PMC

The association of BmNPV BRO-A/C with nuclear structures. Nuclei isolated from BmNPV-infected cells harvested at 14 h p.i. (A) or at 4 h p.i. (B) were extracted with the solutions indicated above the lanes. The arrows indicate a 37-kDa polypeptide detected using anti-BRO-A antibody. Extractions were performed twice.

Interaction of BRO proteins with nucleic acid resins. BmN cell extracts were prepared from infected cells at 14 h p.i. and were subjected to ssDNA-cellulose (A), dsDNA-cellulose (B), and poly(U)-agarose (C) column chromatography. The collected fractions at the indicated concentrations of NaCl were analyzed by SDS–8% PAGE followed by Western blot hybridization.

An ssDNA binding motif in the predicted amino acid sequence of baculovirus BRO proteins. The consensus consists of conserved aromatic and basic amino acids (boldface) separated by variable numbers of unrelated residues (X) (). The ssDNA binding proteins of BmNPV, DBP and LEF-3, also contain this motif. The sequences aligned were as follows: AcMNPV BRO (Ac-BRO) (), LdNPV BRO (Ld-BRO) (), BmNPV BRO (Bm-BRO), DBP, and LEF-3 ().

Identification of a BRO-A region involved in DNA binding. Four fragments of BRO-A were overexpressed in E. coli and used for ssDNA-cellulose batch chromatography. The collected fractions at the indicated concentrations of NaCl were analyzed by SDS–15% PAGE followed by Western blot hybridization. Data are presented for the following fragments: 1 to 159 aa (A), 160 to 317 aa (B), 1 to 78 aa (C), and 79 to 159 aa (D). (E) Detection of four expressed fragments using anti-BRO-A antibody. The cell extracts used in panels A, B, C, and D were loaded in lanes 1 to 4, respectively.

Posttranslational modifications of BmNPV BRO proteins. (A) Inhibition of proteasome activity by using the inhibitor MG-132. Infected cells were incubated in the absence or presence of MG-132. The asterisks in panels A and B show extra protein bands (40 to 60 kDa). (B) Investigation of main substrate for ubiquitination using BmNPV recombinants. BmN cells infected with wild-type BmNPV (WT) and mutant viruses lacking the bro-a, bro-b, bro-c, or bro-e gene (ΔA, ΔB, ΔC, and ΔE, respectively) were harvested at 8 and 14 h p.i. and analyzed by SDS–8% PAGE followed by Western blot hybridization. (C) Phosphorylation of BmNPV BRO-A/C in the course of infection. In vivo ³²P-labeled cells were harvested at the indicated times p.i. and were immunoprecipitated by using anti-BRO antibodies. Immunoprecipitated and labeled proteins were separated by SDS–8% PAGE followed by autoradiography. The arrow indicates a 37-kDa polypeptide (BRO-A/C).

Involvement of BmNPV BRO-A/C in nucleosome organization. (A) MN treatment. Nuclei isolated from infected cells harvested at 4 (lanes 1 and 2), 8 (lanes 3 and 4), and 14 (lanes 5 and 6) h p.i. were incubated with nuclease buffer in the absence (−) (lanes 1, 3, and 5) or presence (+) (lanes 2, 4, and 6) of MN. The arrows indicate major polypeptides detected using anti-BRO-A antibody. The positions of markers (Sigma Aldrich) are shown on the right. (B) Treatment of nuclear fractions with MN. Nuclei isolated from infected cells at 14 h p.i. were extracted with the solutions indicated above the lanes. MN treatment was introduced between the 350 and 600 mM NaCl extraction steps (lanes 5 to 8). Extractions were performed twice. (C) Interaction of BRO-A/C with core histones. The protein fraction was prepared by extracting the nuclei of infected cells with 600 mM NaCl and was analyzed by histone-agarose column chromatography. The eluates were collected at the indicated concentrations of NaCl and subjected to Western blot analysis.