title: A DNA Binding Protein Is Required for Viral Replication and Transcription in Bombyx mori Nucleopolyhedrovirus.
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.
authors.affiliation: College of life sciences, Zhejiang Sci-Tech University, Zhejiang Province, Hangzhou 310018, China.|Zhejiang Provincial Key Laboratory of Silkworm Bioreactor and Biomedicine, Zhejiang Province, Hangzhou 310018, China.
authors.forename: Cui
authors.initials: C
authors.lastname: Zhao
authors.affiliation: College of life sciences, Zhejiang Sci-Tech University, Zhejiang Province, Hangzhou 310018, China.|Zhejiang Provincial Key Laboratory of Silkworm Bioreactor and Biomedicine, Zhejiang Province, Hangzhou 310018, China.
authors.forename: Chen
authors.initials: C
authors.lastname: Zhang
authors.affiliation: College of life sciences, Zhejiang Sci-Tech University, Zhejiang Province, Hangzhou 310018, China.|Zhejiang Provincial Key Laboratory of Silkworm Bioreactor and Biomedicine, Zhejiang Province, Hangzhou 310018, China.
authors.forename: Bin
authors.initials: B
authors.lastname: Chen
authors.affiliation: College of life sciences, Zhejiang Sci-Tech University, Zhejiang Province, Hangzhou 310018, China.|Zhejiang Provincial Key Laboratory of Silkworm Bioreactor and Biomedicine, Zhejiang Province, Hangzhou 310018, China.
authors.forename: Yanghui
authors.initials: Y
authors.lastname: Shi
authors.affiliation: College of life sciences, Zhejiang Sci-Tech University, Zhejiang Province, Hangzhou 310018, China.|Zhejiang Provincial Key Laboratory of Silkworm Bioreactor and Biomedicine, Zhejiang Province, Hangzhou 310018, China.
authors.forename: Yanping
authors.initials: Y
authors.lastname: Quan
authors.affiliation: College of life sciences, Zhejiang Sci-Tech University, Zhejiang Province, Hangzhou 310018, China.|Zhejiang Provincial Key Laboratory of Silkworm Bioreactor and Biomedicine, Zhejiang Province, Hangzhou 310018, China.
authors.forename: Zuoming
authors.initials: Z
authors.lastname: Nie
authors.affiliation: College of life sciences, Zhejiang Sci-Tech University, Zhejiang Province, Hangzhou 310018, China.|Zhejiang Provincial Key Laboratory of Silkworm Bioreactor and Biomedicine, Zhejiang Province, Hangzhou 310018, China.
authors.forename: Yaozhou
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: College of life sciences, Zhejiang Sci-Tech University, Zhejiang Province, Hangzhou 310018, China.|Zhejiang Provincial Key Laboratory of Silkworm Bioreactor and Biomedicine, Zhejiang Province, Hangzhou 310018, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Yu
mesh_terms: D000818:Animals; D012831:Bombyx / Q000648:ultrastructure / Q000821:virology*; D004261:DNA Replication / Q000235:genetics / Q000502:physiology; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000502:physiology*; D055786:Gene Knockout Techniques; D005814:Genes, Viral; D046529:Microscopy, Electron, Transmission; D017924:Nucleopolyhedroviruses / Q000235:genetics* / Q000502:physiology* / Q000648:ultrastructure; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000235:genetics* / Q000502:physiology*; D014779:Virus Replication / Q000235:genetics / Q000502:physiology
chemical_list: D004268:DNA-Binding Proteins; D014764:Viral Proteins