We previously showed that HBV spherical SVPs use the cellular COPII machinery for ER export and secretion [29]. Since intracellular trafficking pathways of HBV subviral and viral particles are supposed to differ [2,21,45], a common exploitation of COPII transport carriers is ambiguous. Therefore, we reasoned to investigate the role of COPII factors in the production and release of infectious HBV particles. For RNA interference (RNAi), HuH-7 cells were treated with siRNA duplexes for 48 h prior to transfection with the pHBV* replicon construct. After an additional 72 h, cell lysates and supernatants were harvested. The used siRNAs comprised control duplexes (siCon) and single or siRNA pools targeting Sec24A, Sec24B, Sec23A, Sec23B, Sar1A or Sar1B. Sec24 is the cargo adaptor protein of COPII and associates with Sec23 to form the inner coat layer, while the Sar1 GTPase recruits the Sec23/Sec24 dimer to the ER membrane. To monitor depletion, COPII-specific transcripts were measured by qRT-PCR, which revealed an almost complete knockdown (KD) of each targeted COPII component (Figure 3). Cell lysates were probed for the expression and stability profiles of the viral L envelope and core proteins by specific WBs. As shown in Figure 3, neither knockdown (KD) affected the intracellular steady-state levels of either of the two viral proteins. The formation and release of HBV particles were analyzed by a virion production (VP) assay. For this, intracellular NCs and extracellular virions were immunoprecipitated with capsid- or envelope-specific antibodies, respectively, followed by particle disruption and real-time, multiplex PCR quantification of the number of HBV progeny genomes. Neither RNAi had a substantial effect on the formation of intracellular, replication-competent NCs. However, upon the silencing of Sec24A and Sec23B, the amounts of extracellular virions decreased in a highly significant manner, as compared to siCon-treated cells (Figure 3). Conversely, the loss of the two closely related Sec24B [46] or Sec23A [47] isoforms had no inhibitory effects on virus production and release. This indicates that HBV strictly depends on the specific functions provided by Sec24A and Sec23B in the same manner as HBV SVPs. In case of the KDs of the two Sar1 isoforms, the inhibition of the HBV release was obvious, albeit less pronounced (Figure 3). Since the Sar1A and Sar1B isoforms share 89% sequence identity concomitant with overlapping functions [48], the individual loss of either isoform may be partly compensated by its paralog. Together, these results demonstrate that HBV viral particles, similar to spherical subviral particles, require the COPII machinery for exiting the host cell, involving functions of Sec24A, Sec23B and Sar1. Hence, a COPII-guided export out of the ER appears to be a mandatory step in HBV biology.