Bombyx mori nucleopolyhedrovirus ORF56 encodes an occlusion-derived virus protein and is not essential for budded virus production

Abstract

Bombyx mori nucleopolyhedrovirus ORF56 (Bm56) is a baculovirus core gene that is highly conserved in all baculoviruses that have had their genomes sequenced to date. Its transcripts in BmNPV-infected cells could be detected from 12 h post-infection (p.i.) and the encoded protein could be detected at 16 h p.i. by using a polyclonal antibody against glutathione S-transferase-Bm56 fusion protein. Western blot analysis showed that Bm56 is a structural component of the occlusion-derived virus nucleocapsid. Subsequent confocal microscopy revealed that Bm56 was distributed in the outer nuclear membrane and the intranuclear region of infected cells. To investigate the role of Bm56 in virus replication, a Bm56-knockout bacmid of BmNPV was constructed via homologous recombination in Escherichia coli. The Bm56 deletion had no effect on budded virus (BV) production in cultured cells; however, the deletion affected occlusion-body morphogenesis. A larval bioassay demonstrated that the Bm56 deletion did not reduce infectivity, whereas it resulted in a 50 % lethal time that was 16-18 h longer than that of the wild-type bacmid at every dose used in this study. These results indicate that Bm56 facilitates efficient virus production in vivo; however, it is not essential for BV production in vitro.

Analysis of the Autographa californica Multiple Nucleopolyhedrovirus Overlapping Gene Pair lef3 and ac68 Reveals that AC68 Is a Per Os Infectivity Factor and that LEF3 Is Critical, but Not Essential, for Virus Replication

“…It was previously reported that deletion of ac68 did not impact the production of OBs, whereas deletion of the BmNPV homolog bm56 resulted in defective OB morphogenesis (25,52). All of our ac68 deletion constructs produced OBs with a normal appearance, as determined by light microscopy, similar to previously reported results.…”

“…All ac68 mutant viruses, ac68KO1, -2, -3, and -4, produced similar levels of BV as lef3-ac68Rep and the WT AcBac. This suggests that ac68 is not required for the normal production of BV which has been previously reported (25,52). BV titers were, however, significantly reduced, as expected for both lef3-ac68 2ϫKO and lef3KO, showing that LEF3 is critical for virus replication.…”

“…Adjacent to ac68 and bm56 is the gene encoding LEF3, the single-stranded DNA binding (SSB) protein. The deletions of ac68 and bm56 in the previous studies involved the lef3 or ac69 promoter, and therefore expression of these genes could have been impacted and affected the observed phenotype; however, this was not thoroughly investigated (24,25,52).lef3 encodes a late-gene expression factor found in all the …”

“…Bioassays of BmNPV bm56KO virus via budded virus (BV) injection showed no difference in LD 50 values, but similar to the AcMNPV ac68KO, there was an extended LT 50 . The AcMNPV ac68KO produced normal OBs, but the bm56KO virus produced OB-like structures that did not contain any embedded ODV (52). The functional analyses of ac68 and bm56, closely related homologues from group I alphabaculoviruses, are therefore contradictory in terms of when the genes are expressed, the protein sizes, and the effects on OB development.…”

Enhanced antitumor activity by the combination of dasatinib and combretastatin A-4 in vitro and in vivo

“…In addition, we observed that the combination of dasatinib and CA-4 enhanced cytochrome c release from mitochondria into the cytosol in HO-8910 cells. IMS protein release can result from the mitochondrial outer membrane permeabilization (MOMP), which is thought to be regulated by proteins of the Bcl-2 family (38). The Bcl-2 protein family includes proapoptotic members, such as Bax, Bad and Bcl-Xs, and antiapoptotic members, such as Bcl-2, Bcl-XL and Mcl-1.…”