Within the last year a new coronavirus (SARS-CoV) has been identified as the etiological agent of severe acute respiratory syndrome (SARS). Given the high mortality, the lack of effective treatment and the uncertainties of when the disease might return and at what frequency, a SARS vaccine and the development of antivirals are of high priority. In order to develop these strategies, we need to understand the basic biology of replication as well as the function of individual viral genes in the viral life cycle. All coronaviruses contain the structural proteins, spike (S), nucleocapsid (N), membrane (M) and small envelope (E). In addition, each group of coronaviruses expresses proteins encoded in small ORFs, unique to that group of viruses. These are, in some cases, nonessential for replication, but may have important accessory roles. Thus, our goals here include establishing a reverse genetics system for SARS-CoV and selecting viruses to be used for more detailed studies of viral replication and pathogenesis. The specific aims of this project are: 1) to establish a reverse genetics system for SARS-CoV, using targeted recombination and 2) to identify the gene products encoded in the small ORFs, unique to SARS-CoV, and to probe the role of these proteins in replication and, in the long term, in pathogenesis. The reverse genetics system will provide the basis for long-term studies on the structure/function analysis of viral structural proteins as well as the small proteins encoded in the small ORFs unique to SARS-CoV. We have a long-term interest in the replication and pathogenesis of coronaviruses. Much of the work we propose here is based on the knowledge and expertise we have accumulated in working with the murine coronavirus, mouse hepatitis virus (MHV). The targeted recombination system has greatly expanded our ability to understand replication and pathogenesis of MHV and we expect it will do the same for SARS-CoV.