Analysis Of ORF5, ORF28 And ORF29 Of Helicoverpa Armigera Single-nucleocapsid Nucleopolyhedrovirus

The family Baculoviridae is a group of enveloped double-stranded DNA viruses that are highlyselective pathogens in arthropods, mainly in insects of the order Lepidoptera. Baculoviruses have beenextensively studied and developed as alternatives to chemical insecticides in agriculture and forestry, inboth natural and engineered forms, because they possess several properties as biological pest controlagents, including higher efficacy in controlling insect pests and a less negative impact on theenvironment and nontarget species than the chemical pesticides. To date, the genomes of 26baculoviruses including 16 lepidopteran nucleopolyhedroviruses and 7 granuloviruses have beensequenced. The baculovirus expression vector system, recombinant baculovirus as bioinsecticide,baculovirus as gene transfer vector and baculovirus surface display have been developed to explore itsusefulness for human being. The cotton bollworm, Helicoverpa armigera, is a worldwide pest causingserious damage to a variety of agricultural crops such as cotton, tobacco, pepper, tomato, maize,sorghum and soybean. A baculovirus, H. armigera single nucleocapsid nucleopolyhedrovirus(HearSNPV), is a selective, highly infectious pathogen to this insect and has been extensively used forthe control of this pest since the first isolation of this pathogen in Hubei province of China. So far, greatprogress has been made on characterization of several genes from completely sequenced HearSNPV Cland G4 isolates. This paper presents the characterization of open reading frame (ORF) 5, ORF28 andORF29 genes from HearSNPV C1 isolate, and the biological comparison between C1 and G4 isolates.The results are shown as follows.1. The ORF5 is 192 nucleotides long, potentially encoding a protein of 63 amino acids (aa) with a predicted molecular weight of 7.4 kDa. A baculovirus consensus early transcriptional initiation motif CAGT is found at 14nts upstream of the ATG. Two GATA boxes are observed at 210 and 227 nts upstream of the ATG The product of ORF5 has homologues of MacoNPV-90/2 ORF8, MacoNPV-96B ORF8, AcMNPV ORF152, SpltMNPV ORF5 and RoMNPV ORF147 with aa identity of 53%, 58%, 50%, 54% and 47%, respectively. The GST-ORF5 fusion protein has been expressed in Escherichia coli BL21 (DE3) LysS cells and anti-GST-ORF5 antiserum has been prepared. Characterization of ORF5product needs to be carried out further.2. The ORF28 gene is 252 nucleotides long, potentially encoding a protein of 83 aa with a predicted molecular weight of 9.2 kDa. Two TATA boxes are found at 175 and 228 nts upstream of the ATG codon. Two baculovirus consensus late transcriptional initiation motif, A/T/GTAAG are also observed at 30 and 107 nts upstream of the ATG. The product of ORF28 is a ubiquitin protein with several putative phosphorylation sites of Thr9, Thr25, Thr55 and Ser78. This protein includes an N-terminal ubiquitin core sequence with 76 aa and 7 aa C-terminal peptide. Great differences are found in the regions of aa 15-32 and aa 51-57 among ORF28 product and its homologues in other baculoviruses and eukaryote ubiquitin protein. The ORF28 ubiquitin protein is detected in infected cells at 36 h post-infection, by Western blot using a polyclonal anti-GST-ORF28 antiserum absorbed by uninfected-cell total proteins. The size of this product is about 14 kDa, which is larger than the presumed size of 9.4 kDa, suggesting it is modified posttranslationally. By localization analysis, this protein is attached to the BV particle. An unknown protein encoded by a host gene is found to interact with ORF28 ubiquitin protein, by GST fusion protein pulldown technique. The interaction needs to be confirmed by other methods, and the sequence of this unknown protein needs to be determined further.3. The ORF29 is a gene specific for HearSNPV, 507 nucleotides long, potentially encoding a protein of 168 aa with a predicted molecular weight of 20.4 kDa. Two TATA boxes are found at 185 and 46 nts upstream of the ATG codon. A baculovirus consensus early transcriptional start motif, CAGT is also observed at 15 nts up...