Envelopment of retrovirus and vaccinia virus

Abstract: This thesis describes envelopment of retrovirus and vaccinia virus. Retrovirus envelopment, i.e. budding, occurs from the plasma membrane (PM) of infected cells in an environment that is abundant with cellular proteins. In general, the viral budding process has been assumed to displace cellular membrane proteins with viral proteins, thus producing viral particles that are free from cellular components. Although this appears to be true for tightly organised viruses such as alphavirus, a number of host proteins have been reported to be incorporated in retroviral particles. Our aim was to study host protein incorporation on a general basis in Moloney- Murine Leukaemia virus Gag particles. Therefore, we developed a method by which it was possible to directly and quantitatively compare the protein composition of the PM used for budding, with the viral envelope. Our results demonstrated that most cellular proteins present in PM were recovered in the retrovirus membrane in almost similar concentrations. This indicated that the lateral associations of Gag proteins, while forming the internal shell, did not exclude any significant amount of host proteins. Since the presence of host proteins in retrovirus particles was never studied in depth until now, the presence of a certain host protein in the particles was thought to be specific. Our results demonstrated that there is no specific sorting of host proteins during budding. In fact, retroviruses randomly included host proteins that were located in the area of budding. Structural studies of the matrix protein of Gag revealed a lattice of loosely packaged hexameric rings. This structure appears to give plenty of room to accommodate foreign proteins, as long as they do not exert significant sterical hindrance for the formation of the Gag shell. We have also demonstrated an interaction in vivo between the Gag and Env protein, since the Env protein was five times more concentrated in viral particles, when compared to the PM. This amount was the same for the wild type retroviral particles. The Env-Gag interaction had no effect on the incorporation of host proteins in the viral particles. One of the host proteins present in the Gag particles was identified by immunolabeling to be actin. By studying Gag particles in immunoelectron microscopy, we found that actin was located underneath the membrane, on top of the Gag-shell. We do not think that actin is a structural component of the particles, but rather we hypothesise that actin is an accidental stowaway from scissioning and release of Gag particles from the PM. Intracellular mature virus (IMV) of Vaccinia virus (VV) is thought to be wrapped by a double membrane derived from the intermediate compartment between ER and Golgi. The membranes are modified by the insertion of VV membrane proteins to form two tightly apposed membranes, which line up as rigid crescents around the virosome. The virosome contains material, which is engulfed by the membrane crescents forming a spherical, immature virus, which matures into the brick-shaped IMV. The IMV membrane protein p21 (gene product of A17L) is essential for formation of the viral crescents, since in its absence, the precursor membranes do not form the rigid crescents. By using immuno-electron microscopy and biochemical methods, we demonstrated that p21 is located on the surface of IMV in the outer membrane, in addition to the internal membrane. Its localisation and topology makes it possible for p21 to form disulphide-linked homodimers between both leaflets of the double membrane. Thus, p21 might function as the merging force to flatten the compartment membranes and form the tightly apposed double membrane of IMV