Impact of ESCRT-II subunits EAP30 and EAP45 on HBV maturation.

<p>HuH-7 cells were treated with control siRNA (siC) or the siRNA duplexes siEAP30#2 or siEAP45#2. After 48 h, cells were retransfected with pHBV, and cell lysates and supernatants were harvested 72 h later. <b>A.</b> Lysates were prepared with detergent and assayed for the synthesis of the HBV core and L envelope (L) proteins by specific Western blotting. <b>B.</b> Lysates were probed for the synthesis of the secretory precore (preC) and S envelope (S) proteins by specific ELISAs. Intracellular amounts of preC and S are expressed as mean units of optical density at 492 nm (<i>n = </i>2) and demonstrated in percent amount relative to siC-treated cells. <b>C.</b> Following treatment with siC, siEAP30#2, or siEAP45#2, cells were transfected with the vector pCore encoding solely the HBV core protein. Cytoplasmic capsids were concentrated by PEG precipitation and separated in a native agarose gel, blotted, and detected with anti-capsid antibodies. <b>D.</b> Cells were treated with the indicated siRNAs followed by transfection with pHBV. Cells were lysed by repetitive freeze-thaw cycles, and intracellular nucleocapsids and enveloped virions were precipitated with capsid- or envelope-specific antibodies, respectively, and assayed by quantitative real time PCR. Error bars indicate the standard deviations from the mean of two experiments measured in duplicates. <b>E.</b> Cells were transfected exactly as outlined in <b>D</b>. For a control, a replication-defective pHBVΔC replicon was included in the Southern blot analysis. Intracellular viral DNA was isolated and processed by Southern blotting using a digoxigenin-dUTP-labeled HBV-specific probe. Numbers to the right refer to marker DNA in kb.