Baculovirus per os infectivity : a complex matter

Insect larvae are infected by baculoviruses when they consume plant material that is contaminated with occlusion bodies (OBs). OBs are polyhedral shaped particles of crystallized protein (polyhedrin or granulin), in which the occlusion derived viruses (ODVs) are embedded. Upon ingestion of the OBs, the ODVs are released in the alkaline lumen of the midgut and infect the midgut epithelial cells. To be infectious for these cells under alkaline conditions, the ODV envelope contains a set of proteins, which are called per os infectivity factors or PIFs. Eight different PIFs had been identified at the start of this research, of which six were known to participate in the formation of a large complex: the ODV entry complex. Presently, ten different PIFs are known, of which nine form the entry complex. This complex has a stable core that is formed by PIF1 to 4, which even resists treatment with denaturing and reducing agents (SDS and β-mercaptoethanol), and five additional PIFs (PIF0 and PIF6 to 9), which are associated to the complex more loosely. All these individual components are essential for complex formation. PIF5 is the only PIF-protein that is not a constituent of the entry complex.   Our initial studies with pif deletion mutant viruses were hampered by the disappearance of not only the targeted, but also non-targeted PIFs, when the OBs were produced in insect larvae, while the various PIFs were all detectable for  the wild type virus. However, those remaining PIFs were found again in these mutant viruses when the larval derived OBs were treated with heat prior to ODV isolation, or when the OBs were produced in cultured insect cells. These observations showed that proteases from the host, which had been reported to be co-occluded in the OBs, are able to degrade PIF proteins when complex formation is disrupted, as was the case in the various pif deletion mutant viruses. These observations provided a first clue on why the PIFs form a complex, which might be important to resist proteolytic degradation by proteases in the gut of the host. Previous research in our laboratory had identified viral protein AC108 as a PIF1 interaction partner in a co-immunoprecipitation study, indicating that this protein might be involved in oral infectivity as well. The research presented in this thesis demonstrated that mutation of the ac108 gene from the viral genome completely abolished the oral infectivity of ODVs, which was recovered when this gene was repaired. Further protein analyses revealed that AC108 is a loosely associated but nevertheless an essential component of the ODV entry complex. This protein was therefore designated as PIF9. The biological function of PIF9 was further investigated by the generation of fluorescently labelled ODVs of the pif9 mutant. These virus particles were then combined with isolated midgut cells of Spodoptera exigua larvae and monitored by confocal microscopy in a time lapse experiment. It was observed that in absence of PIF9, the ODVs still bind to the midgut cell brush border, but that the nucleocapsids failed to enter the cell, in contrast to fluorescent ODVs that have all PIFs. Fluorescent ODVs were also generated for a pif3 deletion mutant as it had been shown previously by others that this (complex deficient) mutant is able to bind and fuse with the host cell plasma membrane as the wild type virus, but nevertheless fails to establish a midgut infection. Our analysis with the confocal microscope showed that this mutant displays the same phenotype as the pif9 deletion mutant virus: ODV binding, but no entry of the nucleocapsids. We therefore proposed a two-step fusion process, in which first the outer leaflets of the lipid bilayers fuse and subsequently the inner leaflets. PIF3 might be important to complete the fusion process by aiding fusion of the inner leaflets of the lipid bilayers.   PIF1 and 2 had been reported to be important for ODV binding and were later also shown to be crucial for the formation of the ODV entry complex. Furthermore, ODV binding had been shown to be one of the viral host range determinants and as most baculoviruses have a narrow host range, we hypothesized that the more variable C-terminal parts of PIF1 and 2 are important for host interaction, while the highly conserved N-terminal parts are needed for complex formation. However, limited C-terminal truncation of either PIF1 or 2 abolished formation of the entry complex as PIF0 and 8 failed to bind the stable core in the truncation mutants. The lost ability to form the entry complex in the truncation mutants was accompanied by a severely hampered oral infectivity in S. exigua larvae. Some larvae incidentally got infected by one of the truncation mutants despite abolished complex formation. It was therefore speculated that although the ODV entry complex is the main determinant for the infectivity of ODVs, additional factors are present in the ODV that is able to incidentally cause infections in absence of the entry complex. To determine the context in which the PIFs function, the interaction partners were mapped of three loosely associated components of the entry complex (PIF6, 8 and 9) and the single solitary PIF, PIF5. These analyses revealed that the PIFs interact with a plethora of viral proteins with a wide variety of functions, from nucleocapsid assembly to OB formation. PIF5 also interacted with two components of the entry complex, PIF0 and 1. This indicated that PIF0 and 1 are not only present in the ODV envelope as part of the entry complex, but also occur outside the complex where these proteins interact with PIF5. This study shows that the PIFs not only interact with each other to form the entry complex, but are part of a network of protein interactions in ODVs. The biological significance of these interactions for midgut infection remains enigmatic

Phenomenological modeling of anisotropy induced by evolution of the dislocation structure on the macroscopic and microscopic scale \ud

This work focuses on the modeling of the evolution of anisotropy induced by the development of the dislocation microstructure. A model formulated at the engineering scale in the context of classical metal plasticity and a model formulated in the context of crystal plasticity are presented. Images obtained by transmission-electron microscopy (TEM) show the influence of the strain path on the evolution of anisotropy for the case of two common materials used in sheet metal forming, DC06 and AA6016-T4. Both models are capable of accounting for the transient behavior observed after changes in loading path for fcc and bcc metals. The evolution of the internal variables of the models is correlated with the evolution of the dislocation structure observed by TEM investigations

The baculovirus Ac108 protein is a per os infectivity factor and a component of the ODV entry complex

Wild-type ODVs (Wt) have an intact ODV entry complex in their envelope and are orally infectious towards insect larvae (left panel). In the absence of Ac108 (mut ac108), the stable core is still present but nevertheless fails to form an entry complex, affecting the ODV oral infectivity (right panel). The components of the core complex are depicted in yellow and the loosely associated components are depicted in red. PIF7 is depicted in green as its affinity with the complex is currently not known.Baculoviruses orally infect insect larvae when they consume viral occlusion bodies (OBs). OBs consist of a crystalline protein matrix in which the infectious virus particles, the occlusion-derived viruses (ODVs), are embedded. The protein matrix dissolves in the alkaline environment of the insect's midgut lumen. The liberated ODVs can then infect midgut endothelial cells through the action of at least nine different ODV-envelope proteins, called per os infectivity factors (PIFs). These PIF proteins mediate ODV oral infectivity, but are not involved in the systemic spread of the infection by budded viruses (BVs). Eight of the known PIFs form a multimeric complex, named the ODV entry complex. In this study, we show for Autographa californica multiple nucleopolyhedrovirus that mutation of the ac108ORF abolishes the ODV oral infectivity, while production and infectivity of the BVs remains unaffected. Furthermore, repair of the ac108 mutant completely recovered oral infectivity. With an HA-tagged repair mutant, we were able to demonstrate by Western analysis that the Ac108 protein is a constituent of the ODV entry complex, where the formation was abolished in the absence of this protein. Based on these results, we conclude that ac108 encodes a per os infectivity factor (PIF9) that is also an essential constituent of the ODV entry complex.</p

An advanced view on Baculovirus per Os infectivity factors

Baculoviruses are arthropod-specific large DNA viruses that orally infect the larvae of lepidopteran, hymenopteran and dipteran insect species. These larvae become infected when they eat a food source that is contaminated with viral occlusion bodies (OBs). These OBs contain occlusion-derived viruses (ODVs), which are released upon ingestion of the OBs and infect the endothelial midgut cells. At least nine different ODV envelope proteins are essential for this oral infectivity and these are denoted per os infectivity factors (PIFs). Seven of these PIFs form a complex, consisting of PIF1, 2, 3 and 4 that form a stable core complex and PIF0 (P74), PIF6 and PIF8 (P95) that associate with this complex with lower affinity than the core components. The existence of a PIF complex and the fact that the pif genes are conserved in baculovirus genomes suggests that PIF-proteins cooperatively mediate oral infectivity rather than as individual functional entities. This review therefore discusses the knowledge obtained for individual PIFs in light of their relationship with other members of the PIF complex.</p

Functional analysis of the baculovirus per os infectivity factors 3 and 9 by imaging the interaction between fluorescently labelled virions and isolated midgut cells

Baculovirus occlusion-derived viruses (ODVs) contain ten known per os infectivity factors (PIFs). These PIFs are crucial for midgut infection of insect larvae and form, with the exception of PIF5, an ODV entry complex. Previously, R18-dequenching assays have shown that PIF3 is dispensable for binding and fusion with midgut epithelial cells. Oral infection nevertheless fails in the absence of PIF3. PIF9 has not been analysed in much depth yet. Here, the biological role of these two PIFs in midgut infection was examined by monitoring the fate of fluorescently labelled ODVs when incubated with isolated midgut cells from Spodoptera exigua larvae. Confocal microscopy showed that in the absence of either PIF3 or PIF9, the ODVs bound to the brush borders, but the nucleocapsids failed to enter the cells. Finally, we discuss how the results obtained for PIF3 with dequenching assays and confocal microscopy can be explained by a two-phase fusion process.</p

The adhesion G protein-coupled receptor G2 (ADGRG2/GPR64) constitutively activates SRE and NFκB and is involved in cell adhesion and migration

Adhesion G protein-coupled receptors (ADGRs) are believed to be activated by auto-proteolytic cleavage of their very large extracellular N-terminal domains normally acting as a negative regulator of the intrinsically constitutively active seven transmembrane domain. ADGRG2 (or GPR64) which originally was described to be expressed in the epididymis and studied for its potential role in male fertility, is highly up-regulated in a number of carcinomas, including breast cancer. Here, we demonstrate that ADGRG2 is a functional receptor, which in transfected HEK293 cells signals with constitutive activity through the adhesion- and migration-related transcription factors serum response element (SRE) and nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) presumably via coupling to Gα12/13 and Gαq. However, activation of these two pathways appears to occur through distinct molecular activation mechanisms as auto-proteolytic cleavage is essential for SRE activation but not required for NFκB signaling. The overall activation mechanism for ADGRG2 is clearly distinct from the established ADGR activation mechanism as it requires the large extracellular N-terminal domain for proper intracellular signal transduction. Knockdown of ADGRG2 by siRNA in the highly motile breast cancer cell lines Hs578T and MDA-MB-231 resulted in a strong reduction in cell adhesion and subsequent cell migration which was associated with a selective reduction in RelB, an NFκB family member. It is concluded that the adhesion GPCR ADGRG2 is critically involved in the adhesion and migration of certain breast cancer cells through mechanisms including a non-canonical NFkB pathway and that ADGRG2 could be a target for treatment of certain types of cancer.Toxicolog

Acetaminophen reduces the protein levels of high affinity amino acid permeases and causes tryptophan depletion

In yeast, toxicity of acetaminophen (APAP), a frequently used analgesic and antipyretic drug, depends on ubiquitin-controlled processes. Previously, we showed a remarkable overlap in toxicity profiles between APAP and tyrosine, and a similarity with drugs like rapamycin and quinine, which induce degradation of the amino acid permease Tat2. Therefore, we investigated in yeast whether APAP reduced the expression levels of amino acid permeases. The protein levels of Tat2, Tat1, Mup1 and Hip1 were reduced, while the expression of the general permease Gap1 was increased, consistent with a nutrient starvation response. Overexpression of Tat1 and Tat2, but not Mup1, Hip1 and Gap1 conferred resistance to APAP. A tryptophan auxotrophic strain trp1Δ was more sensitive to APAP than wild-type and addition of tryptophan completely restored the growth restriction of trp1∆ upon APAP exposure, while tyrosine had an additive effect on APAP toxicity. Furthermore, intracellular aromatic amino acid concentrations were reduced upon APAP exposure. This effect was less prominent in ubiquitin-deficient yeast strains that were APAP resistant and showed a reduced degradation of high affinity amino acid permeases. APAP-induced changes in intracellular amino acid concentrations were also detected in hepatoma HepG2 cells indicating significance for humans

Baculoviruses require an intact ODV entry-complex to resist proteolytic degradation of per os infectivity factors by co-occluded proteases from the larval host

Baculoviruses orally infect caterpillars in the form of occlusion-derived viruses (ODVs). The ODV-envelope contains a number of proteins which are essential for oral infectivity, called per os infectivity factors (PIFs). Most of these PIFs are involved in the formation of an ODV-entry complex that consists of a stable core, formed by PIF1, PIF2, PIF3 and PIF4, and the more loosely associated PIFs P74 (PIF0) and P95 (PIF8). PIF1, PIF2 and PIF3 are essential for formation of the stable core, whereas deletion of the pif4 gene results in the formation of a smaller complex. P74 is not needed for formation of the stable core. We show here in larva-derived ODVs of the Autographa californica multicapsid nucleopolyhedrovirus that PIF-proteins are degraded by host-derived proteases after deletion of a single pif-gene. Constituents of the stable core-complex appeared to be more resistant to proteases as part of the complex than as monomer, as in ODVs of a p74 deletion mutant only the stable core was found but no PIF monomers. When the stable core lacks PIF4, it lost its proteolytic resistance as the resulting smaller core complex was degraded in a pif4 deletion mutant. We also identified PIF6 as a loosely associated component of the entry complex that appeared nevertheless important for the proteolytic resistance of the stable core, which was degraded after deletion of pif6. We conclude from these results that an intact entry-complex in the ODV-envelope is prerequisite for proteolytic resistance of PIF-proteins under the alkaline conditions of the larval midgut