Such alterations in ERGIC-53 trafficking could not be detected in HBV-replicating cells unless the ER export was blocked. Moreover, we were unable to verify a colocalization between ERGIC-53 and the viral envelope under natural settings, which was unanticipated given its pro-viral role in HBV propagation. ERGIC-53 is known to capture its cargoes in a pH- and Ca2+-dependent manner at the ER and releases them in the ERGIC, where the lower pH and Ca2+ concentrations diminish its affinity for glycoproteins [50,53]. Hence, within the ERGIC, the HBV envelope may rapidly dissociate from ERGIC-53, thereby rendering their colocalization below the limit of detection. Similarly, we failed to detect HBV envelope structures within Golgi stacks, although the N-glycan pattern of the extracellular virions implicate a transit through this compartment. One likely interpretation may be that HBV assembly and budding are inextricably linked and rate-limiting processes. Once disseminated from the host membrane, HBV viral particles may be rapidly exported out of the cell.