||Protein 3a is the largest group-specific protein of severe acute respiratory syndrome (SARS) coronavirus, which is distributed in endoplasmic reticulum-Golgi intermediate compartment (ERGIC) and the plasma membrane in infected or transfected cells. The association of the 3a protein with the SARS coronavirus structure and its involvement in cellular responses of the host cells implicate that it is involved in the pathogenesis of the SARS infectious disease. In the current study, the intracellular and extracellular trafficking behaviors of the 3a protein in relation to its cellular effects were investigated. Firstly, it has been found that neither of the tyrosine-based motif nor the adjacent diacidic motif, which has been reported to direct the 3a protein transporting to cell plasma membrane, is essential for the subcellular localization of the 3a protein. Using the site-directed mutagenesis and truncation mutants, another two tyrosine-based signals (Y200VVV and Y211YQL) and a novel phenylalanine rich tetrapeptide signal (F230FIF) have been identified to act synergistically to escort protein 3a to the cell surface. However, these signals have no significant influence on the pro-apoptotic activity of 3a protein and its extracellular release, which seems to be depended on a mechanism different from the cell surface translocation. An adenoviral expression system has been established to investigate the immune response of the 3a protein and found that it stimulates the production of chemokines RANTES and IL-8 by real-time PCR. To understand how the 3a protein exerts these cellular effects, an unknown human protein, DKFZP434H132, which was previously identified from a lymph node cDNA library to interact with the cytoplasmic domain of protein 3a using a yeast two-hybrid system. The expression and subcellular localization of this protein has been characterized in different cell lines, and it is found as a universal protein with abundant expression in mammalian cells with phylogenic specificity. Immunofluorescence studies have demonstrated that DKFZP434H132 protein co-localizes with protein 3a only when the later is in absent of the carboxy terminal fusion, providing unequivocal evidence for in vivo interaction between the two proteins. These studies shed new light on protein 3a trafficking, subcellular localizations and cellular effects that are related to its potential roles in SARS pathogenesis and host-virus interaction.