The PE2 cleavage signal in a full-length cDNA clone of the alphavirus Venezuelan equine encephalitis virus (VEE) was ablated by site-directed mutagenesis. RNA transcripts derived from the resulting plasmids programmed the production of nonviable particles upon transfection of baby hamster kidney (BHK) cells. However, the mutant RNAs also gave rise to a small proportion of viable revertants. Analysis of these biological revertants and their molecularly cloned homologs demonstrated that second-site suppressor mutations at either E2 position 243 or E1 position 253 were able to restore viability to PE2 cleavage signal mutants. The viable revertants incorporated unprocessed PE2 into particles which showed normal infectivity for BHK cells, but reduced ability to grow in C6/36 mosquito cells. A mutant carrying a lethal PE2 cleavage signal mutation in combination with a suppressor at El 253 was either avirulent or highly attenuated in adult mice when inoculated by the subcutaneous, intracerebral, or intranasal route and conferred complete protection against both intraperitoneal and intranasal challenge with virulent VEE. These results indicate the close functional association of the E2 and El proteins in the alphavirus spike. They also have implications for the design of recombinant live virus vaccines for VEE, for other alphaviruses, and for other viruses that use a similar mechanism for glycoprotein maturation. © 1995 Academic Press, Inc.