Our investigations have demonstrated that BmNPV BRO proteins, especially BRO-A, BRO-C, and BRO-D, have nucleic acid binding activities and are involved in nucleoprotein complexes in the nuclei of infected cells. BmNPV BRO-A, -C, and -D proteins were reported to be localized in the nucleus; however, BRO-B and -E showed only cytoplasmic distributions (13
). This also supports the notion that BRO-A, -C, and -D might be nuclear proteins. We have been concentrating on BRO-A and BRO-C due to their apparent abundance in infected cells. Although it was difficult to separate BRO-A from BRO-C by SDS-PAGE in most experiments because of their similar molecular weights, anti-BRO-A antibodies were able to recognize the BRO-C protein in the experiments using bro-a
deletion mutants and vice versa. The failure to obtain double-deletion mutants of bro-a
) suggests not only that this group plays an important role in infection but also that they may carry out the same function(s). Thus, we postulated that our data are true for both BRO-A and BRO-C. The extraction of nuclei by following a histone extraction protocol and MN treatment analyses indicated that BRO-A/C are involved in nucleosome organization by binding to nucleic acids directly. Similar concentrations (500 to 600 mM) of NaCl are required for eluting BRO-A/C from either the chromatin of infected cells or DNA columns, suggesting that BRO-A/C interact with DNA in a sequence-independent manner.
Wilson and Miller reported that viral DNA acquired a chromatinlike structure in the nuclei of Sf-21 cells infected with AcM
). They also showed that this nucleosome structure contained two major virus-induced proteins with molecular masses of 15 and 39 kDa. BmNPV BRO-A/C revealed similar molecular weights, nuclear localization, and binding affinities for nuclear structures, suggesting that they are the counterparts of the 39-kDa nucleoprotein in AcM
NPV. Polyclonal antibodies against BmNPV BRO-A recognize a single polypeptide of 35 kDa that corresponds relatively well to the predicted molecular mass of AcM
NPV BRO protein (37.8 kDa) (E. Zemskov, unpublished data). This polypeptide was specific for infected cells, appeared no later than 4 h p.i., and persisted through at least 26 h p.i. The expression pattern and molecular mass of AcM
NPV BRO suggested that BRO could be the 39-kDa protein in the nucleosome structure of infected Sf-21 cells as described by Wilson and Miller (23
). Although it remains unclear whether these two are the same, our data support the idea that baculoviral proteins are involved in nucleoprotein complexes in the nuclei of infected cells.
DNA-cellulose chromatography experiments using overexpressed BRO-A fragments in E. coli
indicate that the DNA binding ability lies in the N-terminal region of BRO-A containing 80 amino acid residues. Further alignment by computer confirmed the presence of an ssDNA binding motif in this region. This motif was originally found in SSB proteins from prokaryotic and eukaryotic organisms (22
). We also found that most BRO proteins contain this motif. The N-terminal localization of the motif is common to all BmNPV BRO proteins, AcM
NPV BRO protein, and some BRO proteins from LdNPV; however, several BRO proteins of LdNPV and X. c-nigrum
granulovirus contain the motif in a central or C-terminal region. Baculoviral LEF-3 and DBP, which have also been described as SSB proteins, contain this motif (20
). Interestingly, LEF-3 and DBP have no homology with any known SSB proteins. Thus, this consensus seems important for baculoviral SSB proteins.
Due to the limited number of viral proteins, one protein could have several functions in infected cells. This has already been demonstrated for LEF-3 of AcM
NPV. It functions as an SSB protein in DNA replication and also participates in the translocation of virus-encoded DNA helicase from the cytoplasm to the nucleus (8
). Among the products of the five BmNPV bro
genes, at least three BRO proteins, BRO-A, BRO-C, and BRO-D, are nucleic acid binding proteins. Before the onset of viral DNA replication, these proteins are already associated with nuclear structures, most likely with chromatin. BRO-A/C especially showed very strong affinity for ssDNA. Based on these data, we propose a number of possible functions for these proteins. They could block cellular replication and/or transcription and switch host machinery to viral DNA or RNA synthesis by binding to host chromosomal DNA. In addition, RNA binding activity of BRO-A/C demonstrated by poly(U)-agarose chromatography suggests that they could participate in the nuclear export of mRNA. The presence of such proteins is known in eukaryotic cells and some viruses (3
). BmNPV BRO proteins seem to be abundant in the early stage of infection. Therefore, their ubiquitination and involvement in proteasome-directed cleavage could protect other viral proteins from degradation and increase the efficacy of the infection. Phosphorylation of BRO proteins may also regulate DNA and RNA binding activity as shown in many DNA binding proteins as well as LEF-3 and DBP of baculovirus (5
; Zemskov, unpublished). A switch of functions might be modulated by factors such as the ratio of host DNA to viral DNA, interaction with specific proteins, and posttranslational modifications (phosphorylation and ubiquitination). Although a distinct feature of the bro
gene family is its extensive repetition in baculovirus genomes (up to 17 copies in the LdNPV genome [16
]), the necessity for this amplification is unclear. It could be involved in binding to a variety of different DNA-protein conformations that may be present in most cells or specific for different cell types.