Evidence for Nucleic Acid Binding Ability and Nucleosome Association of Bombyx mori Nucleopolyhedrovirus BRO Proteins

Abstract

The Bombyx mori nucleopolyhedrovirus (BmNPV) genome contains five related members of the bro gene family, all of which are actively expressed in infected BmN cells. Although their functions are unknown, their amino acid sequences contain a motif found in all known viral and prokaryotic single-stranded DNA binding proteins. To determine if they bind to nucleic acids, we fractionated the nuclei of BmNPV-infected BmN cells using a histone extraction protocol. We detected BRO-A, BRO-C, and BRO-D in the histone H1 fraction using anti-BRO antibodies. Micrococcal nuclease treatment released these BRO proteins from the chromatin fraction, suggesting their involvement in nucleosome structures. Chromatographic fractionation showed that BRO-A and/or BRO-C interacted with core histones. Expression of partial sequences of BRO-A proved that the N-terminal 80 amino acid residues were required for DNA binding activity. We also demonstrated that BmNPV BRO proteins underwent phosphorylation and ubiquitination followed by proteasome degradation, which may explain their distribution in the cytoplasm as well as the nucleus. We propose that BRO-A and BRO-C may function as DNA binding proteins that influence host DNA replication and/or transcription.Bombyx mori nucleopolyhedrovirus (BmNPV) belongs to the Baculoviridae, a large family of viruses with double-stranded (ds) DNA genomes that are pathogenic mainly for lepidopteran insects. The BmNPV genome is about 128 kb in length and is predicted to contain 136 open reading frames (ORFs) (9). Among these ORFs, five genes (bro-a, bro-b, bro-c, bro-d, and bro-e) were found to belong to a unique baculovirus multigene family, since they demonstrated high homology to each other (13). Multiple members of this gene family have been reported in the genomes of the Orgyia pseudotsugata NPV, Lymantria dispar NPV (LdNPV), and Xestia c-nigrum granulovirus (1,11,16). However, the well-characterized Autographa californica MNPV (AcMNPV) genome contains only a single member (ORF2), which is related to BmNPV bro-d (2, 13) with 80% amino acid sequence identity. This is much lower than the average identity of predicted proteins from these two viruses, which is over 93% (9). In addition, NPV pathogenic for Spodoptera exigua lacks a bro homolog (12).Most bro genes share a related core sequence and demonstrate differing degrees of similarity in other regions (16). Although BmNPV BRO proteins show high homology within the family and with other baculovirus BROs, they have no strong similarity with any known proteins. Thus, it has been difficult to predict their function during the viral infection cycle.Recently, we reported that all bro genes of BmNPV are actively transcribed as delayed-early genes and that BRO proteins are produced at high levels between 8 and 14 h postinfection (p.i.) (13). We also reported that one BmNPV bro gene (bro-d) is essential for viral infection and that bro-a and bro-c may functionally complement each other (13). Since our immunohistochemical analysis using confocal microscopy showe...

Diversity and evolution of the Cydia pomonella granulovirus

“…Cp63 is a homologous to the baculovirus repeat ORFs (bro) and cp66 is homologous to ptp-2, whereas cp64 and cp65 are unique to CpGV at this position. The bro genes are a multi-gene family in baculoviruses and are thought to have an important function in gene transcription and genome replication (Kang et al, 1999;Zemskov et al, 2000;Bideshi et al, 2003). The bro gene loci have been repeatedly identified in different baculovirus genomes as sites of recombination events and genome heterogeneity (Ló pez-Ferber et al, 2003;Li et al, 2002Li et al, , 2005.…”

Comparative analysis of the genomes of Rachiplusia ou and Autographa californica multiple nucleopolyhedroviruses

“…Homologues of the bro ORF are present in multiple copies in other baculovirus genomes. The BRO proteins of BmNPV are associated with nucleoprotein complexes and bind nucleic acids (Zemskov et al, 2000). Kang et al (1999) were unable to produce a viable BmNPV bro-d single deletion mutant or a bro-a/bro-c double deletion mutant, suggesting that these ORFs may be essential for BmNPV replication in cell culture (Kang et al, 1999).…”

Genomic sequence analysis of a fast-killing isolate of Spodoptera frugiperda multiple nucleopolyhedrovirus

“…The bro genes constitute a multigene family that is widespread among insect DNA viruses (Bideshi et al, 2003). Although gene expression, nucleic acid binding activity, nucleosome association, and protein localization and trafficking have been characterized for some Bombyx mori NPV bro genes and proteins (Kang et al, 1999(Kang et al, , 2006Zemskov et al, 2000), the functions of NPV bro genes are unclear. SfMNPV-3AP2 bro shared the greatest degree of amino acid sequence identity with MacoNPV-B bro-d, MacoNPV-A bro-e and AgseNPV brob.…”