Isolation and analysis of vaccinia virus previrions

Vaccinia virus (VV) virion morphogenesis is a complex sequence of events that occurs late in viral infection that is essential for the production of mature progeny. Electron microscopy studies have identified multiple morphogenic forms of virus particles, apparently assembled in a sequence from immature to mature particles that correlates with distinct physical changes. This assembly process is, however, rather poorly understood at the molecular level. To better characterize the multiple forms of VV previrions, sucrose log gradient fractionation of VV-infected cells was used to separate radiolabeled immature and mature forms of the virus. Depending on time postinfection that the infected cells were harvested, four distinct peaks of acid-precipitable counts could be detected that displayed different rates of sedimentation. Using pulse-chase analysis procedures, the labeled peaks were shown to have precursor-product relationships as slower sedimenting entities chased to faster sedimenting ones with time. These peaks were referred to as A, B, C, and V particles, with A being the initial precursor form found near the top of the gradient and V being the fastest sedimenting product. As the previrions mature, they migrated faster in the gradient and became infectious and resistant to treatment with DNase I. The core protein composition of the A particles was predominantly uncleaved precursors, with only small amounts of the mature core proteins 4a, 4b, 25K, and 23K evident. However, as the sedimentation rate of the particles increased, proteolytic maturation proceeded such that C particles were composed almost exclusively of mature core proteins. Together these results indicate that several distinct and separable forms of VV previrions exist, that VV core protein precursors are associated with the previrions prior to cleavage, and that maturation of the core proteins is coordinately linked to the conversion from noninfectious previrions to infectious viral particles.