Molecular genetics of the Spodoptera exigua multicapsid nucleopolyhedrovirus genome.

Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV) is an attractive biological control agent for the beet army worm S. exigua . This baculovirus has a narrow host range and is relatively, compared to other baculoviruses, virulent for beet army worm larvae. The molecular principles that specify the host range and virulence of SeMNPV are unknown. This thesis describes studies aimed at the unravelling of the molecular genetics of this baculovirus and the key steps in the infection and genome replication process.As a first step SeMNPV was multiplied in an established cell line of S. exigua to obtain a better understanding of the replication process. Polyhedra derived from cell culture were unable to infect S. exigua larvae, although the hemolymph isolated from these larvae did contain budded virus able to infect insect cell lines (Chapter 2). This suggested the generation of a genetic defect in the SeMNPV genome during replication and/or maintenance in cell culture. A partial plasmid library and complete cosmid library were made to construct a physical map of the SeMNPV genome and locate the genetic defect(s). The position of the polyhedrin gene and the transcriptional direction of the p10 gene allowed the pin-pointing of the zero point and orientation of the circular SeMNPV genome. A large deletion (20-25 kilobase pairs), turned out to be the genetic defect, arising upon limited passaging of the virus in cell culture. This deletion was located between map unit 12.9 and 31.3 in the SeMNPV genome. The occurrence of this deletion implied that the construction of SeMNPV recombinant viruses still virulent in vivo cannot be achieved via conventional techniques, i.e. homologous recombination in cell culture (Chapter 2). Alternative recombinationstrategies involving yeast artificial chromosomes and in vivo cloning were considered and partially tested (Chapter 7).In Chapters 3 and 4 the identification and characterization of cis -acting elements in SeMNPV DNA replication are described. Transient DNA replication assays, sequence analyses and hybridization experiments identified one non- hr ( hr homologous region) and six hr origins of DNA replication. SeMNPV hr s contained one ( hr 4) up to nine ( hr 1) repeated, near-identical 68-bp long palindromes. The SeMNPV hr s, located in non-coding regions, were found dispersed in the viral genome as observed in the genomes of two other baculoviruses, Autographa californica MNPV and Orgyia pseudotsugata MNPV. Transient DNA replication assays in AcMNPV-infected insect cells revealed no replication of SeMNPV- hr s and, in SeMNPV-infected insect cells no replication of AcMNPV- hr s could be observed, suggesting that these elements display specificity (Chapter 3). In the SeMNPV- Xba I library one additional genomic fragment unrelated to hr s and reminiscent of AcMNPV and OpMNPV non- hr origins of DNA replication that underwent SeMNPV dependent DNA replication was identified. By deletion analysis the core of this non- hr origin was mapped within a 800 bp region of non-coding sequence. This sequence contained also several motifs such as multiple palindromes, direct repeats, putative transcriptional factor binding sites and multiple polyadenylation signals, characteristic for baculovirus non- hr and other eukaryotic origins of DNA replication. In contrast to the hr s, the SeMNPV non- hr origin could be replicated by the replication machinery of the heterologous AcMNPV (Chapter 4).The putative helicase is the most intriguing trans -acting DNA replication factor of baculoviruses, since it may be involved in both DNA replication and host range determination. An open reading frame (ORF) potentially encoding a polypeptide of 143 kDa (p143) with considerable amino acid identity to the putative helicases of AcMNPV, BmNPV and OpMNPV was identified in SeMNPV. Sequence alignment of the SeMNPV p143 indicated that it is somewhat diverged from its AcMNPV, BmNPV and OpMNPV homologs. Whether the protein is also involved in the host range of SeMNPV determination remained unsolved. The ORF is expressed as a 4 kb transcript between 4 and 24 h p.i., starting from an unusual transcriptional initiation site present eleven nucleotides upstream of the translational start. Transient plasmid dependent DNA replication assays showed that not only helicase plays a crucial role in SeMNPV and AcMNPV replication specificity, but also one or more of the other previously mentioned essential trans -acting DNA replication factors. Apparently the interaction between the origins of DNA replication and/or the assembly of the replisome is a highly virus specific process (Chapter 5).Complete sequence and transcriptional analysis of the 11.3 kb SeMNPV- Xbal-C fragment containing the p143 gene revealed twelve ORFs that all showed high amino acid identity to AcMNPV and OpMNPV homologs. The genetic organization of the SeMNPV- Xba I-C fragment was identical to the AcMNPV and OpMNPV helicase region, although in an antigenomic orientation. In line with recent observations in herpes- and poxvirus genomes, which contain conserved central parts of their genomes and more diverged termini, it is hypothesized that baculoviruses genomes could also contain a highly conserved gene block centered around the p143 gene and a more diverged region at the polyhedrin - p10 loci. This hypothesis is further supported by partial sequence and hybridization data from other baculovirus genomes. The organization of the less conserved polyhedrin-p10 region could be a marker for the genetic relatedness of baculoviruses (Chapter 6). The state of the art on the sequence analysis of the complete SeMNPV genome is described in Chapter 7.The availability of a physical map, the insght in the genetic organization of the SeMNPV genome and the occurence of the spontaneous deletion mutants in cell culture prompted the development of alternative recombination strategies to bypass the use of the insect cell lines (Chapter 7). To this end a recombination strategy using yeast genetics was employed. Deletion of the yeast autonomous replicating sequences prior to the application of the recombinant baculoviruses in the field is recommended and can be achieved for SeMNPV using a direct in vivo recombination and selection protocol. The strategy proposed is based on the occurrence of homologous recombination of baculoviruses in the insect (Chapter 7).The research on SeMNPV described in this thesis, has created a good starting point to study the molecular basis of virulence and host range. The development of the recombination system described in chapter 7 could offer a tool for the insertion and deletion of specific (viral) genes for this purpose. Moreover exploiting the proposed recombination system, the improvement of the insecticidal properties of SeMNPV can be pursued