The severe acute respiratory syndrome coronavirus (SARS-CoV) genome contains open reading frames (ORFs) that encode for several genes that are homologous to proteins found in all known coronaviruses. These are the replicase gene 1a/1b and the four structural proteins, nucleocapsid (N), spike (S), membrane (M), and envelope (E), and these proteins are expected to be essential for the replication of the virus. In addition, this genome also contains nine other potential ORFs varying in length from 39 to 274 amino acids. The largest among these is the first ORF of the second longest subgenomic RNA, and this protein (termed U274 in the present study) consists of 274 amino acids and contains three putative transmembrane domains. Using antibody specific for the C terminus of U274, we show U274 to be expressed in SARS-CoV-infected Vero E6 cells and, in addition to the full-length protein, two other processed forms were also detected. By indirect immunofluorescence, U274 was localized to the perinuclear region, as well as to the plasma membrane, in both transfected and infected cells. Using an N terminus myc-tagged U274, the topology of U274 and its expression on the cell surface were confirmed. Deletion of a cytoplasmic domain of U274, which contains Yxx and diacidic motifs, abolished its transport to the cell surface. In addition, U274 expressed on the cell surface can internalize antibodies from the culture medium into the cells. Coimmunoprecipitation experiments also showed that U274 could interact specifically with the M, E, and S structural proteins, as well as with U122, another protein that is unique to SARS-CoV.

Besides genes that are homologous to proteins found in other coronaviruses, the severe acute respiratory syndrome coronavirus genome also contains nine other potential open reading frames. Previously, we have characterized the expression and cellular localization of two of these "accessory" viral proteins, 3a (previously termed U274) and 7a (previously termed U122). In this study, we further examined whether they can induce apoptosis, which has been observed clinically. We showed that the overexpression of 7a, but not of 3a or the viral structural proteins, nucleocapsid, membrane, and envelope, induces apoptosis. 7a induces apoptosis via a caspase-dependent pathway and in cell lines derived from different organs, including lung, kidney, and liver.

A new coronavirus (severe acute respiratory syndrome coronavirus [SARS-CoV]) has been identified to be the etiological agent of severe acute respiratory syndrome. Given the highly contagious and acute nature of the disease, there is an urgent need for the development of diagnostic assays that can detect SARS-CoV infection. For determination of which of the viral proteins encoded by the SARS-CoV genome may be exploited as diagnostic antigens for serological assays, the viral proteins were expressed individually in mammalian and/or bacterial cells and tested for reactivity with sera from SARS-CoV-infected patients by Western blot analysis. A total of 81 sera, including 67 from convalescent patients and seven pairs from two time points of infection, were analyzed, and all showed immunoreactivity towards the nucleocapsid protein (N). Sera from some of the patients also showed immunoreactivity to U274 (59 of 81 [73%]), a protein that is unique to SARS-CoV. In addition, all of the convalescent-phase sera showed immunoreactivity to the spike (S) protein when analyzed by an immunofluorescence method utilizing mammalian cells stably expressing S. However, samples from the acute phase (2 to 9 days after the onset of illness) did not react with S, suggesting that antibodies to N may appear earlier than antibodies to S. Alternatively, this could be due to the difference in the sensitivities of the two methods. The immunoreactivities to these recombinant viral proteins are highly specific, as sera from 100 healthy donors did not react with any of them. These results suggest that recombinant N, S, and U274 proteins may be used as antigens for the development of serological assays for SARS-CoV.