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. 2020 Aug 14;9:e57277. doi: 10.7554/eLife.57277

Figure 5. Opposite charges at DHBc positions 109 and 124 warrant stable capsid formation in hepatoma cells.

(a) Capsid formation. Cytoplasmic lysates from LMH cells transfected with the indicated constructs (pCD16: mutations present in cis; pCD16_core- + trans-DHBc: core-deficient DHBV genome co-transfected with DHBc vectors) were analyzed for capsid formation by NAGE and anti-DHBc immunoblot; recDHBc, E. coli derived CLPs. All samples except i95 -Nuc and WT -Nuc (lanes 14, 15) were treated with micrococcal nuclease to destroy non-packaged nucleic acids prior to NAGE. (b) Viral nucleic acid encapsidation. Cytoplasmic lysates separated as in (a) were examined by hybridization with a 32P-labeled DHBV probe after opening the blotted capsids via alkali treatment for 15 s (leaving RNA and DNA intact) and, thereafter, for 15 min (hydrolyzing RNA but not DNA). Signals from pCD16_YMhD (encoding a reverse transcription-deficient polymerase) and trans-DHBc E110R (lanes 2, 10; marked by **) showed strong and moderate, respectively, reductions upon longer treatment. § in lane one indicates poor transfer of the left part of the main band. (c) Absence of grossly aberrant reverse transcription products. DNAs from cytoplasmic capsids were analyzed by Southern blotting using the same probe as in (b). Labels WT vs. WTopt refer to the DHBc nucleotide sequence from DHBV16 vs. that from the E. coli optimized vectors. The presence of signals for DHBc_R124Q and E109,110R_R124E likely relates to the larger input of capsids for Southern blotting than for NAGE. M1-M3, marker DNAs; pla., pCD16 plasmid. The complete blot is shown in Figure 5—figure supplement 1.

Figure 5.

Figure 5—figure supplement 1. Extension-less DHBc variants fail to support stable nucleocapsid formation in hepatoma cells.

Figure 5—figure supplement 1.

(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.
Figure 5—figure supplement 2. Lack of stable capsid formation by variant DHBc_R124E in hepatoma cells correlates with increased proteolytic sensitivity.

Figure 5—figure supplement 2.

LMH cells were cotransfected, in 10 cm dish format, with pCD16_core- and the indicated trans-DHBc constructs; for sample E109R_R124E lo only one fifth the amount of plasmid DNA was used. For sample GFP cells were solely transfected with the GFP vector pTRUF5. On day four post transfection cells were lysed using 750 µl lysis buffer containing NP40 detergent and protease inhibitor cocktail (Complete, EDTA-free; Roche). (a) NAGE capsid immunoblot. 20 µl of each lysate were analyzed by NAGE and immunoblotting as in Figure 5a. (b) NAGE capsid-DNA blot. 20 µl of each lysate were analyzed for capsid-borne DHBV DNA as in Figure 5b. (c) Steady-state levels and integrity of DHBc proteins irrespective of their assembly status. To enrich DHBc proteins, 400 µl of each lysate were subjected to immunoprecipitation using polyclonal rabbit anti-DHBc antibodies (rabbit serum 12/99, Vorreiter et al., 2007) immobilized on Protein A sepharose beads (GE Healthcare); after extensive washing the beads (about 40 µl gel bed each) were boiled with 80 µl SDS-containing sample buffer and 20 µl thereof were separated by SDS-PAGE (15% polyacrylamide), transferred to PVDF membrane and detected using the same polyclonal antiserum followed by an Fc-specific anti-rabbit IgG peroxidase conjugate and a chemiluminescent substrate. Approximately 30–50 ng of the indicated recombinant DHBc proteins served as markers. The 55 kDa band in the cell culture samples represents the heavy chains of the IP antibodies. The weak bands below the 25 kDa marker position (arrowheads) were present in all IP samples, including the GFP-only transfected lysates; hence they are not DHBc-specific. Note the multiple faster migrating DHBc-specific bands exclusively in the R124E sample (lane 2**), indicative of a much increased proteolytic sensitivity caused by the mutation.