Genomics and genetic engineering of Helicoverpa armigera nucleopolyhedrovirus

The single nucleocapsid nucleopolyhedrovirus (SNPV) of the bollworm Helicoverpa armigera has been extensively used to control this insect around the world, especially in China. However, in order to compete with chemical insecticides - mainly for speed of action -novel approaches are sought to improve the efficacy of HaSNPV either by selection of superior natural variants or by genetic engineering. Prior to the development of improved HaSNPV by genetic engineering, understanding of the structure and expression strategy of the HaSNPV genome is required. This thesis describes studies aimed at the unraveling of the genetic properties of the HaSNPV genome. Furthermore, this research can provide molecular information on the taxonomic status of baculovirus morphotypes, i.e . single nucleocapsid NPVs (SNPV) versus multiple nucleocapsid NPVs (MNPV), and ultimately on the phylogenetic relationship among baculoviruses in general.The polyhedrin gene, a highly conserved gene among baculoviruses and encoding the major structural protein of viral polyhedra, was localized on the HaSNPV genome and then characterized (Chapter 2). This indicated that the HaSNPV polyhedrin has a high degree of sequence similarity to that of H. zea SNPV. From this preliminary analysis is appeared that SNPVs are not a separate group from the MNPVs. The position of the HaSNPV polyhedrin gene was chosen as the zero point of the circular physical map of the viral genome (Chapters 5 and 6). The polyhedrin promoter, with a typical baculovirus late transcription initiation motif, was used to drive the expression of a green fluorescent protein (GFP) and a toxin in recombinant HaSNPV (Chapter 8).In the larval stages the enzyme ecdysteroid UDP-glucosyltransferase (EGT) catalyzes the conjugation of ecdysteroid with sugars and is involved in the prevention of molting and pupation. Baculoviruses generally encode such an enzyme, resulting in the prevention of molting of infected larvae and enhanced polyhedra production. The HaSNPV egt gene was located on the Hin d-D fragment and characterized (Chapter 3). Phylogenetic analysis of this gene confirmed that HaSNPV belongs to the Group II NPVs. To further analyze the relationship between HaSNPV and other baculoviruses, a late expression factor 2' gene ( lef- 2) was identified and characterized (Chapter 4). This gene is essential for viral DNA replication and most likely functions as a DNA primase processivity factor. This is the first lef -2 gene characterized in any SNPV to date. Even though lef- 2, an essential gene, and egt , an auxiliary gene, most likely have been under different pressure in their evolutionary past, the phylogenetic tree of baculovirus LEF-2 appeared to be comparable in form to that of EGT. The positive correlation of the genomic location of the lef-2 genes relative to polyhedrin/granulin genes and the clade structure of the gene trees ( lef-2 , egt ) suggest that genome organization and gene phylogeny represent independent parameters to study the evolutionary history of baculoviruses.In order to study the genome organization and phylogenetic status of HaSNPV, a plasmid library of its 130.1 kb-long DNA genome was made and a detailed physical map of the viral DNA was constructed (Chapter 5). From about 45 kb of dispersed sequence data generated from the plasmid library, fifty-three putative open reading frames (ORFs) with homology to ORFs of other baculoviruses were identified and their locations on the genome of HaSNPV were determined. The basic gene content of HaSNPV appeared to be quite similar to that of AcMNPV, BmNPV, and OpMNPV (group I NPVs). However, the arrangement of the ORFs along the HaSNPV genome differed significantly from that of the Group I NPVs, which all have a highly collinear genome, or that of the granulovirus XcGV. In contrast, the genomes of HaSNPV and SeMNPV (Group II NPVs) are highly collinear, both in gene content and organization. This close relatedness between an MNPV and an SNPV is supported by the phylogeny of selected genes (Chapters 2 and 3) of these two viruses and suggests that the NPV morphotype (S or M) has only a taxonomic but not a phylogenetic meaning. Homologous regions ( hr s), a common feature of baculovirus MNPV genomes, were also located dispersed on the HaSNPV genome suggesting that their presence in common in all NPVs.So far, only MNPV and GV genomes have been sequenced to completion, but no SNPV genome to date. Therefore the entire HaSNPV genome sequence was determined (Chapter 7). The circular, double-stranded DNA genome contains 131,403 bp and has a G+C content of 39.1 %, the lowest value among baculoviruses to date. Of 135 potential ORFs predicted from the sequence, 115 have a homologue in other baculoviruses; twenty are unique to HaSNPV and are subject to further investigation. Upon comparison with the available genomic sequences, sixty-five ORFs were found present in all baculoviruses, and hence they are considered as 'core' baculovirus genes. The HaSNPV genome lacks a homologue of the major budded virus (BV) glycoprotein gene gp64 of group I NPVs. Instead, a functional homologue (Ha133) of gp64 was identified after comparison with SeMNPV. The mean overall amino acid identity of the HaSNPV ORFs was the highest with SeMNPV and LdMNPV homologues. This is in accordance with their common genome organization and confirmed, that HaSNPV together with SeMNPV and LdMNPV cluster into Group II NPVs, while AcMNPV, BmNPV and OpMNPV belong to the Group I NPVs. In this analysis GV behaved like a separate group. The clade structure based on selected genes ( lef-2 and egt ) is further strongly support by genome trees based on all conserved ORFs together and based on gene content as well as gene order on the genomes compared.HaSNPV and HzSNPV share many common biological features such as the same heliothine host range (Chapter 1). Sequence analysis of the complete HzSNPV genome revealed that HaSNPV and HzSNPV have a high degree of ORF identity, which is in line with the view that they are two different isolates of the same virus species (Chapters 6 and 7). The HzSNPV genome potentially encodes 139 potential ORFs of which 135 have homologous in HaSNPV. Four ORFs are unique to HzSNPV. However, these unique ORFs are small, are always found adjacent to hr regions and their functionality remains to be determined. Alignment of the genome sequences indicated that overall ORFs of HzSNPV have a high degree of identity with the homologues of HaSNPV genome on nucleotide (99%) and amino acid (98%) level. The 65 baculovirus core genes among these two viruses have the lowest nucleotide substitution rate, but the hr s showed the highest variation. Two 'baculovirus repeat orfs' ( bro ) genes in these two viruses have the highest sequence divergence and might have a different evolutionary history.Deletion of egt from the baculoviral genome has been shown to increase the speed of kill of the virus and hence to reduce the crop damage by infected insects. This approach, along with the insertion of a scorpion neurotoxin gene, was used to generate recombinant HaSNPV with potentially improved insecticidal activity. The egt gene was deleted from the genome and replaced by the GFP and / or by an insect-specific toxin gene, AaIT (Chapter 8). Bioassay data indicated a significant reduction in the time (LT50) required for each of the HaSNPV recombinants to kill second instar H. armigera larvae. The LT 50 of the egt deletion recombinants was about 27% shorter than that of wild type HaSNPV. The largest reduction in LT 50 (32%) was observed when the egt gene was replaced by the scorpion neurotoxin AaIT gene.The genetic and genomic analysis presented in this thesis shows that HaSNPV and HzSNPV are different variants of the same virus species. Alignment of the known baculovirus genome sequences did not clearly show the molecular basis for the baculovirus S and M NPV morphotype. Phylogenic analysis of genes and of genome organization, such as gene content and gene order, confirmed that baculoviruses can be separated into Group I and II NPV and into a GV group. Based on the investigation of the HaSNPV genome, HaSNPV recombinants with enhanced insecticidal properties were Successfully constructed providing alternative agents to bollworm control in China and elsewhere in the world.