Sindbis Virus Budding: The Making of Enveloped Virus and Virus-Like Particles from Pre-assembled Nucleocapsids

Sindbis Virus is an enveloped animal virus that obtains its viral envelope upon budding from the infected host at the last stage of the viral life cycle. During infection, the replicated viral RNA self-assembles with the capsid protein in the cytoplasm to form icosahedrally-symmetric structures known as nuclecapsids. It has been predominantly believed that nucleocapsid particles---that accumulate in great numbers in the cytoplasm of infected cells---leave the host as mature budded particles when the capsid protein in the nucleocapsid interacts with the cytoplasmic tail of the viral E2 glycoprotein in the plasma membrane. An alternative model, however, suggests that the pre-assembled nucleocasids are not necessary for the formation of the mature, enveloped virions, and that budding occurs in one step at the plasma membrane when the glycoproteins interact with the monomeric capsid proteins complexed with the viral RNA.;This work aimed at studying Sindbis Virus budding and its mechanism through in vitro and in vivo experiments. In vitro reconstitution experiments were carried out using pre-assembled nucleocapsids and viral membranes with the goal of mimicking the budding process and of making infectious, enveloped virions from purified components. These attempts were not successful, and the studies were therefore directed to determining the budding competency of pre-assembled nucleocapsids in cells expressing the viral glycoproteins. In vivo studies were launched, using the following approach: by providing permissive cellular conditions for budding and introduction of pre-assembled nucleocapsids into the cytoplasm of these cells, we could directly study the budding competency of pre-assembled nucleocapsids. The cellular environment permissive to budding was prepared by expressing Sindbis virus glycoproteins produced in levels comparable to the levels expressed in a viral infection, using a Sindbis vector containing a glycoprotein replicon RNA. The utilization of this vector not only induced cellular conditions similar to a viral infection, but it also inhibited any further infections from occurring, leading to elimination of false positives that could arise if the nucleocapsids disassembled upon entry---a scenario that normally leads to an infection in naive cells. Additionally, special nucleocapsids were also engineered, containing a reporter RNA instead of the viral genome as a second safety measure to eliminate the possibility of any infection. The nucleocapsids were introduced into glycoprotein-expressing cells using microinjection, electroporation, and lipid-based transfection techniques, and budding was assayed by a functional, biological transduction assay: The medium on top of nucleocapsid-recipient cells were collected and overlaid on naive cells. If pre-assembled nucleocapsids had budded and were enveloped, they could enter naive cells, delivering their reporter RNA cargo, leading to the expression of the reporter protein in the overlaid cells. We find that the nucleocapsids introduced into glycoprotein-expressing cells are capable of budding regardless of the technique used for their insertion. Additionally, we find that the majority of the delivered nucleocapsids disassemble in the cytoplasm of cells, lowering the yield of budded particles. Future experiments are suggested, aiming at the inhibition of this disassembly process with the prediction that such a strategy will increase the yield of budded particles.