<p>Nuclear polyhedrosis viruses belong to the family <em>Baculoviridae</em> and cause fatal diseases in arthropods predominantly in insects of the order <em>Lepidoptera</em> . These viruses were first reported in silk worms where viral infections could have devastating effects on the production of silk. In nature these viruses regulate the size of insect populations. Each nuclear polyhedrosis virus can only infect a small number of host insects and these viruses are harmless for mammals and birds. Therefore, these viruses are used as biological insecticides. The interest in these insect viruses was further augmented by the observation that they could be used for the high level expression of foreign genes.<p>The exploitation of nuclear polyhedrosis viruses requires understanding of their biology and molecular organization. Infected insect cells produce two proteins in large quantities in the late phase of infection: polyhedrin (approximately 30 kDa), the matrix protein of the viral occlusion bodies, and a protein of 10 kDa (p10). This p10 protein is not present in virions, but is associated with fibrillar structures observed in the nucleus and cytoplasm of infected cells. The study described in this thesis focused on the functional analysis of the p10 protein.<p>Polyhedrin and p10 genes have promoters with a similar structure and hence, a similar regulation mechanism was expected. Both promoters might compete for transcription factors, resulting in reduced expression levels of each individual gene. The effect of the simultaneous activity of the p10 and polyhedrin promoter on the level of p10 gene expression was examined by analysing <em>Autographa californica</em> (Ac) MNPV recombinants with various deletions in the polyhedrin gene (Chapter 2). None of the deletions in the polyhedrin gene resulted in higher expression from the p10 promoter and no significant difference in level of expression was observed between the p10 and polyhedrin promoter. The results suggest that, the p10 and polyhedrin gene, despite the homology in their promoters, are independently regulated.<p>At the beginning of this research only the amino acid sequence of the p10 proteins of the closely related AcMNPV and <em>Orgyia pseudotsugata</em> (Op) MNPV were available. To benefit from amino acid sequence data for the prediction of conserved domains, the p10 gene of the distantly related baculovirus <em>Spodoptera exigua</em> (Se) MNPV was characterized (Chapter 3). The deduced SeMNPV p10 protein showed limited amino acid sequence identity to the p10 proteins of OpMNPV and AcMNPV (39% and 26%, respectively). The three p10 proteins, however, had similar hydrophilicity profiles and most notably, they all had a positively-charged carboxy terminal sequence.<p>On the basis of sequence comparisons, AcMNPV p10 deletion mutants were constructed to identify putative functional domains (Chapter 4). Several domains were found in the p10 protein : (i) The hydrophilic carboxy terminus (position 87 to 94) is required for the assembly of p10 into fibrillar structures. Without this stretch of amino acids p10 molecules form aggregates that lack the morphological features of fibrillar structures. This domain might be involved in the interaction with tubulin. (ii) Amino acids 1-52 have a selfaggregation fucntion (see also VIak <em>et al.,</em> 1988; <em>J.</em><em>Gen. Virol.</em> 69: 765-776). (iii) P10 is involved in the disintegration of infected-cell nuclei, which leads to the release of individual polyhedra. This function is located in the amino-terminal 79 amino acids, and is independent of fibrillar structure formation. It is likely that these 79 amino acids also harbour a binding site for electron-dense spacers and polyhedron envelopes.<p>The p10 protein of SeMNPV shows only limited homology to the p10 protein of AcMNPV (Chapter 3), although it probably has the same functions. To investigate the functional homology between these two p10 proteins, a recombinant AcMNPV virus was constructed, which encoded the SeMNPV p10 protein instead of the authentic p10 protein (Chapter 5). In an AcMNPV environment the SeMNPV p10 protein assembled into fibrillar structures in <em>S.</em><em>frugiperda</em> cells, and is therefore functionally homologous to AcMNPV p10. These structures had the same morphology as in <em>Spodoptera exigua</em> cells infected with SeMNPV P10 has a preference to aggregate with p10 molecules of the same species, as was concluded from simultaneous expression of SeMNPV and AcMNPV p10. The SeMNPV p10 protein was not able to induce nuclear disintegration, when expressed from the AcMNPV genome, regardless of the type of host cells used. This indicates that at least one other viral protein is involved in this process, which specifically recognizes p10 of the same species.<p>The carboxy-terminal amino acid sequence (RRGKRSSK), involved in the formation of fibrillar structures, contains two serine residues, which are prone to phosphorylation, for instance by cAMP-dependent protein kinase (Cheley <em>et al.,</em> 1992. <em>J. Cell. Sci.</em> 102: 739-752). We hypothesized that aggregates of p10 might form fibrillar structures due to the presence of a small fraction of phosphorylated p10 molecules. To test this hypothesis one or both serines were replaced with alanines (Chapter 6). These p10 mutants normally induced fibrillar structures. Phosphorylation on these serines is therefore not involved in this process.<p>The disintegration of infected-cell nuclei at the end of the infection seems to be the major function of p10 proteins. Due to this disintegration polyhedra are released as individual particles, resulting in an efficient dissemination of progeny virus into the environment. Wildtype viruses may therefore have a selective advantage over genetically-modified viruses with altered p10 genes, when these modified viruses are used in biological control.
|Qualification||Doctor of Philosophy|
|Award date||8 Jun 1994|
|Place of Publication||S.l.|
|Publication status||Published - 1994|
- nuclear polyhedrosis viruses
- amino acids
- amino acid sequences