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PubMed1993

Analysis of hepatitis C virus capsid, E1, and E2/NS1 proteins expressed in insect cells.

The baculovirus/insect cell expression system was used to express the capsid protein and glycoproteins (e1 and e2/NS1) of the hepatitis C virus (HCV). Each polypeptide domain was expressed individually using two different constructs varying at their carboxy termini in order to retain or delete hydrophobic domains that may be involved in membrane association. The capsid proteins were transported to the nucleus where they formed a single large crystal-like inclusion. The capsid proteins were phosphorylated in insect cells. The e1 and e2 polypeptides were present in both the soluble and insoluble cellular fractions. Deletion of a hydrophobic domain in the carboxy terminus of e2 resulted in the polypeptide becoming soluble but not secreted. Deletion of the carboxy terminus of e1 had no effect on solubility. Both e1 and e2 were glycosylated, with variable glycosylation of e1 giving rise to a series of polypeptides varying in apparent molecular weight. Co-infection of insect cells with viruses expressing e1 and e2 resulted in a complex that permitted the coimmunoprecipitation of e1 with antibodies to e2 and vice versa. Immunofluorescence staining of insect cells expressing e1 and e2 indicated that reactivity to e2 was more prevalent in anti-HCV positive human sera.

Animals↗
PubMed1994

Processing of E1 and E2 glycoproteins of hepatitis C virus expressed in mammalian and insect cells.

Processing of the envelope glycoproteins (E1 and E2) of hepatitis C virus (HCV) was investigated by using cDNA clones covering the structural and part of the nonstructural (NS) protein regions. The cDNA clones expressed in mammalian and insect cells were immunoprecipitated by serum of a hepatitis C patient and by monoclonal and polyclonal antibodies raised against the recombinant proteins expressed in insect cells or Escherichia coli. The E2 protein expressed in both insect and mammalian cells was a glycoprotein of 60 kDa (gp60) and removal of the sugar residues by N-glycanase yielded 38- and 40-kDa proteins. Pulse-chase experiments revealed that efficient expression and processing of the envelope proteins required coexpression with the flanking core and NS2 proteins. Not only E1 and E2 proteins but also NS2 and NS3 proteins were coprecipitated by anti-E1 or anti-E2 monoclonal antibody in the cells infected with the recombinant baculovirus expressing structural and NS proteins (NS2 and NS3), while only the NS3 protein was precipitated by anti-NS3 antibody. The association of E1 and E2 proteins was not influenced by the presence of a reducing agent and was still observed in the cells coinfected with the deletion mutants lacking both internal and C-terminal hydrophobic regions of each protein. Furthermore, the truncated forms of the E1 and E2 proteins were secreted into the culture supernatant and some of them were still associated with each other.

Animals↗
PubMed1995

Characterization of human papillomavirus type 16 E2 protein and subdomains expressed in insect cells.

The E2 open reading frame of human papillomavirus type 16 (HPV-16) encodes a DNA-binding protein which modulates papillomavirus transcription and replication. To investigate the biological and biochemical properties of the HPV-16 E2 protein, we have constructed recombinant baculoviruses which express the full-length molecule and individual N- and C-terminal domains in Sf21 insect cells. In this system the full-length E2 protein was phosphorylated and targeted to the insect cell nucleus. A 93 amino acid C-terminal fragment encompassing the DNA binding and dimerization functions of E2 was also translocated to the nucleus but was not modified by phosphorylation. The E2 N-terminal protein accumulated in the insect cell cytoplasm but was not efficiently phosphorylated. The formation of heterodimers between full-length and N-terminally truncated E2 species was observed when Sf21 cells were co-infected with recombinant viruses and when homodimers were mixed in vitro, suggesting that the dimer interface is not sufficiently stable to prevent subunit exchange in vivo. Both homo- and heterodimeric E2 species were able to bind specifically and in any combination to tandem E2 binding sites from the HPV-16 regulatory region. Furthermore, the HPV-16 E2 protein bound to DNA exhibited a distinct susceptibility profile to pronase digestion, potentially contrasting with that reported for BPV-1 E2. These observations suggest that significant structural and functional differences may exist between the BPV/HPV E2 proteins and have implications for understanding E2-dependent regulation of transcription and replication.

Amino Acid Sequence↗
PubMed1996

Expression of the 69K movement protein of turnip yellow mosaic virus in insect cells.

The nonstructural 69-kilodalton (K) protein of turnip yellow mosaic virus is necessary for systemic spread of the virus within the plant. To examine the behavior of the 69K protein in vivo, antibodies were raised against the carboxy-terminal region of this protein. The full-length 69K protein was also expressed in insect cells using a recombinant baculovirus. Studies on the posttranslational modifications of the 69K protein in insect cells revealed that the protein is phosphorylated but not glycosylated. Further experiments of subcellular fractionation and indirect immunolocalization in insect cells showed that the 69K protein is localized in the cytoplasm and/or in the plasma membrane.

Animals↗
PubMed1998

The non-permissive infection of insect (gypsy moth) LD-652 cells by Vaccinia virus.

The members of Poxviridae family are among the most complex of animal viruses and subfamily members infect both vertebrate (Chordopoxvirinae) and invertebrate (Entomopoxvirinae) hosts, respectively. Vaccinia virus (VV) is the most commonly studied vertebrate virus and the entomopoxvirus of Amsacta moorei (AmEPV) is the prototypic insect virus. AmEPV, while not able to productively infect vertebrate cells, does enter vertebrate cells and expresses early genes after which the infection aborts although the cells survive (Y. Li, R. L. Hall, and R. W. Moyer. J.Virol. 71(12), 95579562, 1997). We show here that a recombinant VV, containing the lacZ gene regulated by the cowpox virus A-type inclusion (ATI) late promoter, likewise does not productively infect insect cells. Our results suggest that the recombinant VV enters insect cells, host protein synthesis is inhibited, early gene expression is normal, and viral DNA replication occurs as does late protein synthesis. However, little if any proteolytic processing of late viral proteins, typical of morphogenesis, is observed. Electron micrographs of infected cells suggest that while cytoplasmic virosomes (factories) are formed, there is little indication of further morphogenesis or any formation of mature virions. Therefore, while both orthopoxviruses and entomopoxviruses fail to replicate in heterologous hosts, the nature of abortive infections is quite different.

Animals↗
PubMed2001

Autographa californica baculoviruses with large genomic deletions are rapidly generated in infected insect cells.

Defective interfering baculoviruses (DIs) lack considerable portions of the genome, interfere with the replication of helper virus, and cause the so-called "passage-effect" during serial passaging in insect cells and in bioreactor configurations. We investigated their origin by (nested) PCR and demonstrated that DIs lacking approximately 43% (d43) of their DNA are present in low-passage Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV)-E2 virus stocks and in polyhedra, but not in the authentic AcMNPV isolate obtained prior to passage in cell culture. To investigate whether DIs are rapidly generated de novo in Sf21 insect cells, a genetically homogeneous AcMNPV bacmid was serially passaged, resulting in the generation of d43 DIs within two passages. AT-rich sequences of up to 66 nucleotides of partly unknown origin were found at the deletion junctions in the d43 DI genomes. These data suggest that the rapid generation of DIs is an intrinsic property of baculovirus infection in insect cell culture and involves several recombination steps.

Animals↗
PubMed2002

Identification of a nucleocapsid protein (VP35) gene of shrimp white spot syndrome virus and characterization of the motif important for targeting VP35 to the nuclei of transfected insect cells.

To identify the protein encoded by a 687-bp open reading frame (ORF) of a salI genomic DNA fragment of shrimp white spot syndrome virus (WSSV), we expressed the ORF in a baculovirus/insect cell expression system. The apparent molecular mass of the recombinant protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was 35 kDa in insect cells. Antibody raised against bacterially synthesized protein of the ORF identified a nucleocapsid protein (VP35) in the extracts of both the purified WSSV virions and the nucleocapsids which comigrated with the 35-kDa baculovirus-expressed recombinant protein on SDS-PAGE. We also show by transient expression in insect cells (Sf9) that VP35 targets the nucleus. Two potential nuclear localization signals (NLSs) were characterized, but only one of them was important for targeting VP35 to the nuclei of transfected insect cells. Replacement of a cluster of four positively charged residues ((24)KRKR(27)) at the N terminus of the protein with AAAA resulted in mutant proteins that were distributed only in the cytoplasm, thus confirming that this sequence is a critical part of the functionally active NLS of VP35.

Amino Acid Sequence↗
PubMed2005

The role of apoptosis in defense against baculovirus infection in insects.

The baculoviruses make up a large, diverse family of DNA viruses that have evolved a number of fascinating mechanisms to manipulate their insect hosts. One of these is the ability to regulate apoptosis during infection by expressing proteins that can inhibit caspase activation and/or activity, including the caspase inhibitor P35 and its relatives, and the inhibitor of apoptosis (IAP) proteins. Experimental manipulations of the expression of these antiapoptotic genes, either by genetic deletions or by RNAi, have shed light on the effectiveness of apoptosis in combating baculovirus infection. The results of these experiments indicate that apoptosis can be an extremely powerful response to baculovirus infection, reducing viral replication, infectivity, and the ability of the virus to spread within the insect host even if a successful infection is established. Apoptosis is especially effective when it is combined with other innate antiviral defenses, which are largely unexplored in insects to date.

Animals↗
PubMed1993

Molecular studies on insect octopamine receptors.

Octopamine receptors are widely distributed in the insect nervous system and carry out a range of functions equivalent to the adrenergic receptors of the vertebrate sympathetic nervous system. Molecular studies on insect octopamine receptors have concentrated upon molecular pharmacological approaches to identify the particular subtype of octopamine receptor mediating its effects in a particular tissue and on the modes of action of the receptors in a particular tissue. Molecular biological approaches are now being pursued to define the structure of the octopamine receptor. Recent findings in this area will be reviewed, along with promising approaches for future molecular studies on insect octopamine receptors.

Amino Acid Sequence↗
PubMed1996

Transgenic rice established to express corn cystatin exhibits strong inhibitory activity against insect gut proteinases.

Corn cystatin (CC), a phytocystatin, shows a wide inhibitory spectrum against various cysteine proteinases. We produced transgenic rice plants by introducing CC cDNA under CaMV 35S promoter as a first step to obtain a rice plant with insecticidal activity. This attempt was based on the observation that many insect pests, especially Coleoptera, have cysteine proteinases, probably digestive enzymes, and also that oryzacystatin, an intrinsic rice cystatin, shows a narrow inhibition spectrum and is present in ordinary rice seeds in insufficient amounts to inhibit the cysteine proteinases of rice insect pests. The transgenic rice plants generated contained high levels of CC mRNA and CC protein in both seeds and leaves, the CC protein content of the seed reaching ca. 2% of the total heat soluble protein. We also recovered CC activity from seeds and found that the CC fraction efficiently inhibited both papain and cathepsin H, whereas the corresponding fraction from non-transformed rice seeds showed much lower or undetectable inhibitory activities against these cysteine proteinases. Furthermore, CC prepared from transgenic rice plants showed potent inhibitory activity against proteinases that occur in the gut of the insect pest, Sitophilus zeamais.

Animals↗
PubMed1994

Insect resistance of transgenic plants that express modified Bacillus thuringiensis cryIA(b) and cryIC genes: a resistance management strategy.

Tobacco and tomato plants were generated exhibiting insect resistance due to the introduction of modified cryIA(b) and cryIC genes of Bacillus thuringiensis. Limited modifications at selected regions of the coding sequences of both genes are sufficient to obtain resistance against Spodoptera exigua, Heliothis virescens and Manduca sexta. The criteria used to modify both genes demonstrate that the removal of sequence motifs potentially resulting in premature polyadenylation and transcript instability causes increased insect resistance. The expression of a cryIC-cryIA(b) fusion resulting in protection against S. exigua, H. virescens and M. sexta demonstrates the potential of expressing translational fusions, not only to broaden the insect resistance of transgenic plants, but also to simultaneously employ different gene classes in resistance management strategies.

Amino Acid Sequence↗
PubMed1981

Glutamate receptor binding in insects and mammals.

High affinity stereospecific binding sites for L-glutamate have been reported in several regions of mammalian brain. The binding sites in the hippocampus and cerebellum have been studied more extensively than binding in other brain regions. The hippocampal and cerebellar binding sites show similar properties with respect to their pharmacology and their independence of Na+. There is evidence, particularly good in the case of hippocampus, of mechanisms that may regulate the availability of the binding sites in both brain areas. Some progress has been made with the isolation of the hippocampal binding site but the protein has not been extensively characterised. In the case of insect muscle, high-affinity stereospecific binding of L-glutamate to whole membrane preparations, to detergent-solubilised membranes and to isolated proteolipids has been reported. Much greater variability in the binding characteristics is seen than is the case with the mammalian brain preparations. Preliminary experiments suggest that at least four distinct binding sites may be present on insect muscle. The complete characterisation of glutamate binding sites is at present precluded by a lack of potent agonists and antagonists. However, recent advances in the pharmacological classification of receptor sites for the excitatory amino acids in mammalian brain could provide sufficient information to permit the identification of the binding sites as synaptic receptors. Invertebrate toxins whose site of action is the insect neuromuscular junction may well prove to be useful tools with which to isolate and characterise the synaptic receptor proteins.

Animals↗
PubMed1978

A comparative ultrastructural study of blood cells from nine insect orders.

An ultrastructural study of hemocytes from 9 different insect orders has led to the identification of 8 cell types: (1) Plasmatocytes, whose cytoplasm is filled with small dense lysosomes and large heterogeneous structures, are phagocytic cells. (2) Granulocytes, filled with uniformly electron dense granules, are involved in capsule formation. (3) Coagulocytes, which contain granules and structured globules and which possess a well developed RER, are involved in phagocytosis. (4) Spherule cells are filled with large spherical inclusions. (5) Oenocytoids are large cells with few cytoplasmic organelles. These 5 hemocyte types represent the majority of insect blood cells. (6) Prohemocytes, blastic cells which are one of the stem cells a hemocytes, are very few in number in each species investigated. (7) Thrombocytoids and (8) Prodocytes are restricted to a small number of insect species. The ultrastructural characteristics of these hemocyte types are discussed.

Animals↗
PubMed1990

Evidence for a transcellular cisternal route across the caecal epithelium of an insect.

The cells of the mesenteric caeca in the midgut of certain insects possess a labyrinth of transepithelial cisternae. Their existence can be seen in thin sections of lanthanum-incubated tissue, where the tracer enters not only the intercellular clefts but also membranous cisternae which are inpocketings from, and, in continuity with, both the lateral clefts and basal membrane. These infoldings, which are numerous, run from the basal or lateral surfaces into the perinuclear region of the cells, where they are found, laden with lanthanum, as smooth cisternae or vesicles in the peripheral cytoplasm near the plasma membrane. These can be followed in serial sections and are quite distinct from other sub-surface cisternae of the lateral borders which are studded with ribosomes on the cytoplasmic surface. Near the luminal surface, tracer-laden structures in the form of vesicles and granules become increasingly predominant over those in the form of cisternae. Freeze-fracture replicas confirm the above observations, in that the plasma membrane of the intercellular cleft can be characterized as such unequivocally, since it exhibits smooth septate junctional E face grooves and P face ridges. Lateral infoldings, cisternae and vesicles can be seen arising directly from these junction-bearing membranes. The transepithelial cisternae and vesicles may be the morphological basis of an insect transcellular transport system, comparable to the tubulo-cisternal endoplasmic reticulum present in the transporting secretory and absorptive epithelia of vertebrate tissues. However, in insect midgut caecal epithelia, the cisternae appear to be, albeit presumably transiently, in direct continuity with the extracellular space, forming a plasma membrane reticular system which seems not to be the case with the tubulo-cisternal endoplasmic reticulum which terminates in subsurface cisternae.

Animals↗
PubMed1988

Different mitochondrial gene orders among insects: exchanged tRNA gene positions in the COII/COIII region between an orthopteran and a dipteran species.

We have cloned and sequenced a 2.65 kb segment of the mtDNA molecule of the orthopteran insect Locusta migratoria. It harbors the genes for four mitochondrial tRNAs, for cytochrome c oxidase subunits II and III and for ATPase subunits 6 and 8. The order of the locust genes resembles that of Drosophila yakuba: in both insects the genes for COII and ATPase 8 are separated from each other by the genes encoding tRNA(lys) and tRNA(asp), but in the locust, the positions of the two tRNA genes are reversed. This leads to a different mitochondrial gene order in the two insects.

Amino Acid Sequence↗
PubMed1995

The primary structure of Harpalus rufipes 5S ribosomal RNA: a contribution for understanding insect evolution.

The nucleotide sequence of 5S ribosomal RNA from the beetle Harpalus rufipes was determined and compared with primary structures of other insect 5S rRNAs. Sequence differences between two beetle 5S rRNAs may represent phylogenetic markers specific for two groups of Coleoptera - Adephaga and Polyphaga. Analysis of all insect sequences using parsimony allowed us to infer a phylogenetic tree of insects, which is consistent with morphological and paleobiological data.

Animals↗
PubMedDate not supplied

Role of fatty acid-binding protein in lipid metabolism of insect flight muscle.

Since insect flight muscles are among the most active muscles in nature, their extremely high rates of fuel supply and oxidation pose interesting physiological problems. Long-distance flights of species like locusts and hawkmoths are fueled through fatty acid oxidation. The lipid substrate is transported as diacylglycerol in the blood, employing a unique and efficient lipoprotein shuttle system. Following diacyglycerol hydrolysis by a flight muscle lipoprotein lipase, the liberated fatty acids are ultimately oxidized in the mitochondria. Locusta flight muscle cytoplasm contains an abundant fatty acid-binding protein (FABP). The flight muscle FABP of Locusta migratoria is a 15 kDa protein with an isoelectric point of 5.8, binding fatty acids in a 1:1 molar stoichiometric ratio. Binding affinity of the FABP for long-chain fatty acids (apparent dissociation constant Kd = 5.21 +/- 0.16 microM) is however markedly lower than that of mammalian FABPs. The NH2-terminal amino acid sequence shares structural homologies with two insect FABPs recently purified from hawkmoth midgut, as well as with mammalian FABPs. In contrast to all other isolated FABPs, the NH2 terminus of locust flight muscle FABP appeared not to be acetylated. During development of the insect, a marked increase in fatty acid binding capacity of flight muscle homogenate was measured, along with similar increases in both fatty acid oxidation capacity and citrate synthase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

Structure, function, and intracellular localization of glycoprotein B of herpesvirus simian agent 8 expressed in insect and mammalian cells.

The cloned gene of glycoprotein B (gB) of herpesvirus simian agent 8 (SA 8) was expressed with a baculovirus system in insect cells. Expression of gB was easily detectable over the cellular background by Coomassie staining of electrophoretically separated proteins. Endoglycosidase digestion of immunoprecipitated gB revealed that the gene product is N-glycosylated, but only with unprocessed, endoglycosidase-H sensitive carbohydrates. The lack of terminal glycosylation of gB is consistent with the observation that gB expressed in insect cells has a molecular weight slightly lower than gB synthesized during an SA 8 infection in mammalian cells. The truncated carbohydrates of gB from insect cells have no measurable effect on the tertiary structure of gB. Immunofluorescence studies on mammalian cells expressing gB from a simian virus 40 based vector revealed that the glycoprotein is localized to cytoplasmic membranes, to the plasma membrane and to the nuclear envelope. Cells expressing gB were fused to polykaryons, which shows that gB has cell fusing activity in the absence of any other SA 8 gene product.

Animals↗