Characterization of the Spodoptera exigua baculovirus genome: structural and functional analysis of a 20 KB fragment.

Baculoviruses are attractive, biological alternatives to chemical control agents of insect pests. These viruses are natural agents influencing the size insect populations. They are often species- specific and some of them are highly efficacious, though their speed of action cannot meet that of chemical insecticides. In addition, chemical insecticides have a much wider range of target insects. With the advent of genetic engineering the host range of baculoviruses may be altered and their virulence possibly further improved. The principles underlying baculovirus virulence and specificity are not known. Therefore, the genetic organization and expression strategies of the Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV) have been investigated and described in this thesis. This virus is specific to the beet army worm Spodoptera exigua and highly virulent. The genetic information on the baculovirus type species Autographa califomica (Ac) MNPV, which has a broad range of hosts and is less virulent, served as a reference point.As a start the SeMNPV gene for the occlusion body protein, polyhedrin, was characterized. This protein is highly conserved among baculoviruses and comprises a major constituent of polyhedra. In contrast to other baculoviruses, the SeMNPV polyhedrin mRNAs were found to be non- polyadenylated (Chapter 2). The polyhedrin gene serves to set the origin of the physical map of the circular viral genome. By convention, the orientation of the map is set by the position of the other very late baculovirus gene, designated p10, relative to the polyhedrin gene. The presence of abundant amounts of mRNA allowed the identification of the SeMNPV p10 gene via a cDNA probe. Both the SeMNPV polyhedrin and p10 genes are transcribed late in infection and start in the canonical baculovirus late transcription initiation sequence, TAAG. In contrast to the polyhedrin mRNA the p10 mRNA was polyadenylated (Chapters 2 and 3, respectively). The predicted amino acid sequence of SeMNPV p10 showed only low sequence homology with previously characterized baculoviral p10 proteins, but its size and domain structure were very similar (Chapter 3).An equally small, but in contrast to p10, highly conserved gene encountered in the SeMNPV genome was the viral ubiquitin (v-ubi) gene. Ubiquitin is involved in protein degradation and is found associated with the non-occluded virions. The SeMNPV v-ubi gene was expressed late in infection and two baculovirus consensus late start sequences, TAAG, were used as transcriptional initiation sites. One was located only five nucleotides upstream of the translational start site, thereby providing the shortest untranslated leader reported to date for any baculovirus mRNA. The v-ubi gene had an unexpected genomic position in SeMNPV as compared to AcMNPV, suggesting differences in genome organization between baculoviruses (Chapter 4).In constrast to v-ubi, the location of the SeMNPV immediate early gene ie1 was more conserved as compared to AcMNPV. The ie1 gene encodes a multifunctional protein in the baculovirus life cycle and is involved in transcriptional transactivation and in DNA replication. The SeMNPV ie1 gene is larger than previously characterized baculovirus ie1 genes and displayed an unusual transcription pattern and may be lacking splicing (Chapter 5).Since the region between polyhedrin and p10 and encompassing ie1 was much shorter in SeMNPV (111 kb) than in AcMNPV (19 kb), it was of interest to-characterize the entire region and compare its organization with that of AcMNPV. Upstream of the SeMNPV polyhedrin gene an open reading frame (ORF) was identified, encoding the large subunit of ribonucleotide reductase (RR1). This gene was not identified before in baculoviruses. Its product may be involved in nucleic acid synthesis. As expected from its putative function to generate DNA precursors, this gene was expressed as an early gene, prior to DNA replication. S. littoralis MNPV, another baculovirus infecting a Spodoptera species, also appeared to contain this gene. Phylogenetic analysis suggested that both viruses had acquired the rr1 gene independently (Chapter 6). Ribonucleotide reductase is known to function as a virulence factor in other large DNA viruses. However, expression of the SeMNPV rr1 gene in AcMNPV recombinants, using different promoter constructs, gave no indication of altered virulence (data not shown).Finally, the genetic organization of a 20 kb segment of SeMNPV DNA representing 15% of the viral genome, encompassing the polyhedrin and p10 gene, and potentially encoding 20 ORFs, was closely examined (Chapter 7). A non-coding sequence of 900 nucleotides in length, consisting of four imperfect palindromes centered around a Bg/II site and three direct repeats, was identified in this 20 kb segment. This sequence showed structural homology to hr regions found in other baculoviruses and which are well-established transcriptional enhancers and putative origins of DNA replication. All ORFs except for RR1 had a counterpart in AcMNPV, the baculovirus type species. The homology of SeMNPV with AcMNPV and OpMNPV genes was usually low and, except for polyhedrin and ie1 did not span promoter regions. A high level of homology was observed only with v-ubi and polyhedrin. A most remarkable aspect of the genetic organization of this genome segment of SeMNPV is the extensive rearrangement in gene order that must have taken place during baculovirus history.The extremely high degree of conservation of the polyhedrin gene among baculoviruses questions its presumed value as a phylogenetic marker. Parsimony analysis of the large number of occlusion body protein sequences currently available, confirmed the notion that the polyhedrin gene is not suitable for this purpose (Chapter 7). The observation that the nucleotide sequence divergence of this gene had not reached the level of complete redundancy, suggested that it has been acquired by the baculoviruses rather recently, or alternatively, is frequently exchanged between different viruses. Several unresolved aspects of the baculovirus phylogeny, for instance the observed monophyletic grouping of the SNPVs, may better be investigated with other genes, e.g. ie1 (Chapter 5). Finally, the baculovirus gene order itself might be used as an independent phylogenetic marker.The initial characterization of SeMNPV DNA provides the basis for future genetic engineering of this highly specific baculovirus, in order to increase its efficacy as a bioinsecticide. The present data on the genetic structure and organization of the SeMNPV genome showed similarities to the better characterized AcMNPV. At the same time, major differences in gene order, coding capacity, gene sequence and expression were observed. The high divergence between SeMNPV and AcMNPV requires the complete sequencing of the SeMNPV genome to obtain a full view on the structural and genetic relatedness of baculoviruses. Ultimately, these analyses will be valuable in unravelling the issues related to the specificity and virulence in baculoviruses, two parameters of prime importance for their application as biocontrol agents