A DNA Binding Protein Is Required for Viral Replication and Transcription in Bombyx mori Nucleopolyhedrovirus.

Chen Zhang,Cui Zhao,Erjun Ling,Yaozhou Zhang,Bin Chen,Yanghui Shi,Yanping Quan,Zuoming Nie,Wei Yu

doi:10.1371/journal.pone.0159149

Abstract

A DNA-binding protein (DBP) [GenBank accession number: M63416] of Bombyx mori nuclear polyhedrosis virus (BmNPV) has been reported to be a regulatory factor in BmNPV, but its detailed functions remain unknown. In order to study the regulatory mechanism of DBP on viral proliferation, genome replication, and gene transcription, a BmNPV dbp gene knockout virus dbp-ko-Bacmid was generated by the means of Red recombination system. In addition, dbp-repaired virus dbp-re-Bacmid was constructed by the means of the Bac to Bac system. Then, the Bacmids were transfected into BmN cells. The results of this viral titer experiment revealed that the TCID50 of the dbp-ko-Bacmid was 0; however, the dbp-re-Bacmid was similar to the wtBacmid (p>0.05), indicating that the dbp-deficient would lead to failure in the assembly of virus particles. In the next step, Real-Time PCR was used to analyze the transcriptional phases of dbp gene in BmN cells, which had been infected with BmNPV. The results of the latter experiment revealed that the transcript of dbp gene was first detected at 3 h post-infection. Furthermore, the replication level of virus genome and the transcriptional level of virus early, late, and very late genes in BmN cells, which had been transfected with 3 kinds of Bacmids, were analyzed by Real-Time PCR. The demonstrating that the replication level of genome was lower than that of wtBacmid and dbp-re-Bacmid (p<0.01). The transcriptional level of dbp-ko-Bacmid early gene lef-3, ie-1, dnapol, late gene vp39 and very late gene p10 were statistically significantly lower than dbp-re-Bacmid and wtBacmid (p<0.01). The results presented are based on Western blot analysis, which indicated that the lack of dbp gene would lead to low expressions of lef3, vp39, and p10. In conclusion, dbp was not only essential for early viral replication, but also a viral gene that has a significant impact on transcription and expression during all periods of baculovirus life cycle.

Highlights

The Bombyx mori nuclear polyhedrosis virus (BmNPV) is a typical member of the insect baculoviruses, a family of double-stranded DNA viruses with large circular genomes
Most open reading frames (ORFs) of the BmNPV are over 90% homologous with Autographa californica multiple NPV (AcMNPV), but subtle changes often result in significant differences in morphology, infection dynamics, and host range [4,5,6]
The dbp is a conserved gene that is widely present in the baculovirus genome and is considered to be a core gene of the virus

Summary

Introduction

The Bombyx mori nuclear polyhedrosis virus (BmNPV) is a typical member of the insect baculoviruses, a family of double-stranded DNA (dsDNA) viruses with large circular genomes. The potential protein coding regions, gene structure, viral DNA replication initiation site, and the existence of regulatory elements of BmNPV can be predicted by aligning with those of AcMNPV. There are 65 amino acids that the 198 bp BmNPV DBP ORF; the predicted protein molecular weight is 8.08 KD and the isoelectric point is 12.46. The sequence of amino acids appears 3 times in the basic protein DBP of BmNPV, while appearing twice in the AcMNPV and OpNPV [10, 11]. The basic protein DBP is considered to be involved in the neutralization of viral DNA by arginine residues, and plays an important role in depolymerizing the virus through the phosphorylation of serine and threonine during the infection process [12]

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Results

Conclusion