Six monoclonal antibodies were isolated that exhibited specificity for a furin cleavage site deletion mutant (V3526) of Venezuelan equine encephalitis virus (VEEV). These antibodies comprise a single competition group and bound the E3 glycoprotein of VEEV subtype I viruses but failed to bind the E3 glycoprotein of other alphaviruses. These antibodies neutralized V3526 virus infectivity but did not neutralize the parental strain of Trinidad donkey (TrD) VEEV. However, the E3-specific antibodies did inhibit the production of virus from VEEV TrD-infected cells. In addition, passive immunization of mice demonstrated that antibody to the E3 glycoprotein provided protection against lethal VEEV TrD challenge. This is the first recognition of a protective epitope in the E3 glycoprotein. Furthermore, these results indicate that E3 plays a critical role late in the morphogenesis of progeny virus after E3 appears on the surfaces of infected cells.Viruses in the Alphavirus genus of the family Togaviridae are composed of an icosahedral nucleocapsid surrounded by a lipid envelope studded with a distinctive lattice of glycoprotein spikes. The structural proteins of alphaviruses arise through co-and posttranslational processing of a polyprotein encoded by a single 26S mRNA (22,27) in which the order of the gene products is NH 2 -capsid-PE2-6K-E1-COOH. The capsid (C) protein cleaves itself from the nascent polypeptide soon after emergence from the ribosome. The PE2 glycoprotein is a precursor containing the E3 glycoprotein fused to the amino terminus of the E2 envelope glycoprotein. The PE2 glycoprotein is followed by 6K, a small membrane-associated protein, and E1, the second polypeptide component of glycoprotein spikes. Trimerized heterodimers of the E1 and E2 viral glycoproteins form the surface spikes and contain determinants of viral tropism and virulence (1).The E3 glycoprotein acts as a signal for transport of PE2 across the membranes of the rough endoplasmic reticulum (22) and may promote the formation and intracellular transport of E1-PE2 heterodimers (12,23,46) to the cell surface. The E2 glycoprotein promotes specificity of virus binding to the host cell surface and is a target of protective antibodies (7,(18)(19)(20)39). The E1 glycoprotein mediates fusion of the virion envelope with the membranes of acidified endosomes, allowing release of the nucleocapsid into the cytoplasm and the onset of viral replication (21,40,43). Antibodies to the E1 glycoprotein do not typically neutralize virus infectivity in vitro but can protect against lethal challenge in animals (41, 42). During transport to the cell surface, PE2 undergoes a maturational cleavage event by a furin-like protease to produce E2 and E3. The E3 glycoprotein may be subsequently released into the extracellular space (26, 49) or incorporated into the virion (6, 13). At the plasma membrane, trimerized E1-E2 heterodimers are incorporated into the budding virus particle.Mutations that block cleavage of PE2 of Venezuelan equine encephalitis virus (VEEV) are lethal ...