Resistance of a vaccinia virus A34R deletion mutant to spontaneous rupture of the outer membrane of progeny virions on the surface of infected cells

Abstract

The extracellular form of vaccinia virus is referred to as an enveloped virion (EV) because it contains an additional lipoprotein membrane surrounding the infectious mature virion (MV) that must be discarded prior to cell fusion and entry. Most EVs adhere to the surface of the parent cell and mediate spread of the infection to adjacent cells. Here we show that some attached EVs have ruptured envelopes. Rupture was detected by fluorescence microscopy of unfixed and unpermeabilized cells using antibodies to the F13 and L1 proteins, which line the inner side of the EV membrane and the outer side of the MV membrane, respectively. The presence of ruptured EV membranes was confirmed by immunogold transmission electron microscopy. EVs with broken membranes were present on several cell lines examined including one deficient in glycosaminoglycans, which are thought to play a role in breakage of the EV membrane prior to fusion of the MV. No correlation was found between EVs with ruptured membranes and actin tail formation. Studies with several mutant viruses indicated that EV membranes lacking the A34 protein were unbroken. This result was consistent with other properties of A34R deletion mutants including resistance of the EV membrane to polyanions, small plaque formation and low infectivity that can be increased by disruption of the EV membrane by freezing and thawing.

Fig. 1

Detection of EV and MV proteins on the surface of unfixed and unpermeabilized infected cells. HeLa cells were infected with vF13-HA and after 16 h were stained first with mouse anti-HA MAb (top row), mouse anti-L1 MAb (middle row) or rabbit anti-HA polyclonal antibody (bottom row) followed by FITC-conjugated anti-mouse IgG or anti-rabbit IgG antibody. Cells were then stained with rat anti-B5 MAb (top and middle rows), mouse anti-L1 MAb (bottom row) followed by rhodamine red-conjugated anti-rat or anti-mouse IgG antibodies. Stained cells were then analyzed by confocal microscopy. Green, FITC; red, rhodamine red. Bars, 10 μm.

Fig. 2

Detection of EV and MV proteins in fixed and digitonin permeabilized infected cells. HeLa cells infected with vF13-HA for 16 h were fixed with paraformaldehyde and permeabilized with digitonin. Cells were stained with mouse anti-HA MAb (top row), rat anti-B5 MAb (middle row) or rabbit anti-HA polyclonal antibody (bottom row) followed by FITC-conjugated anti-mouse or anti-rat IgG or anti-rabbit IgG antibodies. Cells were then stained with rat anti-B5 MAb (top row) or mouse anti-L1 MAb (middle and bottom rows) followed by rhodamine red-conjugated anti-rat or anti-mouse IgG antibodies. Cells were then analyzed by confocal microscopy. Green, FITC; red, rhodamine red. Bars, 10 μm.

Fig. 3

Detection of ruptured EV particles on the surface of multiple cell lines. BHK, BS-C-1, RK₁₃, L and Sog9 cells were infected with vF13-HA and at 16 h were stained with mouse anti-HA MAb followed by Alexa 488-conjugated anti-mouse IgG antibody. Cells were then fixed and analyzed by confocal microscopy. Bars, 10 μm.

Fig. 4

Detection of ruptured EV particles on the surface of cells by electron microscopy. HeLa cells were infected with vF13-HA for 16 h. Unfixed and unpermeabilized cells were stained with mouse anti-HA MAb followed by protein A gold. After staining cells were fixed, cryosectioned and examined by transmission electron microscopy.

Fig. 5

Staining of EV particles and actin tails. HeLa cells were infected with vF13-HA (rows 1-3) or vA36(Y112,132F) (row 4). At 16 h, cells were stained with rat anti-B5 MAb (row 1), mouse anti-HA MAb (rows 2 and 3) and mouse anti-L1 MAb (row 4) followed by Alexa 488-conjugated anti-rat IgG, anti-mouse IgG and Alexa 594-conjugated anti-mouse IgG antibodies. Cells were then fixed, permeabilized with Triton X-100 and stained with Texas red-conjugated phalloidin (rows 1-3). In row 3, cells were first fixed and permeabilized with Triton X-100 before staining. Cells were analyzed by confocal microscopy. Green, Alexa 488; red, Alexa 594 and Texas red. Bars, 10 μm. Arrows point to representative actin tails.

Fig. 6

Detection of ruptured EVs on the surface of cells infected with VACV mutants. HeLa cells were infected with vA33Δ, vA56Δ or vA34Δ. After 16 h, cells were stained with mouse anti-L1 MAb followed by Alexa 488-conjugated anti-mouse IgG. Cells were then stained with rat anti-B5 MAb followed by Alexa 568 IgG. Cells were then fixed and analyzed by confocal microscopy. Green, Alexa 488; red, Alexa 568 or 594. Bars, 10 μm.

Fig. 7

Absence of ruptured EV particles on the surface of cells infected with vaccinia virus with deleted A34R gene. HeLa cells were infected with vF13-HA(A34Δ) and after 16 h were stained with mouse anti-HA MAb (rows 1, 4), mouse anti-L1 MAb (rows 2, 5) and rat anti-B5 MAb (rows 3,6) followed by Alexa 594-conjugated anti-mouse or anti-rat IgG antibodies. Cells in rows 1-3 were unfixed and unpermeabilized; cells in rows 4-6 were fixed with paraformaldehyde and permeabilized with digitonin. Confocal microscopy images are shown. Green, GFP; red, Alexa 594. Bars, 10 μm.