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| Effect of bro-d Deletion on the Viral Transcription and Cell Apoptosis in Silkworm (Bombyx mori) |
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Abstract bro (baculovirus repeated ORF)-d gene of Bombyx mori nucleopolyhedrovirus (BmNPV) is a kind of repeated open reading frame sequence in baculovirus and is widespread in a variety polyhedrosis virus genome which specially infects Lepidopteran insect. In order to understand the role of bro-d gene in viral infection and the relationship with host cell apoptosis, the bro-d gene was knocked out by Red recombination system to construct the bro-d-deficiency type virus (bro-d-ko-Bacmid), and was encapsulated by liposome and then was transfected into BmN cells, and wild type bacmid (Wt-Bacmid) was used as the control. The effects of bro-d gene on the replication and transcription level of virus genome and inhibition of apoptosis protein 2 (iap2) were detected by qRT-PCR, and the results showed that bro-d gene deletion would lead to a significant decrease on the replication level of viral genome and the transcription levels of viral early gene lef-3, late gene vp39 and very late gene p10 would be also significant decrease (P＜0.05). These results indicated that bro-d gene was a nonessential gene for BmNPV replication and it was involved in the regulatory process of viral gene in different transcription phase. In order to further explore the effects of bro-d gene deletion on virus-induced apoptosis of host cells, the bro-d-ko-Bacmid was co-transfected into BmN cells with the transient expression plasmid pIEx-1-bro-d which contained the IE promoter which only triggered in eukaryon. The results showed that the transcription level of iap2 was decreased significantly (P＜0.05) in the cells transfected with bro-d-ko-Bacmid and was increased after cotransfected with pIEx-1-bro-d. Furthermore, in spite of the viral replication and transcription levels of bro-d-ko-Bacmid were significantly lower than that of Wt-Bacmid (P＜0.05), and bro-d-ko-Bacmid would cause a significant decrease in cell viability compared with Wt-Bacmid (P＜0.05). These results implied that bro-d deficiency would strengthen the virus pathogenicity. Moreover, the B-cell lymphoma-2 (Bcl-2) protein and Bax (Bcl-2 assaciated X protein) expression levels in post-transfected cells and the key regulatory proteins in mitochondrial signaling death pathway were detected by Western blot. The results further confirmed that bro-d gene deletion would lead to significant decrease in the level of Bcl-2 protein (P＜0.05) and significant increase in the level of Bax protein (P＜0.05) compared with wild type virus, which resulted in a increased apoptotic level of host cells. These results indicated that bro-d gene could not only up-regulate the gene expression levels of different phages, but also could down-regulate the apoptotic level of host cells through mitochondrial pathway by regulating the expression of iap2. These results provide a new basis for understanding the function of bro-d gene in virus infection cycle, and a new way to transform the baculovirus expression system by prolonging the host cell life-span, and improve the expression amount of target proteins.
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Received: 24 December 2015
Published: 20 May 2016
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