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Direct confirmation of low DNA content in intact capsids derived from mutant ε constructs.

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posted on 20.08.2013 by Hui Feng, Ping Chen, Fei Zhao, Michael Nassal, Kanghong Hu

(A) DNA detection by molecular hybridization. Cytoplasmic capsids from cells transfected with the indicated constructs were separated by NAGE. After blotting, HBV DNA in the capsids was monitored by hybridization with a minus-strand specific probe, and capsids by immunodetection (panel labeled capsids). β-actin mRNA as determined by RT-PCR (panel labeled β-actin) served as loading control. (B) Endogenous polymerase assays (EPAs). One aliquot each of cytoplasmic capsids was subjected to EPA conditions in the presence of α-32P-dATP or α-32P-dCTP, then separated by NAGE. Labeled products associated with the capsids were visualized by autoradiography. Y63F refers to a replication-defective HBV construct in which the priming Tyr63 residue of P was replaced by Phe. A third aliquot from each sample was used for immunodetection of NAGE-separated capsids (panel labeled capsids). (C) Relative EPA activities. The bar graph shows the signal intensities generated by individual mutants for α-32P-dCTP and α-32P-dATP EPAs relative to that produced by wild-type HBV which was set at 100. Numbers are mean values from at least two independent experiments; error bars indicate standard deviation.