Exploring the role of cholesterol in the Semliki Forest virus life cycle

Abstract

This thesis focuses on the role of cholesterol in the life cycle of the enveloped alphavirus, Semliki Forest virus (SFV). SFV infects cells via a membrane fusion reaction triggered by low pH. For fusion to occur, cholesterol is required in the target membrane as demonstrated in both in vitro fusion assays and in vivo infection of a host cell. Here we examined the role of cholesterol in post-fusion events in the SFV life cycle. Cholesterol-depleted insect cells were transfected with SFV RNA or infected at very high multiplicities to circumvent the fusion block caused by the absence of cholesterol. Under these conditions, the virus subgenomic RNA was translated and the virus spike proteins were synthesized and dimerized with the same efficiency as in control cells. Transport of the spike proteins to both the site of p62 cleavage and to the plasma membrane occurred with normal kinetics. Surprisingly, the exit of virus particles was dramatically slowed compared to cholesterol-containing cells. This inhibition of virus production could be reversed by the addition of cholesterol. In contrast to results with SFV, no cholesterol requirement for virus exit was observed for the production of a rhabdovirus that is cholesterol-independent for fusion. There was also a significantly reduced cholesterol requirement in the exit pathway of three SFV mutants selected for cholesterol-independent fusion. Thus, under these conditions, cholesterol was only critical in the exit pathway of viruses that require cholesterol for fusion. These results demonstrate a specific and unexpected cholesterol requirement in virus exit and suggest that in addition to its role in fusion, cholesterol is involved in the release of SFV from the plasma membrane.;Cell culture conditions were subsequently discovered that enable cholesterol-depleted cells to be efficiently infected by SFV. These cells do not contain cholesterol or any 3{dollar}\beta{dollar}-hydroxysterol and yet, astonishingly, they can support both the entry and exit of the virus nearly as well as cholesterol-containing cells. It is possible that these conditions induce a change in cellullar metabolism such as the upregulation or induction of a lipid or protein that permits virus infection and production in the absence of cholesterol.