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| Replication and Transcription of Bombyx mori Nucleopolyhedrovirus |
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Abstract Bombyx mori Nucleopolyhedrovirus (BmNPV) 39K gene which encodes a phosphoprotein associated with viral gene expression, exists in genomes of all Lepidopteran Baculoviruses. The results of existing studies show that the 39K gene is related to the regulation of viral gene expression, but the specific regulation effects of BmNPV 39K gene on viral replication and transcription is still unknown. Red recombination technique and Bac-to-Bac system were respectively utilized to delete and recover 39K gene to construct a knockout bacmid (39K-ko-Bacmid) and 39K- rescued bacmid (39K-re-Bacmid). Then the constructed bacmids DNA were transfected into BmN cells. The results showed that both types of viruses could yield infectious virion in cells, indicating that 39K gene was not essential for the replication of BmNPV, however, the deletion of 39K gene could reduce virus titer significantly. In late transfection, the replication level of the viral genome was related to infectious budded virus (BV) which was generated by virus. Possibly, the reduced BV led to decreased replication capacity of deficient virus in late transfection. Furthermore, the resultes of qPCR showed that the deletion of 39K gene had no effect on virus early replication but would decrease the transcription level of viral late genes and caused extremely significant reduction of viral gene transcription level (P＜0.01) in different phases. In order to explore the role of 39K gene in the expression regulation of polyhedral promoter, we constructed a recombinant bacmid with BmNPV polyhedral promoter drived-firefly luciferase and Bombyx mori cytoplasm action A3 promoter drived-renilla luciferase. Quantitative detection of luciferase activity proved that 39K gene had significant regulation effect on polyhedral gene promoter, the deletion could enhance the activity of polyhedral gene promoter. In conclusion, 39K gene was not essential for the replication of BmNPV, but the deletion could reduce virus gene transcription level in different phases and enhance the activity of polyhedral promoter. We speculated that the 39K protein which may act as a repressor protein bound to polyhedron gene promoter, so that affected the polyhedron gene promoter combining with other transcription factors, and then resulted in decreasing of polyhedron gene promoter activity. This study will lay the foundation for further investigating the effect of BmNPV 39K gene on viral genome replication and transcription, also provides information on the efficient transcription mechanism of Bombyx mori baculovirus expression system.
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Received: 06 September 2015
Published: 05 February 2016
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