PMC:7464876 / 39842-41738 JSONTXT

{"project":"2_test","denotations":[{"id":"32806600-12427977-20296235","span":{"begin":346,"end":348},"obj":"12427977"}],"text":"To narrow down the roles of ERGIC-53 and Sec24A in the HBV´s life cycle, colocalization studies were performed in RNAi-treated HBV-replicating cells. Aside, cells were treated with BFA, an established fungal compound that inhibits protein trafficking and secretion in mammalian cells via the collapsing of ER exit sites and, hence, of ER export [51]. The double-staining with anti-L and anti-core antibodies showed that neither the loss of ERGIC-53 nor of Sec24A hindered the recruitment of core to the typical CS structure of L (Figure 8A), indicating that both host factors are dispensable for L/core contact formation. Similar results were obtained in BFA-treated cells (Figure 8A), implicating that core is recruited by L prior to ER exit. To test whether ERGIC-53 and Sec24A might play a role in HBV assembly/budding or trafficking, we modified the VP assay in such that we screened for virions in cell extracts. In order to prevent the destruction of the viral envelope during lysis, cells were mechanistically disrupted. Extracts were next subjected to an L-specific IP in the absence of detergents, followed by particle disruption and PCR quantification of the number of HBV progeny genomes (Figure 8B). To assure the IP efficiency under these conditions, extracts precipitated in the same manner were analyzed by L-specific immunoblotting, which demonstrated effective and comparable precipitations of L (Figure 8C). Noteworthy, the pull-downed L pool may comprise both viral particles and subviral filamentous SVPs. Even so, filaments lack nucleocapsids and viral genomes and are not detectable by PCR analysis. In respect thereof, the PCR data, shown in Figure 8B, demonstrated that the depletion of either ERGIC-53 or Sec24A led to a significant increase of intracellular virions as compared to the control cells. This indicates a blockage of viral transport more than viral assembly."}