Functional Analysis Of A Unique Gene (Ha122) And The Envelope Fusion Protein Gene (Ha133) In Helicoverpa Armigera Single Nucleopolyhedrovirus (HaSNPV)

There are four chapters in this thesis, which consists in functional analysis of two Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus (HaSNPV) genes (Hal22 and Hal33).A general introduction of the research on baculovirus is present in Chapter one, including the overview of baculovirus, taxonomy and structure of baculovirus and the life cycle of the phenotype virus (Autographa californica multiple-nucleocapsid nucleopolyhedrovirus, AcMNPV) of baculovirus. We also give a brief introduction on the genomics of HaSNPV in this chapter. The overview of this thesis is also included in.Chapter two is a report on genetic organization of the Hindlll-l fragment of HaSNPV. The fragment that is 7501 bp long and located at map units (mu) 12.3-17.9 of the viral genome was sequenced. The fragment contained ten open reading frames (ORFs) representing homologues of the AcMNPV orflll and orf63, a lef-2 gene, a p24 gene, a gp!6 gene, a pep gene, a 38.7kd gene, a lef-1 gene and two unique genes named as or/591 (Hal22) and orf435(Ha!25). Sequence Alignment revealed that baculoviral P24, PEP and LEF-1 have some conserved motifs and gene structures as those observed in their homologues from other baculoviruses. The genomic arrangement of the ORFs in the HaSNPV Hindlll-l fragment is very different from the arrangement of their homologues in the genome of Autographa californica multiple nucleocapsid (M)NPV.HaSNPV contains 20 open reading frames so far unique among baculoviruses. In Chapter three, we investigated the function of HaSNPV open reading frame 122 (Hal22),which is also present in the closely related H. zea SNPV. Hal22 was transcribed as a polyadenylated transcript from 8 h post infection of H. armigera insect cells onwards. 5'RACE analysis indicated that Ha 122 transcripts start predominantly in the consensus major late transcription initiation motif RTAAG around 47nt upstream of the putative translation start codon with a minor start at position-89. Using 3'RACE the transcription stop site mapped 27nt downstream of the putative translation stop codon. By western analysis using a chicken-derived polyclonal antibody the product of Hal22 was found in infected cells as a 21kDa protein, close to the theoretical size of 21.6kDa. The Ha 122 protein, when fused to GFP was observed in the nuclei of H. armigera cells, but only in conjunction with wild type HaSNPV infection. The 21kDa protein was specifically located in the nucleocapsid of occlusion-derived and not in that of budded virions. The available data suggest that Hal22 is a functional open reading frame of HaSNPV and that the 21kDa protein is a novel specific component of baculovirus occlusion body-derived virions.Chapter four reported the functional analysis of HaSNPV envelope fusion protein gene (Hal33) gene. This gene is functional homologous gene to Ldl30 and Se8. Two specific antibodies against Fl (aa 164- 677) and against F2 (aa 1- 159) were raised in chicken. In this report, we found that the Hal33 encodes the major envelope fusion protein (EFP) Fl(62kDa) and F2(20kDa) in HaSNPV BVs. RT-PCR result demonstrated that Hal33 gene is transcribed relatively later than AcMNPV gp64 gene. HaSNPV fusion protein F2 can be detected from 24 hr p.i. to 72 hr p.i., suggesting that HaSNPV EFP is translated in the later phase in the infection circle and the protein was furin-cleaved immediately after translation. Results of Western analysis based on HaSNPV BVs indicate that the envelope fusion protein is cleaved into two molecules associated by disulfide bridges. Furthermore, there is no oligomerized fusion protein present in HaSNPV BVs, which shares the same situation as Se8 but it is different from GP64. Tunicamycin assay demonstrated that only F2 part is N-glycosylated. Transfection of the gp64-mi\\ and reintroducing Hal33 gene BACmid DNA into Sf9 cells shown that Hal33 can rescue the gp64 null BACmid in Sf9 cells. In the wild type HaSNPV BVs, no detectable multimer was found. However, in the pseudotyping baculovirus BVs, three oligomer bands were foun...