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

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.