SUPPRESSORS OF CLEAVAGE-SITE MUTATIONS IN THE P62 ENVELOPE PROTEIN OFSEMLIKI-FOREST-VIRUS REVEAL DYNAMICS IN SPIKE STRUCTURE AND FUNCTION

The E2 spike glycoprotein of Semliki Forest virus is produced as a p62 precursor protein, which is cleaved by host proteases to its mature f orm, E2. Cleavage is not necessary for particle formation or release b ut is necessary for infectivity. Previous results had shown that pheno typic revertants of cleavage-deficient p62 mutants are generated, and here we show that these may contain second-site suppressor mutations i n the vicinity of the cleavage site. These hot-spot sites were mutated to abolish the generation of such suppressor mutations; however, seco ndary mutations in another distant domain of the E2 protein appeared i nstead, all of which still caused cleavage-deficient mutations. Such m utants grew very poorly and were inefficient in virus entry and releas e. The mutated sites define domains of the spike protein which probabl y interact to regulate its structure and function. Because of their hi ghly attenuated phenotype and the lower probability of reversion, the new mutations close to the cleavage site were used to make new helper vectors for packaging of recombinant RNA into infectious particles, th us increasing further the biosafety of the vector system based on the Semliki Forest virus replicon.