Severe acute respiratory syndrome (SARS) emerged in the Guangdong province of southern China in late 2002, and rapidly spread worldwide in 2003 with significant mortality. The disease is caused by a novel coronavirus (SARS CoV), and developing vaccines and antiviral therapeutics are high priorities. Coronaviruses have the unusual property of assembling at intracellular membranes: they target their envelope proteins to membranes of the endoplasmic reticulum-Golgi intermediate compartment, bud into the lumen of this compartment, and then exit the cell by exocytosis. Targeting and assembly of the envelope proteins from several coronaviruses have been well studied, but the release step has not been examined. Efficient release of particles is essential for economical vaccine production. Virus-like particles (VLPs) from model coronaviruses are formed by co-expression of two of the envelope proteins alone (M and E), and the spike protein (S) is incorporated when present. However, the requirements for formation of SARS VLPs are controversial. VLP vaccines offer many advantages in terms of immunogenicity and safety of production. Efficient release of VLPs from the cells producing them allows purification from the supernatant rather than homogenized cells, which is simpler and yields preparations with higher purity. Our preliminary data for a model coronavirus (infectious bronchitis virus, IBV) suggest that the small envelope protein E is required for efficient particle assembly and for efficient release. The cytoplasmic tail of the IBV E protein is essential for interaction with M and assembly of particles. By contrast, the transmembrane domain of IBV E is required for efficient release of infectious virus. In the experiments proposed here, we will systematically determine the requirements for assembly and release of SARS VLPs, and test the idea that the SARS E protein has two separate functions in this process.
Our specific aims are to (1) test the hypothesis that the SARS E protein is a structural protein that is essential for VLP assembly; and (2) test the hypothesis that the SARS E protein plays a nonstructural role by promoting release of intact particles. These studies will increase our understanding of release of viruses that assemble intracellularly, provide a framework for increasing the efficiency of virus release for vaccine production, and identify new targets for antiviral therapeutics. ? ? ?
| McBride, Corrin E; Machamer, Carolyn E (2010) Palmitoylation of SARS-CoV S protein is necessary for partitioning into detergent-resistant membranes and cell-cell fusion but not interaction with M protein. Virology 405:139-48 |
| McBride, Corrin E; Machamer, Carolyn E (2010) A single tyrosine in the severe acute respiratory syndrome coronavirus membrane protein cytoplasmic tail is important for efficient interaction with spike protein. J Virol 84:1891-901 |
