(
a) NAGE capsid immunoblot. LMH hepatoma cells were co-transfected with the core-deficient DHBV vector pCD16_core
- plus expression vectors for wt-DHBc or the extension-less variants DHBc_ Δ78–122_R124E (well expressed in
E. coli but insoluble) or DHBc_ Δ78–122_R124E (well expressed in
E. coli as soluble CLPs;
Figure 4 and S2, S6). Formation of cytoplasmic capsids was analyzed by NAGE immunoblot as in
Figure 5a, using the polyclonal rabbit anti-DHBc antiserum 12/99 which recognizes various avihepadnavirus core proteins (
Vorreiter et al., 2007), followed by a peroxidase-conjugated secondary antibody and a chemiluminescent substrate. Despite long exposure only a very faint smear was detectable for variant Δ78–122_R124E. The signal from variant Δ78–122_R124E was stronger but still much weaker than that from wt-DHBV transfected cells. (
b) Southern blot. The figure shows the complete version of the Southern blot from
Figure 5, where lanes 9 and 12–14 loaded with redundant samples from repeat experiments (grey lettering, with labels b,c) had been spliced out. As variant Δ78–122_R124E did not form detectable capsids the lack of viral DNA is unsurprising. For variant Δ78–122_R124E the data suggest that if the weak immunoblot band in (
a) represents true capsids they are defective in pgRNA packaging and/or reverse transcription.