CHOLESTEROL IS REQUIRED IN THE EXIT PATHWAY OF SEMLIKI FOREST VIRUS

The enveloped alphavirus Semliki Forest virus (SFV) infects cells via a membrane fusion reaction triggered by low pH. For fusion to occur ch olesterol is required in the target membrane, as demonstrated both in in vitro fusion assays and in vivo for virus infection of a host cell. In this paper we examine the role of cholesterol in postfusion events in the SFV life cycle. Cholesterol-depleted insect cells were transfe cted with SFV RNA or infected at very high multiplicities to circumven t the fusion block caused by the absence of cholesterol. Under these c onditions, the viral spike proteins were synthesized and transported t o the site of p62 cleavage with normal kinetics. Surprisingly, the sub sequent exit of virus particles was dramatically slowed compared to ch olesterol-containing cells. The inhibition of virus production could b e reversed by the addition of cholesterol to depleted cells. In contra st to results with SFV, no cholesterol requirement for virus exit was observed for the production of either the unrelated vesicular stomatit is virus or a cholesterol-independent SFV fusion mutant. Thus, cholest erol was only critical in the exit pathway of viruses that also requir e cholesterol for fusion. These results demonstrate a specific and une xpected lipid requirement in virus exit, and suggest that in addition to its role in fusion, cholesterol is involved in the assembly or budd ing of SFV.