To study the dynamic of RSP production, we performed a pulse chase experiment on overnight starved HeLa-prME cells that were metabolically labeled with S 35 -methionine for 1 hour and subsequently chased for 24 hours in normal medium. Supernatant and cell lysate were collected at the specified time points after chase; RSPs were immuno-precipitated using the 4E11 anti-E monoclonal antibody and then analyzed by autoradiography. Detection of prM or M in this assay results from co-immunoprecipitation and, therefore, indicates efficient interaction with E protein. Secretion of E and M proteins was detected from the 8 hours time point on, with a significant increase at 24 hours postchase ( Figure 5A ). Consistently, high levels of E and M proteins were found in cell lysates at all time points with a substantial Figure 5B ). Two major protein products were found in cell lysates, with an apparent molecular weight corresponding to E and prM proteins. Bands of higher molecular weight were also present and could be oligomeric forms of E and prM or M. Interestingly, the pattern of immunoprecipitated proteins from cell supernatant was slightly different. Higher proportions of E dimers were found and a band corresponding to the molecular weight of M but not prM was detected at the 24 hours time point, indicating that most secreted RSPs are mature. Our data demonstrate that newly synthesized proteins need 8 hours to be translocated through the secretory pathway and released into the supernatant as mature RSPs.