INTRACELLULAR AND EXTRACELLULAR VACCINIA VIRIONS ENTER CELLS BY DIFFERENT MECHANISMS

Vaccinia virus (VV) produces two antigenically distinct infectious vir ions, intracellular mature virus (IMV) and extracellular enveloped vir us (EEV), Structurally, EEV consists of an IMV with an additional oute r membrane containing proteins that are absent from IMV, EEV is import ant for virus dissemination both in vitro and in vivo, Studies of EEV entry have been hampered by having two infectious virions and by the r upture of the EEV outer membrane in the majority of EEV virions during their purification, To overcome these problems, we have developed a n ovel approach to study VV entry that is based on confocal microscopy a nd does not require EEV purification, This assay relies on immunofluor escent staining and detection of individual, intracellular, uncoated v irus cores, By this method, we show that EEV entry, in contrast to IMV , is dependent on a low-pH pathway and that the IMV enwrapped inside t he EEV exhibits a low-pH fusogenic activity, Together with neutralizat ion data demonstrating that exposure to low pH disrupts the EEV outer membrane, this study strongly supports a model for EEV entry which con sists of binding, endocytosis, low-pH-induced disruption of the EEV ou ter membrane and fusion of the exposed IMV with the endosomal membrane releasing the core into the cytosol, The roles of the EEV outer membr ane in virus dissemination and virus entry are discussed in relation t o this model.