Figure 5.

P protein variants in the context of complete DHBV genomes. (A) DHBV nucleocapsid formation. LMH cells were transfected with expression plasmids for DHBV genomes encoding wild-type P protein, the indicated variants, an encapsidation-deficient construct (ɛ^–) or with empty plasmid vector (mock). Equal aliquots from cytoplasmic lysates were analyzed in duplicate by native agarose gel electrophoresis. After blotting DHBV core protein was detected using an anti-DHBV capsid antiserum (top) and a secondary antibody-peroxidase conjugate, followed by a chemiluminescent substrate. The faster migrating band (asterisk) corresponds to a non-specific cross-reaction product. DHBV DNA was detected by Southern hybridization using a DIG-labeled DNA probe (bottom) and a chemiluminescent substrate. (B) Encapsidation competence of variant DHBV P proteins. Capsids were enriched from cytoplasmic lysates of LMH cells transfected with the indicated constructs by immunopreciptiation with anti-DHBV capsid antiserum, and core-associated RNA was isolated. For comparison, total RNA was prepared from the same transfections. RNAs were separated by agarose gel electrophoresis, and hybridized with a DIG-labeled DNA probe specific for DHBV pgRNA. The position of a 3.5 kb marker RNA is indicated on the right.