The SBASE protein domain library, release 5.0: a collection of annotated protein sequence segments.

SBASE 5.0 is the fifth release of SBASE, a collection of annotated protein domain sequences that represent various structural, functional, ligand-binding and topogenic segments of proteins. SBASE was designed to facilitate the detection of functional homologies and can be searched with standard database-search programs. The present release contains over 79863 entries provided with standardized names and is cross-referenced to all major sequence databases and sequence pattern collections. The information is assigned to individual domains rather than to entire protein sequences, thus SBASE contains substantially more cross-references and links than do the protein sequence databases. The entries are clustered into >16 000 groups in order to facilitate the detection of distant similarities. SBASE 5.0 is freely available by anonymous 'ftp' file transfer from <ftp.icgeb.trieste.it >. Automated searching of SBASE with BLAST can be carried out with the WWW-server <http://www.icgeb.trieste.it/sbase/ >. and with the electronic mail server <[email protected] >which now also provides a graphic representation of the homologies. A related WWW-server <http://www.abc.hu/blast.html > and e-mail server <[email protected] > predicts SBASE domain homologies on the basis of SWISS-PROT searches

The genome of Spodoptera exigua multicapsid nucleopolyhedrovirus : a study on unique features

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 .</p

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.</p