Mixtures of Insect-Pathogenic Viruses in a Single Virion: towards the Development of Custom-Designed Insecticides.

Alphabaculoviruses (Baculoviridae) are pathogenic DNA viruses of Lepidoptera that have applications as the basis for biological insecticides and expression vectors in biotechnological processes. These viruses have a characteristic physical structure that facilitates the transmission of groups of genomes. We demonstrate that coinfection of a susceptible insect by two different alphabaculovirus species results in the production of mixed-virus occlusion bodies containing the parental viruses. This occurred between closely related and phylogenetically more distant alphabaculoviruses. Approximately half the virions present in proteinaceous viral occlusion bodies produced following coinfection of insects with a mixture of two alphabaculoviruses contained both viruses, indicating that the viruses coinfected and replicated in a single cell and were coenveloped within the same virion. This observation was confirmed by endpoint dilution assay. Moreover, both viruses persisted in the mixed-virus population by coinfection of insects during several rounds of insect-to-insect transmission. Coinfection by viruses that differed in genome size had unexpected results on the length of viral nucleocapsids, which differed from those of both parental viruses. These results have unique implications for the development of alphabaculoviruses as biological control agents of insect pests.IMPORTANCE Alphabaculoviruses are used as biological insecticides and expression vectors in biotechnology and medical applications. We demonstrate that in caterpillars infected with particular mixtures of viruses, the genomes of different baculovirus species can be enveloped together within individual virions and occluded within proteinaceous occlusion bodies. This results in the transmission of mixed-virus populations to the caterpillar stages of moth species. Once established, mixed-virus populations persist by coinfection of insect cells during several rounds of insect-to-insect transmission. Mixed-virus production technology opens the way to the development of custom-designed insecticides for control of different combinations of caterpillar pest species.

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.

The C-termini of the baculovirus per os infectivity factors 1 and 2 mediate ODV oral infectivity by facilitating the binding of PIF0 and PIF8 to the core of the entry complex.

Oral infection of caterpillars by baculoviruses is initiated by occlusion-derived virus particles (ODVs) that infect midgut epithelium cells. The ODV envelope therefore contains at least ten different proteins, which are called per os infectivity factors (PIFs). Nine of these PIFs form the so-called ODV entry complex that consists of a stable core formed by PIF1, 2, 3 and 4, to which the other PIFs [PIF0, 6, 7, 8 and 9 (ac108)] bind with lower affinity. PIF1 and 2 are not only essential for complex formation, but also mediate ODV-binding to the epithelial brush border, probably via the C-termini. To study the involvement of these PIFs during midgut infection in greater detail, we assessed the oral infectivity and the ability to form the complex of a series of PIF1 and PIF2 C-terminal truncation mutants of Autographa californica multiple nucleopolyhedrovirus (AcMNPV), which were constructed in this study. Limited truncation of either PIF1 or 2 already severely impaired the ODV oral infectivity, but did not affect the formation of the core complex. However, the entry complex as a whole was not assembled in these mutants as PIF0 and 8 failed to bind to the core. This suggests that the interactions between the core and the loosely associated PIFs are important for the ODV infectivity and that complex formation complicates the determination of the exact roles of PIF1 and 2 during midgut infection. We also showed that the presence of PIF0, 6 and the ZF-domain of PIF8 are crucial for complex formation.

Herpes Simplex Virus 1 Can Enter Dynamin 1 and 2 Double-Knockout Fibroblasts.

Dynamin GTPases, best known for their role in membrane fission of endocytic vesicles, provide a target for viruses to be exploited during endocytic uptake. Recently, we found that entry of herpes simplex virus 1 (HSV-1) into skin cells depends on dynamin, although our results supported that viral internalization occurs via both direct fusion with the plasma membrane and via endocytic pathways. To further explore the role of dynamin for efficient HSV-1 entry, we utilized conditional dynamin 1 and dynamin 2 double-knockout (DKO) fibroblasts as an experimental tool. Strikingly, HSV-1 entered control and DKO fibroblasts with comparable efficiencies. For comparison, we infected DKO cells with Semliki Forest virus, which is known to adopt clathrin-mediated endocytosis as its internalization pathway, and observed efficient virus entry. These results support the notion that the DKO cells provide alternative pathways for viral uptake. Treatment of cells with the dynamin inhibitor dynasore confirmed that HSV-1 entry depended on dynamin in the control fibroblasts. As expected, dynasore did not interfere with viral entry into DKO cells. Electron microscopy of HSV-1-infected cells suggests viral entry after fusion with the plasma membrane and by endocytosis in both dynamin-expressing and dynamin-deficient cells. Infection at low temperatures where endocytosis is blocked still resulted in HSV-1 entry, although at a reduced level, which suggests that nonendocytic pathways contribute to successful entry. Overall, our results strengthen the impact of dynamin for HSV-1 entry, as only cells that adapt to the lack of dynamin allow dynamin-independent entry.IMPORTANCE The human pathogen herpes simplex virus 1 (HSV-1) can adapt to a variety of cellular pathways to enter cells. In general, HSV-1 is internalized by fusion of its envelope with the plasma membrane or by endocytic pathways, which reflects the high adaptation to differences in its target cells. The challenges are to distinguish whether multiple or only one of these internalization pathways leads to successful entry and, furthermore, to identify the mode of viral uptake. In this study, we focused on dynamin, which promotes endocytic vesicle fission, and explored how the presence and absence of dynamin can influence viral entry. Our results support the idea that HSV-1 entry into mouse embryonic fibroblasts depends on dynamin; however, depletion of dynamin still allows efficient viral entry, suggesting that alternative pathways present upon dynamin depletion can accomplish viral internalization.

Kinetic analysis of the influenza A virus HA/NA balance reveals contribution of NA to virus-receptor binding and NA-dependent rolling on receptor-containing surfaces.

Interactions of influenza A virus (IAV) with sialic acid (SIA) receptors determine viral fitness and host tropism. Binding to mucus decoy receptors and receptors on epithelial host cells is determined by a receptor-binding hemagglutinin (HA), a receptor-destroying neuraminidase (NA) and a complex in vivo receptor-repertoire. The crucial but poorly understood dynamics of these multivalent virus-receptor interactions cannot be properly analyzed using equilibrium binding models and endpoint binding assays. In this study, the use of biolayer interferometric analysis revealed the virtually irreversible nature of IAV binding to surfaces coated with synthetic sialosides or engineered sialoglycoproteins in the absence of NA activity. In addition to HA, NA was shown to be able to contribute to the initial binding rate while catalytically active. Virus-receptor binding in turn contributed to receptor cleavage by NA. Multiple low-affinity HA-SIA interactions resulted in overall extremely high avidity but also permitted a dynamic binding mode, in which NA activity was driving rolling of virus particles over the receptor-surface. Virus dissociation only took place after receptor density of the complete receptor-surface was sufficiently decreased due to NA activity of rolling IAV particles. The results indicate that in vivo IAV particles, after landing on the mucus layer, reside continuously in a receptor-bound state while rolling through the mucus layer and over epithelial cell surfaces driven by the HA-NA-receptor balance. Quantitative BLI analysis enabled functional examination of this balance which governs this dynamic and motile interaction that is expected to be crucial for penetration of the mucus layer and subsequent infection of cells by IAV but likely also by other enveloped viruses carrying a receptor-destroying enzyme in addition to a receptor-binding protein.

Identification of sialic acid-binding function for the Middle East respiratory syndrome coronavirus spike glycoprotein.

Middle East respiratory syndrome coronavirus (MERS-CoV) targets the epithelial cells of the respiratory tract both in humans and in its natural host, the dromedary camel. Virion attachment to host cells is mediated by 20-nm-long homotrimers of spike envelope protein S. The N-terminal subunit of each S protomer, called S1, folds into four distinct domains designated S1A through S1D Binding of MERS-CoV to the cell surface entry receptor dipeptidyl peptidase 4 (DPP4) occurs via S1B We now demonstrate that in addition to DPP4, MERS-CoV binds to sialic acid (Sia). Initially demonstrated by hemagglutination assay with human erythrocytes and intact virus, MERS-CoV Sia-binding activity was assigned to S subdomain S1A When multivalently displayed on nanoparticles, S1 or S1A bound to human erythrocytes and to human mucin in a strictly Sia-dependent fashion. Glycan array analysis revealed a preference for α2,3-linked Sias over α2,6-linked Sias, which correlates with the differential distribution of α2,3-linked Sias and the predominant sites of MERS-CoV replication in the upper and lower respiratory tracts of camels and humans, respectively. Binding is hampered by Sia modifications such as 5-N-glycolylation and (7,)9-O-acetylation. Depletion of cell surface Sia by neuraminidase treatment inhibited MERS-CoV entry of Calu-3 human airway cells, thus providing direct evidence that virus-Sia interactions may aid in virion attachment. The combined observations lead us to propose that high-specificity, low-affinity attachment of MERS-CoV to sialoglycans during the preattachment or early attachment phase may form another determinant governing the host range and tissue tropism of this zoonotic pathogen.

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.

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.

A Novel Virus Causes Scale Drop Disease in Lates calcarifer.

From 1992 onwards, outbreaks of a previously unknown illness have been reported in Asian seabass (Lates calcarifer) kept in maricultures in Southeast Asia. The most striking symptom of this emerging disease is the loss of scales. It was referred to as scale drop syndrome, but the etiology remained enigmatic. By using a next-generation virus discovery technique, VIDISCA-454, sequences of an unknown virus were detected in serum of diseased fish. The near complete genome sequence of the virus was determined, which shows a unique genome organization, and low levels of identity to known members of the Iridoviridae. Based on homology of a series of putatively encoded proteins, the virus is a novel member of the Megalocytivirus genus of the Iridoviridae family. The virus was isolated and propagated in cell culture, where it caused a cytopathogenic effect in infected Asian seabass kidney and brain cells. Electron microscopy revealed icosahedral virions of about 140 nm, characteristic for the Iridoviridae. In vitro cultured virus induced scale drop syndrome in Asian seabass in vivo and the virus could be reisolated from these infected fish. These findings show that the virus is the causative agent for the scale drop syndrome, as each of Koch's postulates is fulfilled. We have named the virus Scale Drop Disease Virus. Vaccines prepared from BEI- and formalin inactivated virus, as well as from E. coli produced major capsid protein provide efficacious protection against scale drop disease.

Supramolecular Virus-Like Nanorods by Coassembly of a Triblock Polypeptide and Reversible Coordination Polymers.

We investigate a new case of a self-assembly-stimulated self-assembly in which a triblock polypeptide is combined with a anionic coordination polymer of a dipicolinic acid bis-ligand, and d- or f- block metal ions like ZnII or EuIII . The polypeptide not only has a silk-like domain that can fold and stack, but also a C-terminal cationic sequence by which it can interact with the supramolecular (coordination) polyanion. In the presence of all three ingredients (polypeptide, bis-ligand, and metal ions), we observe the initiation and slow growth of well-defined metal-containing nanorods of up to 150 nm in length, proving that self-assembly of the polypeptide is triggered by the self-assembly of the coordination polyelectrolyte and vice versa. The particles, which have a striking resemblance to rod-like viruses, are stable up to 1.2 m NaCl, and can be made fluorescent when lanthanides like EuIII are used, showing the potential to exploit functional properties and applications of virus-like supramolecular structures.

Revealing and tuning the core, structure, properties and function of polymer micelles with lanthanide-coordination complexes.

Controlling self-assembly processes is of great interest in various fields where multifunctional and tunable materials are designed. We here present the versatility of lanthanide-complex-based micelles (Ln-C3Ms) with tunable coordination structures and corresponding functions (e.g. luminescence and magnetic relaxation enhancement). Micelles are prepared by charge-driven self-assembly of a polycationic-neutral diblock copolymer and anionic coordination complexes formed by Ln(III) ions and the bis-ligand L2EO4, which contains two dipicolinic acid (DPA) ligand groups (L) connected by a tetra-ethylene oxide spacer (EO4). By varying the DPA/Ln ratio, micelles are obtained with similar size but with different stability, different aggregation numbers and different oligomeric and polymeric lanthanide(III) coordination structures in the core. Electron microscopy, light scattering, luminescence spectroscopy and magnetic resonance relaxation experiments provide an unprecedented detailed insight into the core structures of such micelles. Concomitantly, the self-assembly is controlled such that tunable luminescence or magnetic relaxation with Eu-C3Ms, respectively, Gd-C3Ms is achieved, showing potential for applications, e.g. as contrast agents in (pre)clinical imaging. Considering the various lanthanide(III) ions have unique electron configurations with specific physical chemical properties, yet very similar coordination chemistry, the generality of the current coordination-structure based micellar design shows great promise for development of new materials such as, e.g., hypermodal agents.

Proteomic analysis of the plasma membrane-movement tubule complex of cowpea mosaic virus.

Cowpea mosaic virus forms tubules constructed from the movement protein (MP) in plasmodesmata (PD) to achieve cell-to-cell movement of its virions. Similar tubules, delineated by the plasma membrane (PM), are formed protruding from the surface of infected protoplasts. These PM-tubule complexes were isolated from protoplasts by immunoprecipitation and analysed for their protein content by tandem mass spectrometry to identify host proteins with affinity for the movement tubule. Seven host proteins were abundantly present in the PM-tubule complex, including molecular chaperonins and an AAA protein. Members of both protein families have been implicated in establishment of systemic infection. The potential role of these proteins in tubule-guided cell-cell transport is discussed.

Budded baculovirus particle structure revisited.

Baculoviruses are a group of enveloped, double-stranded DNA insect viruses with budded (BV) and occlusion-derived (ODV) virions produced during their infection cycle. BVs are commonly described as rod shaped particles with a high apical density of protein extensions (spikes) on the lipid envelope surface. However, due to the fragility of BVs the conventional purification and electron microscopy (EM) staining methods considerably distort the native viral structure. Here, we use cryo-EM analysis to reveal the near-native morphology of two intensively studied baculoviruses, Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) and Spodoptera exigua MNPV (SeMNPV), as models for BVs carrying GP64 and F as envelope fusion protein on the surface. The now well-preserved AcMNPV and SeMNPV BV particles have a remarkable elongated, ovoid shape leaving a large, lateral space between nucleocapsid (NC) and envelope. Consistent with previous findings the NC has a distinctive cap and base structure interacting tightly with the envelope. This tight interaction may explain the partial retaining of the envelope on both ends of the NC and the disappearance of the remainder of the BV envelope in the negative-staining EM images. Cryo-EM also reveals that the viral envelope contains two layers with a total thickness of ≈ 6-7 nm, which is significantly thicker than a usual biological membrane (<4 nm) as measured by X-ray scanning. Most spikes are densely clustered at the two apical ends of the virion although some envelope proteins are also found more sparsely on the lateral regions. The spikes on the surface of AcMNPV BVs appear distinctly different from those of SeMNPV. Based on our observations we propose a new near-native structural model of baculovirus BVs.

Live imaging of baculovirus infection of midgut epithelium cells: a functional assay of per os infectivity factors.

The occlusion-derived viruses (ODVs) of baculoviruses are responsible for oral infection of insect hosts, whereas budded viruses (BVs) are responsible for systemic infection within the host. The ODV membrane proteins play crucial roles in mediating virus entry into midgut epithelium cells to initiate infection and are important factors in host-range determination. For Autographa californica multiple nucleopolyhedrovirus (AcMNPV), seven conserved ODV membrane proteins have been shown to be essential for oral infectivity and are called per os infectivity factors (PIFs). Information on the function of the individual PIF proteins in virus entry is limited, partly due to the lack of a good in vitro system for monitoring ODV entry. Here, we constructed a baculovirus with EGFP fused to the nucleocapsid to monitor virus entry into primary midgut epithelium cells ex vivo using confocal fluorescence microscopy. The EGFP-labelled virus showed similar BV virulence and ODV infectivity as WT virus. The ability to bind and enter host cells was then visualized for WT AcMNPV and viruses with mutations in P74 (PIF0), PIF1 or PIF2, showing that P74 is required for ODV binding, whilst PIF1 and PIF2 play important roles in the entry of ODV after binding to midgut cells. This is the first live imaging of ODV entry into midgut cells and complements the genetic and biochemical evidence for the role of PIFs in the oral infection process.

Synergistic stiffening in double-fiber networks.

Many biological materials are composite structures, interpenetrating networks of different types of fibers. The composite nature of such networks leads to superior mechanical properties, but the origin of this mechanical synergism is still poorly understood. Here we study soft composite networks, made by mixing two self-assembling fiber-forming components. We find that the elastic moduli of the composite networks significantly exceed the sum of the moduli of the two individual networks. This mechanical enhancement is in agreement with recent simulations, where it was attributed to a suppression of non-affine deformation modes in the most rigid fiber network due to the reaction forces in the softer network. The increase in affinity also causes a loss of strain hardening and an increase in the critical stress and stain at which the network fails.

Correlation between structure, protein composition, morphogenesis and cytopathology of Glossina pallidipes salivary gland hypertrophy virus.

The Glossina pallidipes salivary gland hypertrophy virus (GpSGHV) is a dsDNA virus with rod-shaped, enveloped virions. Its 190 kb genome contains 160 putative protein-coding ORFs. Here, the structural components, protein composition and associated aspects of GpSGHV morphogenesis and cytopathology were investigated. Four morphologically distinct structures: the nucleocapsid, tegument, envelope and helical surface projections, were observed in purified GpSGHV virions by electron microscopy. Nucleocapsids were present in virogenic stroma within the nuclei of infected salivary gland cells, whereas enveloped virions were located in the cytoplasm. The cytoplasm of infected cells appeared disordered and the plasma membranes disintegrated. Treatment of virions with 1 % NP-40 efficiently partitioned the virions into envelope and nucleocapsid fractions. The fractions were separated by SDS-PAGE followed by in-gel trypsin digestion and analysis of the tryptic peptides by liquid chromatography coupled to electrospray and tandem mass spectrometry. Using the MaxQuant program with Andromeda as a database search engine, a total of 45 viral proteins were identified. Of these, ten and 15 were associated with the envelope and the nucleocapsid fractions, respectively, whilst 20 were detected in both fractions, most likely representing tegument proteins. In addition, 51 host-derived proteins were identified in the proteome of the virus particle, 13 of which were verified to be incorporated into the mature virion using a proteinase K protection assay. This study provides important information about GpSGHV biology and suggests options for the development of future anti-GpSGHV strategies by interfering with virus-host interactions.

Characterization of novel components of the baculovirus per os infectivity factor complex.

Baculovirus occlusion-derived virus (ODV) infects insect midgut cells under alkaline conditions, a process mediated by highly conserved per os infectivity factors (PIFs), P74 (PIF0), PIF1, PIF2, PIF3, PIF4, and PIF5 (ODV-E56). Previously, a multimolecular complex composed of PIF1, PIF2, PIF3, and P74 was identified which was proposed to play an essential role during ODV entry. Recently, more proteins have been identified that play important roles in ODV oral infectivity, including PIF4, PIF5, and SF58, which might work in concert with previously known PIFs to facilitate ODV infection. In order to understand the ODV entry mechanism, the identification of all components of the PIF complex is crucial. Hence, the aim of this study was to identify additional components of the PIF complex. Coimmunoprecipitation (CoIP) combined with proteomic analysis was used to identify the components of the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) PIF complex. PIF4 and P95 (AC83) were identified as components of the PIF complex while PIF5 was not, and this was confirmed with blue native PAGE and a second CoIP. Deletion of the pif4 gene impaired complex formation, but deletion of pif5 did not. Differentially denaturing SDS-PAGE further revealed that PIF4 forms a stable complex with PIF1, PIF2, and PIF3. P95 and P74 are more loosely associated with this complex. Three other proteins, AC5, AC68, and AC108 (homologue of SF58), were also found by the proteomic analysis to be associated with the PIF complex. Finally the functional significance of the PIF protein interactions is discussed.

Proteomic footprints of a member of Glossinavirus (Hytrosaviridae): an expeditious approach to virus control strategies in tsetse factories.

The Glossinavirus (Glossina pallidipes salivary gland hypertrophy virus (GpSGHV)) is a rod-shaped enveloped insect virus containing a 190,032 bp-long, circular dsDNA genome. The virus is pathogenic for the tsetse fly Glossina pallidipes and has been associated with the collapse of selected mass-reared colonies. Maintenance of productive fly colonies is critical to tsetse and trypanosomiasis eradication in sub-Saharan Africa using the Sterile Insect Technique. Proteomics, an approach to define the expressed protein complement of a genome, was used to further our understanding of the protein composition, morphology, morphogenesis and pathology of GpSGHV. Additionally, this approach provides potential targets for novel and sustainable molecular-based antiviral strategies to control viral infections in tsetse colonies. To achieve this goal, identification of key protein partners involved in virus transmission is required. In this review, we integrate the available data on GpSGHV proteomics to assess the impact of viral infections on host metabolism and to understand the contributions of such perturbations to viral pathogenesis. The relevance of the proteome findings to tsetse and trypanosomiasis management in sub-Sahara Africa is also considered.

In situ cleavage of baculovirus occlusion-derived virus receptor binding protein P74 in the peroral infectivity complex.

Proteolytic processing of viral membrane proteins is common among enveloped viruses and facilitates virus entry. The Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) occlusion-derived virus (ODV) protein P74 is part of a complex of essential peroral infectivity factors (PIFs). Here we report that P74 is efficiently cleaved into two fragments of about equal size by an occlusion body (OB) endogenous alkaline protease during ODV release when AcMNPV OBs are derived from larvae. The cleavage is specific for P74, since the other known peroral infectivity factors in the same complex (PIF1, PIF2, and PIF3) were not cleaved under the same conditions. P74 cleavage was not observed in OBs produced in three different insect cell lines, suggesting a larval host origin of the responsible protease. P74 in OBs produced in larvae of two different host species was cleaved into fragments with the same apparent molecular mass, indicating that the virus incorporates a similar alkaline protease from different hosts. Coimmunoprecipitation analysis revealed that the two P74 subunit fragments remain associated with the recently discovered PIF complex. We propose that under in vivo ODV infection conditions, P74 undergoes two sequential cleavage events, the first one being performed by an ODV-associated host alkaline protease and the second carried out by trypsin in the host midgut.

Baculovirus per os infectivity factors form a complex on the surface of occlusion-derived virus.

Five highly conserved per os infectivity factors, PIF1, PIF2, PIF3, PIF4, and P74, have been reported to be essential for oral infectivity of baculovirus occlusion-derived virus (ODV) in insect larvae. Three of these proteins, P74, PIF1, and PIF2, were thought to function in virus binding to insect midgut cells. In this paper evidence is provided that PIF1, PIF2, and PIF3 form a stable complex on the surface of ODV particles of the baculovirus Autographa californica multinucleocapsid nucleopolyhedrovirus (AcMNPV). The complex could withstand 2% SDS-5% beta-mercaptoethanol with heating at 50 degrees C for 5 min. The complex was not formed when any of the genes for PIF1, PIF2, or PIF3 was deleted, while reinsertion of these genes into AcMNPV restored the complex. Coimmunoprecipitation analysis independently confirmed the interactions of the three PIF proteins and revealed in addition that P74 is also associated with this complex. However, deletion of the p74 gene did not affect formation of the PIF1-PIF2-PIF3 complex. Electron microscopy analysis showed that PIF1 and PIF2 are localized on the surface of the ODV with a scattered distribution. This distribution did not change for PIF1 or PIF2 when the gene for PIF2 or PIF1 protein was deleted. We propose that PIF1, PIF2, PIF3, and P74 form an evolutionarily conserved complex on the ODV surface, which has an essential function in the initial stages of baculovirus oral infection.