|Title||The genome of Spodoptera exigua multicapsid nucleopolyhedrovirus : a study on unique features|
|Source||Wageningen University. Promotor(en): R.W. Goldbach; J.M. Vlak; D. Zuidema. - S.l. : S.n. - ISBN 9789058084194 - 150|
Laboratory of Virology
|Publication type||Dissertation, internally prepared|
|Keyword(s)||kernpolyedervirussen - baculovirus - genomen - moleculaire genetica - spodoptera exigua - nuclear polyhedrosis viruses - baculovirus - genomes - molecular genetics - spodoptera exigua|
|Categories||Viruses of Invertebrates|
The Baculoviridae are a family of rod-shaped viruses with large circular double-stranded DNA genomes (Chapter 1). The family is subdivided into two genera, Granulovirus (GV) and Nucleopolyhedrovirus (NPV) on the basis of the type of body occluding the virions. NPVs are further subdivided in group I and II based on phylogenetic evidence of the DNA polymerase protein. Baculoviruses almost exclusively infect insects and are, therefore, attractive biological alternatives to chemical insecticides for insect pest control. The baculovirus Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV) infects the beet army worm S. exigua (Lepidoptera: Noctuidae) and has been successfully used as a bioinsecticide to control this world-wide insect pest of agricultural importance. SeMNPV differs from many other baculoviruses in that it is mono-specific and highly virulent for S. exigua larvae. The research described in this thesis aimed at the molecular characterization of the baculovirus SeMNPV to gain insight in its gene content and organization in comparison to those of other baculoviruses. At the same time this study will support or reject its current taxonomic position using gene and genome phylogeny analyses and might reveal insight in the molecular mechanisms associated with the biological properties of SeMNPV.
As a start the complete nucleotide sequence of the DNA genome of SeMNPV, a putative group II NPV, was determined and analyzed (Chapter 2). The genome was composed of 135,612 bp containing 138 putative genes or open reading frames (ORFs). Major differences in SeMNPV gene content and arrangement were found compared with the group I NPVs Autographa californica (Ac), Bombyx mori (Bm ), Orgyia pseudotsugata (Op) and the group II NPV Lymantria dispar (Ld). Sixteen ORFs were unique to SeMNPV, while the remaining ORFs (122) all had a homolog in one or more of the nine baculoviruses sequenced to date (Chapter 7). Sixty-three ORFs were conserved among all nine baculoviruses and are likely to be essential for NPV multiplication and survival. Strikingly, two of these NPV 'core' genes, odv-e66 and p26 , were found duplicated in SeMNPV. Gene parity analysis of baculoviral genomes indicated that SeMNPV and LdMNPV are closely related and that they are only distantly related to group I NPVs. Therefore, SeMNPV can be considered as a group II NPV.
Two of the 16 unique SeMNPV genes, Se116 and Se117, share similarity on amino acid level, but are not related on nucleotide level. To investigate the function, if any, of the unique SeMNPV genes in general, Se116 and Se117 were analyzed and characterized (Chapter 3). Se116 and Se117 were expressed from early till late in infection both in cultured cells and in larvae of S. exigua. Their transcripts were polyadenylated and initiated from typical baculovirus early promoter motifs. Se116 and Se117 encoded proteins of 27 and 23 kDa, respectively, which were localized in the virogenic stroma of the nucleus. While the function of the Se116 protein remains enigmatic, the Se117 protein appeared to be a structural protein associated with nucleocapsids of occlusion-derived virus (ODV), but not of budded virus (BV). Further investigation will reveal if and how these proteins are involved in the SeMNPV virulence or host range determination.
The research on unique SeMNPV genes was extended (Chapter 4) by the characterization of another gene, Se17/18, unique among NPVs, but strikingly having a homolog (ORF129) in the granulovirus Xestia c-nigum (XcGV), which is only distantly related to SeMNPV. Se17/18 was transcribed in cultured S. exigua 301 cells from early till late in infection. However, in vivo transcripts could only be detected late in infection. These polyadenylated transcripts started in a region containing a baculovirus consensus early promoter motif. In contrast to the Se116 and Se117 proteins, the Se17/18 protein was primarily localized in the cytoplasm. A chicken polyclonal antiserum was raised that reacted specifically to Se17/18 protein expressed in E. coli . However, no immunoreactive protein was detected in SeMNPV-infected insect cells. The absence of immunoreactive Se17/18 protein implies that it is rapidly turned over in insect cell culture or that the gene is only active in larvae and possibly has a regulatory function.
A thorough analysis of the complete SeMNPV genome revealed that it lacked a homolog of the major budded virus glycoprotein gene gp64, that is found in AcMNPV and other group I NPVs. Upon infection, by representatives of this group, acidification of the endosome triggers fusion of the viral and endosomal membrane, which is mediated by the BV envelope glycoprotein GP64. Therefore, the entry mechanism of SeMNPV in cultured cells was examined. SeMNPV budded virus (BV) entered insect cells by endocytosis like BVs of group I NPVs. Furthermore, a functional homolog of the envelope fusion protein GP64 was identified in Se8 (76 kDa) and appeared to be the major envelope protein of SeMNPV BVs. Surprisingly, a 60 kDa cleavage product of this protein was present in the BV envelope. A furin-like proprotein convertase cleavage site was identified immediately upstream of the N-terminus of the mature Se8 protein and this site was also conserved in the LdMNPV homolog (Ld130) of Se8. Syncytium formation assays showed that Se8 and Ld130 alone were sufficient to mediate membrane fusion. Both proteins were primarily localized in the plasma membrane of insect cells, which was consistent with their fusogenic activity. If Se8 is cleaved by a cellular convertase the host could also play a role in the determination of virus host range and virulence.
The research on function of single SeMNPV genes and also the engineering of this virus for improved insecticidal activity or as expression vector have been hampered as defective viruses are quickly generated when using insect cell culture. These defective viruses lack 25 kb sequence information and are no longer active in vivo upon oral feeding. A novel procedure to isolate SeMNPV recombinants was adopted by alternate cloning between insect larvae and cultured cells. In this way a SeMNPV recombinant (SeXD1) was obtained infectious both in vivo and in cell culture and with an improved speed of kill. This recombinant lacked 10.6 kb of sequence information, including ecdysteroid UDP glucosyl transferase ( egt ), gp37 , chitinase and cathepsin genes, as well as several genes unique to SeMNPV. One of these unique genes was Se17/18. The result indicated, however, that these genes are dispensable for virus replication both in cell culture and in vivo . A mutant with a similar deletion was identified by PCR in the parental wild type SeMNPV isolate suggesting that genotypes with differential biological activities exist in field isolates of baculoviruses.
The research on SeMNPV described in this thesis, has provided a complete overview of its coding potential and insight in several features common to lepidopteran baculoviruses, such as 'core' genes, unique genes and clustering of conserved genes (Chapter 7). The initial characterization of several SeMNPV genes resulted in the identification of a novel ODV-specific nucleocapsid protein unique to SeMNPV and a novel major BV envelope fusion protein. The latter is the first baculovirus protein reported to be cleaved by a cellular furin-like proprotein convertase. The development of a novel procedure to generate recombinants in vivo is presumably applicable to many baculovirus species in order to obtain biologically active recombinants. Exploitation of this technique will enable the further characterization of (unique) SeMNPV genes by deletion, insertion and mutation by in vivo recombination. Understanding the function of SeMNPV genes will ultimately lead to the unravelling of the molecular basis underlying the mono-specificity and high virulence of SeMNPV for the beet army worm Spodoptera exigua .