Native agarose gel electrophoresis assay as a reliable method for determining A3G incorporation into HBV nucleocapsids. HepG2 cells were cotransfected with plasmids expressing either wild-type HBV or a polymerase-deficient HBV mutant (ΔRT) along with A3G. Cell lysates were prepared 72 h after transfection and subjected to ultracentrifugation over a sucrose gradient to separate HBV capsids from most cellular proteins. The unfractionated lysates (lanes 1 to 3) and the resuspended pellets (lanes 4 to 6) were loaded onto an SDS-PAGE gel (A) and a native agarose gel (B). Western blot analysis was used to detect the levels of HBV core protein (A, bottom panel), capsid (B, bottom panel), or A3G protein (A and B, upper panels). For native agarose gel analysis (B), A3G was detected by immunoblotting with the anti-A3G antibodies (B, upper panel), followed by subsequent reprobing of the same membrane for HBV capsids using the anti-HBV core protein antibody (B, bottom panel).

A3G incorporation was dependent on the viral RT protein and ɛ RNA. HepG2 cells were transfected with plasmids expressing HBV, a polymerase-deficient HBV mutant (ΔRT), or a ɛ⁻ HBV mutant together with A3G. For transcomplementation of HBV RT, HepG2 cells were transfected with plasmids expressing ΔRT and RT in the presence or absence of A3G. Cells were lysed, and A3G packaging into HBV nucleocapsids was measured by native agarose gel analysis. A3G levels (upper panel) and assembled HBV capsids (bottom panel) were detected on the same membrane, as described for Fig. 1B.

A3G interaction with HBV RT in 293T cells. (A) Transfected 293T cells were lysed, and A3G packaging into HBV nucleocapsids was measured by native agarose gel analysis. A3G levels (upper panel) were detected by A3G-specific antibodies, followed by subsequent reprobing of the same membrane for HBV capsids (bottom panel) using the anti-HBV core protein antibody. (B) 293T cells were transfected with plasmids expressing HBV and A3G, cytoplasmic nucleocapsids were extracted from transfected cells, and capsid-associated DNA levels were determined by native agarose gel analysis. HBV DNA was detected by using a minus-strand-specific riboprobe (top). Capsids were detected by probing the same membrane with anti-HBV core protein antibody (bottom). (C) Co-IP of Flag-tagged HBV RT and A3G. Transfected 293T cell lysate was incubated with the anti-Flag antibody, and the immunoprecipitated proteins were analyzed by Western blotting (WB) with anti-A3G (upper panel) or anti-Flag antibodies (lower panel).

Requirement for the A3G N-terminal domain in encapsidation. (A) Schematic representation of the A3G deletion mutants and their expression and incorporation. All deletion constructs contain a Flag tag at the amino terminus of the protein, with the exception of A3G 104-384. (B and C) HepG2 cells were transfected with a plasmid expressing either the full-length A3G or the A3G deletion mutants together with the HBV plasmid. Seventy-two hours posttransfection, cells were lysed in NP-40 and total A3G levels in the lysate (soluble) and pellet (insoluble) (B) and the amount of packaged A3G (C) were determined by SDS-PAGE and native agarose gel assay, respectively. Expression of C-terminal A3G fragments and incorporated A3G levels were detected using anti-A3G antibody, while N-terminal A3G fragments were detected by using anti-Flag antibody. For the native agarose gel analysis (C), A3G was detected by immunoblotting with anti-A3G antibodies (B, upper panel), followed by reprobing of the same membrane for HBV capsids using the anti-HBV core antibody (B, bottom panel). *, protein expressed but insoluble; **, protein expressed but partially degraded; ?, incorporation status uncertain due to protein degradation. The arrow in panel B indicates the intact A3G 104-384 protein.

Protection of nucleocapsid-associated A3G from protease digestion. HepG2 cells were transfected with plasmids expressing HBV and A3G. Three days later, NP-40 cell lysates were prepared and treated with the indicated concentration of proteinase K. Lysates were then resolved on a native agarose gel and transferred onto a nitrocellulose membrane. Western blot analysis was used to detect A3G (upper panel) followed by detection of assembled capsids (bottom panel) on the same membrane using the anti-A3G and anti-HBV core antibodies, respectively.

HBV RT interacts with A3G in HepG2 cells. Plasmids expressing Flag-tagged HBV RT and A3G were cotransfected into HepG2 cells. Forty-eight hours after transfection the interaction between A3G and HBV RT was determined. HBV RT was immunoprecipitated from the cell lysate using the anti-Flag antibody, and the presence of A3G in the immunoprecipitate was detected by immunoblotting with anti-A3G. To determine if the interaction between A3G and HBV RT was dependent on RNA, the cell lysate was treated with 200 μg/ml RNase A prior to IP (lanes 3, 4, 7, and 8).

Correlation between levels of A3G incorporation and levels of pgRNA incorporation and A3G expression. (A) RT titration. HepG2 cells were transfected with constant amounts of RT-defective HBV and A3G plasmid, along with increasing amounts of the HBV RT plasmid. Twenty-four hours after transfection, the cells were treated with 20 μM 3TC to inhibit DNA synthesis. Cytoplasmic nucleocapsids were then isolated from transfected cells and loaded onto an agarose gel. For DNA analysis, the agarose gel was treated with NaOH to remove RNA. A3G (first panel) and capsid levels (fourth panel) were analyzed by Western blotting of the same membrane as described for Fig. 1B, while core-associated RNA (second panel) and DNA (third panel) levels were determined by Southern blotting using a plus-strand-specific riboprobe for HBV plus-strand DNA. (B) A3G titration. HepG2 cells were transfected with plasmids expressing wild-type HBV and increasing amounts of A3G. Cytoplasmic nucleocapsids were extracted from transfected cells and analyzed by native agarose gel assay (panels 1 to 4) as in panel A. Total A3G expression was analyzed by SDS-PAGE and Western blotting (fifth panel). (C and D) Quantitative correlation between incorporated A3G levels, HBV RNA and DNA, capsid protein, and total A3G expression levels. Nucleic acid signals and protein bands were quantified with a phosphorimager and densitometer, respectively.

Proposed model of A3G incorporation into HBV nucleocapsids. Incorporation of A3G into the HBV nucleocapsid is dependent on its interaction with viral RT. During HBV nucleocapsid assembly, A3G binds to RT and/or to the RT-ɛ complex, leading to its specific packaging into the assembling nucleocapsid. Additional A3G proteins can also be packaged by their nonspecific binding to the pgRNA that is packaged via RT interaction or, alternatively, multiple A3G molecules (e.g., as HMW complexes) can be bound by one RT molecule and incorporated into the nucleocapsids. The pgRNA and ɛ RNA packaging signal are indicated.