Relationship Between BP (Baculovirus penaei) Energy Reserves in Larval and Postlarval Pacific White Shrimp Penaeus vannamei

The relationship between energy reserves of the penaeid shrimp Penaeus vannamei and Baculovirus penaei, or BP, were investigated in a series of experiments using mysis stage or early postlarval shrimp. Pre-exposure and post-exposure levels of protein and triacylgycerol (TAG) were determined. The effect of pre-exposure protein and TAG levels on susceptibility to BP infections was also investigated by starving a group of shrimp immediately prior to BP exposure. There was no consistent relationship between either pre-exposure or post-exposure protein levels and the percent of shrimp developing patent BP infections. There was, however, a significant positive correlation between TAG levels immediately prior to viral exposure and prevalence of infection 72 h later. Experimental reduction of TAG reserves prior to BP exposure delayed the development of a patent infection. In some, but not all, experiments there was a significant reduction in TAG levels of infected compared with uninfected shrimp 72 h post-exposure. The effect of patent BP infections on host TAG levels was subordinate to fluctuations in TAG content associated with the ontogeny of the hepatopancreas. Results of this study support histological observations that shrimp lipid levels can be altered by baculovirus infections. Furthermore, high levels of energy reserves in the form of TAG are associated with increased susceptibility to BP infection in larval and postlarval shrimp.

Modularity and evolutionary constraints in a baculovirus gene regulatory network

Abstract Background The structure of regulatory networks remains an open question in our understanding of complex biological systems. Interactions during complete viral life cycles present unique opportunities to understand how host-parasite network take shape and behave. The Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV) is a large double-stranded DNA virus, whose genome may encode for 152 open reading frames (ORFs). Here we present the analysis of the ordered cascade of the AgMNPV gene expression. Results We observed an earlier onset of the expression than previously reported for other baculoviruses, especially for genes involved in DNA replication. Most ORFs were expressed at higher levels in a more permissive host cell line. Genes with more than one copy in the genome had distinct expression profiles, which could indicate the acquisition of new functionalities. The transcription gene regulatory network (GRN) for 149 ORFs had a modular topology comprising five communities of highly interconnected nodes that separated key genes that are functionally related on different communities, possibly maximizing redundancy and GRN robustness by compartmentalization of important functions. Core conserved functions showed expression synchronicity, distinct GRN features and significantly less genetic diversity, consistent with evolutionary constraints imposed in key elements of biological systems. This reduced genetic diversity also had a positive correlation with the importance of the gene in our estimated GRN, supporting a relationship between phylogenetic data of baculovirus genes and network features inferred from expression data. We also observed that gene arrangement in overlapping transcripts was conserved among related baculoviruses, suggesting a principle of genome organization. Conclusions Albeit with a reduced number of nodes (149), the AgMNPV GRN had a topology and key characteristics similar to those observed in complex cellular organisms, which indicates that modularity may be a general feature of biological gene regulatory networks.

On the inheritance and mechanism of baculovirus resistance of the codling moth, Cydia pomonella (L.)

Das Cydia pomonella Granulovirus (CpGV, Baculoviridae) wird seit Ende der 1980er Jahre als hoch-selektives und effizientes biologisches Bekämpfungsmittel zur Kontrolle des Apfelwicklers im Obstanbau eingesetzt. Seit 2004 wurden in Europa verschiedene Apfelwicklerpopulationen beobachtet die resistent gegenüber dem hauptsächlich angewendeten Isolat CpGV-M aufweisen. Die vorliegende Arbeit befasst sich mit der Untersuchung der Vererbung und des Mechanismus der CpGV Resistenz. Einzelpaarkreuzungen zwischen einem empfindlichen Laborstamm (CpS) und einem homogen resistenten Stamm (CpRR1) zeigten, dass die Resistenz durch ein einziges dominantes Gen, das auf dem Z-Chromosom lokalisiert ist, vererbt wird. Massernkreuzungen zwischen CpS und einer heterogen resistenten Feldpopulation (CpR) deuteten zunächst auf einen unvollständig dominanten autosomalen Erbgang hin. Einzelpaarkreuzungen zwischen CpS und CpR bewiesen jedoch, dass die Resistenz in CpR ebenfalls monogen dominant und geschlechtsgebunden auf dem Z-Chromosom vererbt wird. Diese Arbeit diskutiert zudem die Vor- und Nachteile von Einzelpaarkreuzungen gegenüber Massernkreuzungen bei der Untersuchung von Vererbungsmechanismen. Die Wirksamkeit eines neuen CpGV Isolates aus dem Iran (CpGV-I12) gegenüber CpRR1 Larven, wurde in Bioassays getestet. Die Ergebnisse zeigen, dass CpGV-I12 die Resistenz in allen Larvenstadien von CpRR1 brechen kann und fast so gut wirkt wie CpGV-M gegenüber CpS Larven. Daher ist CpGV-I12 für die Kontrolle des Apfelwicklers in Anlagen wo die Resistenz aufgetreten ist geeignet. Um den der CpGV Resistenz zugrunde liegenden Mechanismus zu untersuchen, wurden vier verschiedene Experimente durchgeführt: 1) die peritrophische Membran degradiert indem ein optischer Aufheller dem virus-enthaltenden Futtermedium beigefügt wurde. Das Entfernen dieser mechanischen Schutzbarriere, die den Mitteldarm auskleidet, führte allerdings nicht zu einer Reduzierung der Resistenz in CpR Larven. Demnach ist die peritrophische Membran nicht am Resistenzmechanismus beteiligt. 2) Die Injektion von Budded Virus in das Hämocoel führte nicht zur Brechung der Resistenz. Folglich die die Resistenz nicht auf den Mitteldarm beschränkt, sondern auch in der Sekundärinfektion wirksam. 3) Die Replikation von CpGV in verschiedenen Geweben (Mitteldarm, Hämolymphe und Fettkörper) von CpS und CpRR1 wurde mittels quantitativer PCR verfolgt. In CpS Larven konnte in allen drei Gewebetypen sowohl nach oraler als auch nach intra-hämocoelarer Infektion eine Zunahme der CpGV Genome in Abhängigkeit der Zeit festgestellt werden. Dagegen konnte in den Geweben aus CpRR1 nach oraler sowie intra-hämocoelarer Infektion keine Virusreplikation detektiert werden. Dies deutet darauf hin, dass die CpGV Resistenz in allen Zelltypen präsent ist. 4) Um zu untersuchen ob ein humoraler Faktor in der Hämolymphe ursächlich an der Resistenz beteiligt ist, wurde Hämolymphe aus CpRR1 Larven in CpS Larven injiziert und diese anschließend oral mit CpGV infiziert. Es konnte jedoch keine Immunreaktion beobachtet und kein Faktor in der Hämolymphe identifiziert werden, der Resistenz induzieren könnte. Auf Grundlage dieser Ergebnisse kann festgestellt werden, dass in resistenten Apfelwicklerlarven die virale Replikation in allen Zelltypen verhindert wird, was auf eine Virus-Zell Inkompatibilität hinweist. Da in CpRR1 keine DNA Replikation beobachtet wurde, wird die CpGV Resistenz wahrscheinlich durch eine frühe Unterbindung der Virusreplikation verursacht.Das früh exprimierte Gen pe38 codiert für ein Protein, das wahrscheinlich für die Resistenzbrechung durch CpGV-I12 verantwortlich ist. Interaktionen zwischen dem Protein PE38 und Proteinen in CpRR1 wurden mit Hilfe des Yeast Two-Hybrid (Y2H) Systems untersucht. Die detektierten Interaktionen sind noch nicht durch andere Methoden bestätigt, jedoch wurden zwei mögliche Gene auf dem Z-Chromosom und eines auf Chromosom 15 gefunden, wie möglicherweise an der CpGV Resistenz beteiligt sind.

Rhodopsin kinase: Expression in baculovirus-infected insect cells, and characterization of post-translational modifications*

Structure–function studies of rhodopsin kinase (RK; EC 2.7.1.125) require a variety of mutants. Therefore, there is need for a suitable system for the expression of RK mutant genes. Here we report on a study of expression of the RK gene in baculovirus-infected Sf21 cells and characterization of the enzyme produced as purified to near homogeneity. Particular attention has been paid to the post-translational modifications, autophosphorylation and isoprenylation, found in the native bovine RK. The protein produced has been purified using, successively, heparin-Sepharose, Mono Q, and Mono S FPLC (fast protein liquid chromatography) and was obtained in amounts of about 2 mg from 1 liter of cell culture. The enzyme from the last step of purification was obtained in two main fractions that differ in the level of phosphorylation. The protein peak eluted first carries two phosphate groups per protein, whereas the second protein peak is monophosphorylated. Further, while both peaks are isoprenylated, the isoprenyl groups consist of mixtures of C5, C10, C15, and C20 isoprenyl moieties. From these results, we conclude that the above expression system is suitable for some but not all aspects of structure–function studies.

Reconstitution of human replication factor C from its five subunits in baculovirus-infected insect cells

Human replication factor C (RFC, also called activator 1) is a five-subunit protein complex (p140, p40, p38, p37, and p36) required for proliferating cell nuclear antigen (PCNA)-dependent processive DNA synthesis catalyzed by DNA polymerase δ or ɛ. Here we report the reconstitution of the RFC complex from its five subunits simultaneously overexpressed in baculovirus-infected insect cells. The purified baculovirus-produced RFC appears to contain equimolar levels of each subunit and was shown to be functionally identical to its native counterpart in (i) supporting DNA polymerase δ-catalyzed PCNA-dependent DNA chain elongation; (ii) catalyzing DNA-dependent ATP hydrolysis that was stimulated by PCNA and human single-stranded DNA binding protein; (iii) binding preferentially to DNA primer ends; and (iv) catalytically loading PCNA onto singly nicked circular DNA and catalytically removing PCNA from these DNA molecules.

A Discrete Stage of Baculovirus GP64-mediated Membrane Fusion

Viral fusion protein trimers can play a critical role in limiting lipids in membrane fusion. Because the trimeric oligomer of many viral fusion proteins is often stabilized by hydrophobic 4-3 heptad repeats, higher-order oligomers might be stabilized by similar sequences. There is a hydrophobic 4-3 heptad repeat contiguous to a putative oligomerization domain of Autographa californica multicapsid nucleopolyhedrovirus envelope glycoprotein GP64. We performed mutagenesis and peptide inhibition studies to determine if this sequence might play a role in catalysis of membrane fusion. First, leucine-to-alanine mutants within and flanking the amino terminus of the hydrophobic 4-3 heptad repeat motif that oligomerize into trimers and traffic to insect Sf9 cell surfaces were identified. These mutants retained their wild-type conformation at neutral pH and changed conformation in acidic conditions, as judged by the reactivity of a conformationally sensitive mAb. These mutants, however, were defective for membrane fusion. Second, a peptide encoding the portion flanking the GP64 hydrophobic 4-3 heptad repeat was synthesized. Adding peptide led to inhibition of membrane fusion, which occurred only when the peptide was present during low pH application. The presence of peptide during low pH application did not prevent low pH–induced conformational changes, as determined by the loss of a conformationally sensitive epitope. In control experiments, a peptide of identical composition but different sequence, or a peptide encoding a portion of the Ebola GP heptad motif, had no effect on GP64-mediated fusion. Furthermore, when the hemagglutinin (X31 strain) fusion protein of influenza was functionally expressed in Sf9 cells, no effect on hemagglutinin-mediated fusion was observed, suggesting that the peptide does not exert nonspecific effects on other fusion proteins or cell membranes. Collectively, these studies suggest that the specific peptide sequences of GP64 that are adjacent to and include portions of the hydrophobic 4-3 heptad repeat play a dynamic role in membrane fusion at a stage that is downstream of the initiation of protein conformational changes but upstream of lipid mixing.

Baculovirus inhibitors of apoptosis (IAPs) block activation of Sf-caspase-1

We have investigated the ability of Sf-caspase-1 and two mammalian caspases, caspase-1 and caspase-3, to induce apoptosis in Spodoptera frugiperda Sf-21 insect cells. While the transient expression of the pro-Sf-caspase-1 did not induce apoptosis, expression of the pro-domain deleted form, p31, or coexpression of the two subunits of mature Sf-caspase-1, p19 and p12, induced apoptosis in Sf-21 cells. The behavior of Sf-caspase-1 resembled that of the closely related mammalian caspase, caspase-3, and contrasted with that of the mammalian caspase-1, the pro-form of which was active in inducing apoptosis in Sf-21 cells. The baculovirus caspase inhibitor P35 blocked apoptosis induced by active forms of all three caspases. In contrast, members of the baculovirus inhibitor of apoptosis (IAP) family failed to block active caspase-induced apoptosis. However, during viral infection, expression of OpIAP or CpIAP blocked the activation of pro-Sf-caspase-1 and the associated induction of apoptosis. Thus, the mechanism by which baculovirus IAPs inhibit apoptosis is distinct from the mechanism by which P35 blocks apoptosis and involves inhibition of the activation of pro-caspases like Sf-caspase-1.

Baculovirus-mediated gene transfer into mammalian cells.

This paper describes the use of the baculovirus Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) as a vector for gene delivery into mammalian cells. A modified AcMNPV virus was prepared that carried the Escherichia coli lacZ reporter gene under control of the Rous sarcoma virus promoter and mammalian RNA processing signals. This modified baculovirus was then used to infect a variety of mammalian cell lines. After infection of the human liver cell lines HepG2, >25% of the cells showed high-level expression of the transduced gene. Over 70% of the cells in primary cultures of rat hepatocytes showed expression of beta-galactosidase after exposure to the virus. Cell lines from other tissues showed less or no expression of lacZ after exposure to the virus. The block to expression in less susceptible cells does not appear to result from the ability to be internalized by the target cell but rather by events subsequent to viral entry. The onset of lacZ expression occurred within 6 hr of infection in HepG2 cells and peaked 12-24 hr postinfection. Because AcMNPV is able to replicate only in insect hosts, is able to carry large (>15 kb) inserts, and is a highly effective gene delivery vehicle for primary cultures of hepatocytes, AcMNPV may be a useful vector for genetic manipulation of liver cells.

Efficient gene transfer into human hepatocytes by baculovirus vectors.

Viral vectors are the most efficient tools for gene delivery, and the search for tissue-specific infecting viruses is important for the development of in vivo gene therapy strategies. The baculovirus Autographa californica nuclear polyhedrosis virus is widely used as a vector for expression of foreign genes in insect cells, and its host specificity is supposed to be restricted to arthropods. Here we demonstrate that recombinant A. californica nuclear polyhedrosis virus is efficiently taken up by human hepatocytes via an endosomal pathway. High-level reporter gene expression from heterologous promoters was observed in human and rabbit hepatocytes in vitro. Mouse hepatocytes and some other epithelial cell types are targeted at a considerably lower rate. The efficiency of gene transfer by baculovirus considerably exceeds that obtained by calcium phosphate or lipid transfection. These properties of baculovirus suggest a use for it as a vector for liver-directed gene transfer but highlight a potential risk in handling certain recombinant baculoviruses.

Cloning, baculovirus expression, and characterization of a second mouse prolyl 4-hydroxylase alpha-subunit isoform: formation of an alpha 2 beta 2 tetramer with the protein disulfide-isomerase/beta subunit.

Prolyl 4-hydroxylase (EC 1.14.11.2) catalyzes the posttranslational formation of 4-hydroxyproline in collagens. The vertebrate enzyme is an alpha 2 beta 2 tetramer, the beta subunit of which is a highly unusual multifunctional polypeptide, being identical to protein disulfide-isomerase (EC 5.3.4.1). We report here the cloning of a second mouse alpha subunit isoform, termed the alpha (II) subunit. This polypeptide consists of 518 aa and a signal peptide of 19 aa. The processed polypeptide is one residue longer than the mouse alpha (I) subunit (the previously known type), the cloning of which is also reported here. The overall amino acid sequence identity between the mouse alpha (II) and alpha (I) subunits is 63%. The mRNA for the alpha (II) subunit was found to be expressed in a variety of mouse tissues. When the alpha (II) subunit was expressed together with the human protein disulfide-isomerase/beta subunit in insect cells by baculovirus vectors, an active prolyl 4-hydroxylase was formed, and this protein appeared to be an alpha (II) 2 beta 2 tetramer. The activity of this enzyme was very similar to that of the human alpha (I) 2 beta 2 tetramer, and most of its catalytic properties were also highly similar, but it differed distinctly from the latter in that it was inhibited by poly(L-proline) only at very high concentrations. This property may explain why the type II enzyme was not recognized earlier, as an early step in the standard purification procedure for prolyl 4-hydroxylase is affinity chromatography on a poly(L-proline) column.

A physiological product-release model for baculovirus infected insect cells

Existing models describe the product release from baculovirus infected insect cells as an unspecific protein leakage occurring in parallel with protein production. The model presented here shows that the observed product release of normally non-secreted proteins can be described through cell death alone. This model avoids the implicit non-physiological assumption of previous models that cells permeable to recombinant protein as well as trypan blue continue to produce protein. (c) 2005 Wiley Periodicals, Inc.