Characterization of a nucleopolyhedrovirus with a deletion of the baculovirus core gene Bm67.
Open reading frame (ORF) 67 (Bm67) of the Bombyx mori nucleopolyhedrovirus (BmNPV) is a highly conserved gene that is found in all completely sequenced baculoviruses; its function is unknown. In the present study, a Bm67-knockout virus was generated for studying the role of Bm67 in the BmNPV infection cycle. Furthermore, a Bm67-repair bacmid was constructed by transposing the Bm67 native promoter-promoted Bm67 ORF into the polyhedrin locus of the Bm67-knockout bacmid. After these recombinant bacmids were transfected into BmN cells, the Bm67-knockout bacmid caused defects in the production of infectious budded viruses. However, the Bm67-repair bacmid could rescue the defect, and budded virus titres reached wild-type levels. Quantitative real-time PCR analysis indicated that Bm67 is required for normal levels of DNA synthesis or for the stability of nascent viral DNA at the early stage. Electron microscopic analysis revealed that the formation of normal-appearing nucleocapsids is reduced in Bm67-knockout bacmid-transfected cells, and nucleocapsids are rarely found in the cytoplasm. The presence of 'enveloped' nucleocapsids at the nucleoplasm bilayer indicated that they are enveloped abnormally. These results indicated that Bm67 is required for the production of infectious budded viruses and for assembly of envelope and nucleocapsids.
Ge
Jun-Qing
JQ
Institute of Insect Science, Zhejiang University, Kaixuan Road 268, Hangzhou 310029, PR China.
Yang
Zhang-Nv
ZN
Institute of Insect Science, Zhejiang University, Kaixuan Road 268, Hangzhou 310029, PR China.
Tang
Xu-Dong
XD
Institute of Insect Science, Zhejiang University, Kaixuan Road 268, Hangzhou 310029, PR China.
Xu
Hai-Jun
HJ
Institute of Insect Science, Zhejiang University, Kaixuan Road 268, Hangzhou 310029, PR China.
Hong
Jian
J
Institute of Insect Science, Zhejiang University, Kaixuan Road 268, Hangzhou 310029, PR China.
Chen
Jian-Guo
JG
College of Life Sciences, Peking University, Beijing 100087, PR China.
Zhang
Chuan-Xi
CX
Institute of Insect Science, Zhejiang University, Kaixuan Road 268, Hangzhou 310029, PR China.
D000818:Animals; D012831:Bombyx / Q000821:virology*; D002478:Cells, Cultured; D004279:DNA, Viral / Q000096:biosynthesis* / Q000235:genetics; D017353:Gene Deletion; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D019251:Nucleocapsid / Q000378:metabolism*; D017924:Nucleopolyhedroviruses / Q000145:classification* / Q000235:genetics / Q000472:pathogenicity; D000077183:Occlusion Body Matrix Proteins; D016366:Open Reading Frames / Q000235:genetics; D016133:Polymerase Chain Reaction; D011995:Recombination, Genetic; D014758:Viral Core Proteins / Q000235:genetics* / Q000378:metabolism; D015678:Viral Structural Proteins / Q000235:genetics / Q000378:metabolism; D019065:Virus Assembly
D004279:DNA, Viral; D000077183:Occlusion Body Matrix Proteins; D014758:Viral Core Proteins; D015678:Viral Structural Proteins; C485184:enhanced green fluorescent protein; C009829:polyhedrin protein, Nucleopolyhedrovirus; D049452:Green Fluorescent Proteins