Plasma membrane budding as an alternative release mechanism of the extracellular enveloped form of vaccinia virus from HeLa cells

Abstract

In HeLa cells the assembly of modified vaccinia virus Ankara (MVA), an attenuated vaccinia virus (VV) strain, is blocked. No intracellular mature viruses (IMVs) are made and instead, immature viruses accumulate, some of which undergo condensation and are released from the cell. The condensed particles may undergo wrapping by membranes of the trans-Golgi network and fusion with the plasma membrane prior to their release (M. W. Carroll and B. Moss, Virology 238:198-211, 1997). The present study shows by electron microscopy (EM), however, that the dense particles made in HeLa cells are also released by a budding process at the plasma membrane. By labeling the plasma membrane with antibodies to B5R, a membrane protein of the extracellular enveloped virus, we show that budding occurs at sites that concentrate this protein. EM quantitation revealed that the cell surface around a budding profile was as strongly labeled with anti-B5R antibody as were the extracellular particles, whereas the remainder of the plasma membrane was significantly less labeled. To test whether budding was a characteristic of MVA infection, HeLa cells were infected with the replication competent VV strains Western Reserve strain (WR) and International Health Department strain-J (IHD-J) and also prepared for EM. EM analyses, surprisingly, revealed for both virus strains IMVs that evidently budded at the cell surface at sites that were significantly labeled with anti-B5R. EM also indicated that budding of MVA dense particles was more efficient than budding of IMVs from WR- or IHD-J-infected cells. This was confirmed by semipurifying [(35)S]methionine-labeled dense particles or extracellular enveloped virus (EEVs) from the culture supernatant of MVA- or IHD-J-infected HeLa cells, respectively, showing that threefold more labeled dense particles were secreted than EEVs. Finally, although the released MVA dense particles contain some DNA, they are not infectious, as assessed by plaque assays.

FIG. 1.

Upon overnight infection of HeLa cells with MVA, dense spherical particles are produced. In panels A through D cells were infected with MVA at an MOI of 10 and then fixed at 16 h postinfection, and samples were prepared for Epon embedding. Panel A is an overview image showing the accumulation of many IVs (small arrowheads) and electron-dense spherical particles (large arrowheads). Panel B shows that some of these dense particles (small arrowheads) can also be found at the extracellular side of the plasma membrane. Panel C shows a dense particle beneath the cell surface, and panel D shows a dense particle attached to the plasma membrane via a membrane stalk (arrow). PM, plasma membrane. Bars: A, 1 μm; B to D, 200 nm.

FIG. 2.

Dense particles undergo TGN wrapping and are released from the cell. HeLa cells were infected as described in Fig. 1 and either embedded in Epon (A and B) or prepared for cryosectioning and labeled with anti-B5R (C to F). Panels A and B show that dense particles may undergo wrapping by membranes (large arrowheads) that locate close to the Golgi complex (“G” in panel A). Panel C shows a Golgi complex (G) that is labeled on one side with anti-B5R (small arrows). Dense particles undergo wrapping (large arrowheads) close to the Golgi with membranes that are also labeled with anti-B5R (small arrows; 10-nm gold). Panels D to F show a panel of extracellular dense particles that are abundantly labeled with anti-B5R on their surface. In panel E the extracellular particle is attached to the plasma membrane (PM) via a membrane stalk. Bars, 200 nm.

FIG. 3.

Dense particles may also be released via budding at the plasma membrane. In all images HeLa cells were infected as in Fig. 1, except for panel F, which is from an infection with WR. After infection the cells were fixed, and the cell monolayer was labeled with anti-B5R, rabbit anti-rat antibody, and 10-nm protein A-gold. The cells were then postfixed with 1% glutaraldehyde and embedded in Epon. Panels A, C, and D show dense particles that are in the process of leaving the cell via budding at the plasma membrane (large arrows). The plasma membrane (P) around the buds is significantly labeled with anti-B5R (small arrows). Panel B shows extracellular particles (large arrowheads) that are abundantly labeled with anti-B5R (small arrows). Panels E and F shows putative fusion profiles of MVA (E) and WR (F). The images show a dense particle in panel E or a CEV in panel F that lies in a plasma membrane (P) cavity that has a different electron density (small arrowheads) than the surrounding cell surface. Bars, 200 nm.

FIG. 4.

Quantification of B5R labeling over budding profiles, extracellular particles, and the plasma membrane; the production of the different viral forms in HeLa cells and CEFs; and the quantification of budding and fusion events. (A) HeLa cells were infected with WR, IHD-J, and MVA, and the cells were fixed, labeled with anti-B5R, and embedded as described for Fig. 3. The gold particles over extracellular particles (“free” particles), the cell surface around budding particles, and random pieces of plasma membrane were counted for 30 randomly chosen electron micrographs showing budding profiles and extracellular particles. The values are expressed as number of gold particles per micron of membrane and represent the average and standard deviations. (B) HeLa cells or CEFs were infected at an MOI of 10 and fixed at 16 h postinfection. The total amount of IVs (white bars), IMVs (dense particles in HeLa cells infected with MVA; black bars), IEVs or TGN wrapping profiles (dark gray bars), and CEVs (dense extracellular particles near HeLa cells infected with MVA; light gray bars) were counted in 50 profiles of infected cells and are expressed as a percentage of the total particles counted. (C) HeLa cells were infected and embedded as described for panel A with MVA (black bars), WR (gray bars), and IHD-J (white bars). Fusion and budding profiles, defined asdescribed in the text, at the plasma membrane were counted in 30 randomly chosen electron micrographs and are expressed as events per micron of plasma membrane. Note that the standard deviations are high. This reflects the fact (as described in Results) that in some cells budding is very abundant, whereas in other cells no budding occurs at all. Also note that fusion is barely detectable upon IHD-J infection, which probably reflects the way fusion was defined (see the text).

FIG. 5.

IHD-J and WR are also released via budding at the plasma membrane in HeLa cells. HeLa cells were infected at an MOI of 10 with either WR (A, B, and F) or IHD-J (C to E) and then fixed at 16 h postinfection. The cell surface was labeled with anti-B5R as described in Fig. 3, and cells were embedded in Epon. Panels A and B show the plasma membrane (P) with a collection of IMVs (large arrows) in the process of budding. The sites of budding are labeled with anti-B5R (small arrows). Panels C through E show similar images with IHD-J. Large arrows indicate IMVs that are budding at sites labeled with anti-B5R (small arrows). Although most putative budding profiles lie beneath the cell surface that is slightly elevated, some IMVs appear to be in a “real” plasma membrane bud (B and F) similar to those seen upon MVA infection (see Fig. 3A, C, and D). Panel F shows an EEV that has just left the cell and is attached to a membrane stalk. The surface of this particle is abundantly labeled with anti-B5R. Bars, 200 nm.

FIG. 6.

The secretion of dense particles may be more efficient than the secretion of EEV. (A) HeLa cells were infected at an MOI of 10 with IHD-J or MVA and metabolically labeled from 6 to 24 h postinfection with [³⁵S]methionine (35S) and [³H]thymidine (3H). The culture supernatant was harvested and spun through a 36% sucrose cushion, and the pellets were resuspended in an equal volume. The counts per minute contained in the pellet were determined by liquid scintillation counting. The values are the average and standard deviations of triplicate samples. (B) Similar counts from pelleted material obtained as described for panel A were run on a 15% polyacrylamide gel, and the gel was processed for autoradiography. The asterisks indicate the positions of the core proteins A3L and A10L. In the case of IHD-J, these proteins run as a doublet of the cleaved forms of 65 kDa. In MVA the uncleaved precursor forms are seen. On the right, the position of the molecular weight marker proteins (M) is indicated.

FIG. 7.

The dense particles are not infectious. HeLa cells were infected at an MOI of 10 with MVA (♦), WR (▪), and IHD-J (▴). The culture supernatant (A) and the cells (B) were harvested at the indicated times in hours postinfection (hpi), and the infectivity (i.e., the titer in PFU/milliliter) was determined by plaque assay. The values are the averages of triplicate samples titrated in duplicate.