Coronavirus envelope (E) proteins play an important, not fully understood role(s) in the virus life cycle. All E proteins have conserved cysteine residues located on the carboxy side of the long hydrophobic domain, suggesting functional significance. In this study, we confirmed that mouse hepatitis coronavirus A59 E protein is palmitoylated. To understand the role of the conserved residues and the necessity of palmitoylation, three cysteines at positions 40, 44, and 47 were changed singly and in various combinations to alanine. Double-and triple-mutant E proteins resulted in decreased virus-like particle output when coexpressed with the membrane (M) protein. Mutant E proteins were also studied in the context of a full-length infectious clone. Single-substitution viruses exhibited growth characteristics virtually identical to those of the wild-type virus, while the double-substitution mutations gave rise to viruses with less robust growth phenotypes indicated by smaller plaques and decreased virus yields. In contrast, replacement of all three cysteines resulted in crippled virus with significantly reduced yields. Triplemutant viruses did not exhibit impairment in entry. Mutant E proteins localized properly in infected cells. A comparison of intracellular and extracellular virus yields suggested that release is only slightly impaired. E protein lacking all three cysteines exhibited an increased rate of degradation compared to that of the wild-type protein, suggesting that palmitoylation is important for the stability of the protein. Altogether, the results indicate that the conserved cysteines and presumably palmitoylation are functionally important for virus production.Coronaviruses are medically important viruses that cause primarily respiratory and enteric infections in humans and a broad range of animals. The emergence of severe acute respiratory syndrome coronavirus (SARS-CoV) and the recent identification of new human coronaviruses (HCoVs), HCoV-NL63 and HCoV-HKU1 (10, 21), have significantly increased the importance of understanding key interactions during virus assembly, since these interactions may provide insight into potential targets for antiviral and vaccine development.Coronaviruses are enveloped, positive-stranded RNA viruses that belong to the Coronaviridae family in the Nidovirales order. The virion envelope contains at least three proteins: membrane (M), spike (S), and envelope (E). The genomic RNA is encapsidated by the phosphorylated nucleocapsid. Coronaviruses assemble at and bud into membranes of the endoplasmic reticulum Golgi intermediate compartment (ERGIC) (22,46). The focus of this paper is the E protein. Coronavirus E proteins are small (76 to 109 amino acids) integral membrane proteins bearing long hydrophobic domains. The protein plays an important, but not fully understood role(s) in virus production (7,11,24,38). Virus-like particles (VLPs) are formed only when the E and M proteins are expressed (3,5,50). When E protein is expressed by itself, E protein-containing vesicles are relea...