) Influence of the protease inhibitors E-64 and leupeptin on infection of 17Cl-1 cells with MHV-A59 (gray bar) and MHV-A59 FI (black bars). The data present means ± the standard errors of estimates of three independent experiments unless otherwise indicated (**, n = 2; #, n = 5).
FIG. 2.
FIG. 2.
Low pH triggers fusion of MHV-A59 with the plasma membrane of 17Cl-1 cells. R18-labeled virus was bound to cells. Subsequently, virus-cell complexes were incubated at 37°C under neutral (pH 7.0, A to C) or acidic (pH 5.0, D to F) conditions and observed by fluorescence microscopy. (E) Mild acidic conditions (pH 5.0) mimic the acidic pH of endosomal compartments and enable the virus to directly fuse at the plasma membrane, as indicated by strong labeling of the plasma membrane already after 5 min of incubation at 37°C. No significant fusion of viruses with the plasma membrane was observed at pH 7.0 (panels A to C). After 15 min (see panel C), some intracellular staining was observed. For further explanations, see Results. Bar, 50 μm.
FIG. 3.
FIG. 3.
Interaction of MHV-A59 with 17Cl-1 cells studied by confocal fluorescence microscopy. R18-labeled virus was bound to cells. Subsequently, virus-cell complexes were incubated at 37°C under neutral (pH 7.0, A and B) or at acidic conditions (pH 5.0, C and D) for 30 or 5 min, respectively, and imaged by confocal fluorescence microscopy. To characterize the labeling pattern a three-dimensional stack analysis was performed. In panels A and C selected z-planes are shown, while in panels B and D a plot of fluorescence along the x and y coordinates provide respective side views of the cells. At neutral pH the viruses are delivered to intracellular compartments (B, arrowheads) (for further details, see Results and Discussion). When virus-cell complexes were incubated at low pH, a strong labeling of the plasma membrane was observed (D) that was not seen for incubation at neutral pH. Bars, 10 μm. Asterisks mark the positions of the nuclei.
FIG. 4.
FIG. 4.
Incubation of virus-cell complexes with ammonium chloride prevents intracellular staining at pH 7.0. 17Cl-1 cells were either pretreated (A and B, 20 mM ammonium chloride) or not treated (C and D, control) at 37°C for 30 min (see Materials and Methods). Subsequently, R18-labeled viruses were bound to the cells, and virus-cell complexes were incubated at 37°C for the indicated time. For cells pretreated with ammonium chloride (A and B), only a faint intracellular fluorescence was observed, while in untreated cells (C and D) significantly brighter fluorescence labeling was detected. Bar, 20 μm.
FIG. 5.
FIG. 5.
Fusion of MHV-A59 with 17Cl-1 target cells is triggered by low pH. After binding of R18-labeled viruses to 17Cl-1 cells, virus-cell complexes were suspended, and fluorescence dequenching was measured (see Materials and Methods). (A) Time course of R18 fluorescence dequenching measured at neutral and acidic pH and 37°C. The pH was lowered by the addition of appropriate amounts of citric acid (arrow). At the end of the experiment Triton X-100 (arrow) was added to achieve complete fluorescence dequenching. (B) Extent of fusion after measuring fluorescence dequenching for 30 min at 37°C (▪) and room temperature (□). The data present the means ± the standard errors of estimates of at least three independent experiments.
FIG. 6.
FIG. 6.
Low pH triggers a conformational change of the S protein. (A) Negative-stain electron micrograph of MHV-A59 at pH 7.4 and 37°C. Round-shaped virus with several spike proteins on the surface can be seen. The spike proteins consist of a stalk and a distal head domain. (B to D) Negative staining of the electron micrograph of MHV-A59 after incubation of the virus for 5 min at pH 5 and 37°C. The number of visible spike proteins tends to be reduced. We also observed viruses with no visible spikes on the surface (D) and viruses with thin, elongated spike proteins (C). (E) Cryo-electron micrograph of MHV-A59 (without stain) at neutral pH as a control. Bars, 100 nm.
FIG. 7.
FIG. 7.
The fusion activity of MHV-A59 is sensitive to preincubation at low pH. R18-labeled viruses were preincubated at low pH (pH 5.0) for 15 min at either 37°C (A) or 4°C (B) and after neutralization bound to 17Cl-1 cells. Virus-cell complexes were incubated at pH 5.0 and 37°C for 15 min. (A). On the left are differential interference contrast images; on the right are fluorescence images. Bar, 50 μm. (C) Quantification of cells (see panels A and B) showing fusion between R18-labeled viruses and the plasma membrane. Viruses were preincubated as described and subsequently bound to the cells. Virus-cell complexes were incubated at pH 5.0 (black bars) or at pH 7.0 (gray bars) at 37°C for 15 min, and the percentage of R18-positive cells was determined by fluorescence microscopy. (D) Influence of low-pH treatment of MHV-A59 (pH 5.0, 15 min, 37°C) on infectivity (left panel, plaque assay) and fusion activity (right panel, fluorescence dequenching [FDQ]). For the plaque assay, see the legend to Fig. 1. Fusion activity was measured by the fluorescence dequenching assay as shown in Fig. 5. The extent of fluorescence dequenching was measured at t = 1,800 s (see Fig. 5). The data present the means ± the standard errors of estimates of at least three independent experiments.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Bayer, N., D. Schober, M. Huttinger, D. Blaas, and R. Fuchs. 2001. Inhibition of clathrin-dependent endocytosis has multiple effects on human rhinovirus serotype 2 cell entry. J. Biol. Chem. 276:3952-3962. - PubMed
    1. Blanchard, E., S. Belouzard, L. Goueslain, T. Wakita, J. Dubuisson, C. Wychowski, and Y. Rouille. 2006. Hepatitis C virus entry depends on clathrin-mediated endocytosis. J. Virol. 80:6964-6972. - PMC - PubMed
    1. Blumenthal, R., A. Bali-Puri, A. Walter, D. Covell, and O. Eidelman. 1987. pH-dependent fusion of vesicular stomatitis virus with Vero cells: measurement by dequenching of octadecyl rhodamine fluorescence. J. Biol. Chem. 262:13614-13619. - PubMed
    1. Blumenthal, R., S. A. Gallo, M. Viard, Y. Raviv, and A. Puri. 2002. Fluorescent lipid probes in the study of viral membrane fusion. Chem. Phys. Lipids 116:39-55. - PubMed
    1. Bos, E. C., J. C. Dobbe, W. Luytjes, and W. J. Spaan. 1997. A subgenomic mRNA transcript of the coronavirus mouse hepatitis virus strain A59 defective interfering (DI) RNA is packaged when it contains the DI packaging signal. J. Virol. 71:5684-5687. - PMC - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources