The acylation of the envelope proteins of Semliki Forest virus by palmitic acid in infected mosquito (C6/36) cells was investigated. It is shown that in these cells palmitic acid was incorporated post-translationally via hydroxylamine-labile linkages onto cysteines in the inner domains of the viral envelope proteins. The kinetics of incorporation, however, differed considerably as compared to higher eukaryotic cells. (i) The precursor of the envelope proteins E2 and E3, p62, was weakly and incompletely palmitoylated irrespective of the duration of labeling. (ii) Under all conditions tested complete acylation of E2 was delayed as compared to E1. (iii) Heavy protein complexes were formed consisting of unacylated p62 and partially unacylated E1. From this data, we conclude that during the maturation of SFV glycoproteins in mosquito cells differently acylated intermediates of p62/E2 exist. Furthermore, acylation of p62/E2 and cleavage of p62 are coupled events, occurring in an early compartment and allowing the release of the envelope oligomers for transport.

We have analysed the temperature dependence of the transport of Semliki Forest virus (SFV) envelope proteins in mosquito cells, the natural host cells of alphaviruses. These cells are cultivated at a lower temperature (28 degrees C) and have a different lipid composition as compared to mammalian cells. When the incubation temperature was reduced at early times after infection, the onset of virus shedding was delayed and the maximal titers decreased correspondingly to the temperature. No virus was shed at 12 degrees C. No evidence was observed for a block of virus release due to a shift of the sites of virus maturation. When the incubation temperature was reduced at later times after infection a critical temperature of 12 degrees C was again observed. At this temperature no transport of viral proteins took place, p62 remained uncleaved, the glycan processing of E1 did not occur and the envelope proteins accumulated in a pre-Golgi compartment. We suggest a mathematical formula which allows the extrapolation of transport data to the temperature at which intracellular protein transport becomes blocked.