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At least 505 records · Page 28Linked to original sources
PubMed1996

The E1 protein is mandatory for pore formation by Semliki Forest virus spikes.

Insect cells (Aedes albopictus, clone C6/36) were infected with various variants of Semliki Forest virus including the wild type using the SFV replicon system. The variants included deletion mutants lacking one of the structural proteins and a mutant with a point mutation in p62 (SQL). The latter mutation results in a failure to process p62 to E2 and E3. After infection of the cells with different variant viruses and subsequent expression of viral proteins in the host cell plasma membrane low pH-induced pore formation was detected by measuring the efflux of a radiolabeled compound. The results of these experiments clearly showed that the E1 protein is mandatory for the acid-induced pore formation. A participation of the 6K or C-protein could be excluded. Furthermore, results obtained with the SQL mutant suggest that dissociation of the E1/E2 heterodimer and subsequent homooligomerization of E1 are required for pore formation.

Aedes↗
PubMed2000

Different architectures in the assembly of infectious bursal disease virus capsid proteins expressed in insect cells.

Infectious bursal disease virus (IBDV) capsid is formed by the processing of a large polyprotein and subsequent assembly of VPX/VP2 and VP3. To learn more about the processing of the polyprotein and factors affecting the correct assembly of the viral capsid in vitro, different constructs were made using two baculovirus transfer vectors, pFastBac and pAcYM1. Surprisingly, the expression of the capsid proteins gave rise to different types of particles in each system, as observed by electron microscopy and immunofluorescence. FastBac expression led to the production of only rigid tubular structures, similar to those described as type I in viral infection. Western blot analysis revealed that these rigid tubules are formed exclusively by VPX. These tubules revealed a hexagonal arrangement of units that are trimer clustered, similar to those observed in IBDV virions. In contrast, pAcYM1 expression led to the assembly of virus-like particles (VLPs), flexible tubules, and intermediate assembly products formed by icosahedral caps elongated in tubes, suggesting an aberrant morphogenesis. Processing of VPX to VP2 seems to be a crucial requirement for the proper morphogenesis and assembly of IBDV particles. After immunoelectron microscopy, VPX/VP2 was detected on the surface of tubules and VLPs. We also demonstrated that VP3 is found only on the inner surfaces of VLPs and caps of the tubular structures. In summary, assembly of VLPs requires the internal scaffolding of VP3, which seems to induce the closing of the tubular architecture into VLPs and, thereafter, the subsequent processing of VPX to VP2.

Animals↗
PubMed1988

Expression of simian virus 40 T antigen in insect cells using a baculovirus expression vector.

Simian virus 40 (SV40) large T and small t antigens were synthesized in insect cells using the baculovirus Autographa californica as an expression vector. A recombinant virus containing a genomic copy of the SV40 early region expressed high levels of small t antigen but only low levels of large T antigen. However, very high levels of T antigen synthesis were observed when viruses were constructed with a cDNA copy of the large T antigen mRNA. Insect cells were capable of modifying T antigen by phosphorylation, palmitylation, glycosylation, and oligomerization. Functional assays demonstrated that the origin-specific DNA binding, ATPase, and helicase activities of insect cell-derived T antigen were comparable to T antigen synthesized in mammalian cells. Use of the baculovirus vector system to produce T antigen should facilitate future investigations requiring large quantities of T antigen.

Animals↗
PubMed1997

A role for baculovirus GP41 in budded virus production.

Insect cells infected with tsB1074, a temperature-sensitive mutant of Autographa californica nuclear polyhedrosis virus, exhibit a "single-cell-infection" phenotype whereby the infection progresses through the very late phase culminating in occlusion body formation, but neighboring cells do not become infected. Marker rescue mapping and DNA sequencing correlated a single nucleotide substitution within the baculovirus gp41 gene with the temperature-sensitive phenotype of tsB1074. The product of the gp41 gene, GP41, is an O-glycosylated protein found in occluded but not budded virions [M. Whitford and P. Faulkner (1992) J. Virol. 66, 3324-3329]. However, budded virus was not produced in tsB1074-infected cells at the nonpermissive temperature of 33 degrees, indicating an additional role for GP41 in budded virus formation. Electron microscopy revealed that nucleocapsids were produced but retained in the nucleus of tsB1074-infected cells at 33 degrees. Thus, GP41 was required for the egress of nucleocapsids from the nucleus in the pathway of budded virus synthesis.

Amino Acid Sequence↗
PubMed1997

Baculovirus expression of the respiratory syncytial virus fusion protein using Trichoplusia ni insect cells.

Respiratory syncytial virus (RSV) is a major viral pathogen responsible for severe respiratory tract infections in infants, young children, and the elderly. The RSV fusion (F) protein is highly conserved among RSV subgroups A and B and is the major protective immunogen. A genetically-engineered version of the RSV F protein was produced in insect cells using the baculovirus expression system. To express a secreted form of this protein, the transmembrane domain was eliminated by removing the region of the gene encoding 48 amino acids at the C-terminus. Production of the truncated RSV F protein (RSV-Fs) was compared in two different insect cell lines, Spodoptera frugiperda (Sf9) and Trichoplusia ni (High Five). The yield of RSV-Fs secreted from High Five insect cells was over 7-fold higher than that from Sf9 insect cells. Processing of the RSV-Fs protein was also different in the two insect cell lines. N-terminal sequencing demonstrated that while most of the RSV-Fs protein secreted by High Five cells was correctly processed at the F2-F1 proteolytic cleavage site, most of the RSV-Fs protein secreted by Sf9 cells was unprocessed or incorrectly processed. Antigenicity of the major RSV F neutralization epitopes was maintained in the RSV-Fs protein secreted from High Five cells. The RSV-specific neutralizing antibody titres in the sera of cotton rats immunized with the RSV-Fs protein were equivalent to those in the sera of animals intranasally inoculated with live RSV. Animals immunized with either live RSV or the immunoaffinity purified RSV-Fs protein from High Five cells were completely protected against live virus challenge.

Animals↗
PubMed2003

Functionality of the 5'- and intergenic IRES elements of cricket paralysis virus in a range of insect cell lines, and its relationship with viral activities.

Cricket paralysis virus (Dicistroviridae: Cripavirus) (CrPV) naturally has a wide range of insect hosts which is reflected in its ability to infect several cultured insect cell lines. The expression of viral gene products is controlled by two kinds of internal ribosome entry site (IRES) elements, 5' and intergenic (IG). Using seven cultured cell lines we tested the functionality of both IRES elements by transfection with bi-cistronic RNA constructs. In six of the seven cell lines, expression initiated from both IRES's was significantly higher than that from a control construct and in five of these six lines the expression from the 5'-IRES was higher than that from the IG-IRES. Permissiveness of each of the cell lines for replication of CrPV was tested by infection with purified virions and transfection with viral RNA. Only three of the cell lines were fully permissive for CrPV replication and no correlation between permissiveness and IRES activity was apparent. These results suggest that while IRES function is required for permissiveness, additional cellular and/or viral factors, involved in processing of viral products, packaging of viral particles and interacting with the cap-dependent translation machinery of host cells, are necessary for CrPV to be able to replicate in any given cell.

5' Untranslated Regions↗
PubMed2001

Proteolytic activity and recombinant protein production in virus-infected Sf-9 insect cell cultures supplemented with carboxyl and cysteine protease inhibitors.

In insect cell-baculovirus expression systems for recombinant protein production, it is sometimes necessary to supplement cultures with protease inhibitors to protect recombinant proteins against proteolysis. To date, however, there is no information available concerning protease activities in inhibitor-supplemented cultures. The aim of the present study was to investigate intracellular and extracellular protease activities in cultures of virus-infected Sf-9 insect cells which were supplemented with inhibitors against carboxyl and cysteine proteases produced during culture. Prior to the supplementation culture, the cell toxicity of several protease inhibitors was determined. As a result, pepstatin A (carboxyl protease inhibitor) and E64, cystatin, leupeptin, and antipain (cysteine protease inhibitors) tested in this study showed no apparent negative effects on the growth and viability of noninfected Sf-9 insect cells at low concentrations. In addition, E64 and pepstatin A could rapidly permeate virus-infected Sf-9 cells and inhibit the respective intracellular protease activities. A virus-infected culture with a multiplicity of infection of 1 was carried out with E64 and pepstatin A which were added to the culture medium at 2 d post-infection. As a result of inhibitor supplementation, the cellular activity for recombinant protein biosynthesis was reduced by 5-30%. However, a significant reduction in carboxyl and cysteine protease activities was observed not only in the medium but also intracellularly. This is the first study that directly demonstrates a reduction in extracellular and intracellular protease activities in protease inhibitor-supplemented cultures of virus-infected insect cells.

Journal Article↗
PubMed1990

Characterization of baculovirus-expressed Rift Valley fever virus glycoproteins synthesized in insect cells.

A cDNA corresponding to the complete coding region of the M RNA of the M12 mutant of Rift Valley fever virus (RVFV) strain ZH548 (K. Takehara, M-K. Min, J.K. Battles, K. Sugiyama, V.C. Emery, J.M. Dalrymple, and D.H.L. Bishop, Virology, 169, 452-457, 1989) has been inserted into the baculovirus transfer vector pAcYM1. By comparison with the parent RVFV, the M RNA of the M12 mutant has a new small open reading frame (ORF1) upstream of the one that initiates the precursor of the viral glycoproteins (ORF2, gene order: NS(M)-G2-G1). A derivative of the M12 cDNA was prepared from which most of the upstream sequences (including a polyT tract and ORF1) were removed. Other cDNA constructs were made from this derivative, constructs in which most of the G1 sequences were also removed, or most of the NS(M) coding sequences, or all of the NS(M) and most of G2 coding sequences. Each RVFV M cDNA construct was inserted into a pAcYM1 transfer vector and recombinant baculoviruses were produced (RVM1-5). The derived viruses were employed to study the expression and properties of the RVFV glycoproteins in Spodoptera frugiperda insect cells. For each recombinant virus evidence was obtained which indicated that the RVFV glycoproteins were produced and processed in the insect cells. Although four of the recombinants gave low expression levels of the RVFV glycoproteins, for the vector that made only the G1 product, the expression level was significantly higher. Immunofluorescence analyses established that the RVFV glycoproteins were present both at intracellular locations and on the surface of the recombinant baculovirus infected insect cells.

Animals↗
PubMed1993

Activation and detection of a latent baculovirus resembling Mamestra brassicae nuclear polyhedrosis virus in M. brassicae insects.

A laboratory culture of Mamestra brassicae insects (MbLC) has been found to harbor a latent baculovirus infection. The latent virus was activated by feeding the M. brassicae larvae with either the closely related Panolis flammea nuclear polyhedrosis virus (NPV), or the distantly related Autographa californica NPV. Restriction fragment profiles of the activated virus DNA showed that it is very closely related, if not identical, to M. brassicae NPV. Polymerase chain reaction amplification of polyhedrin gene sequences demonstrated that the latent virus was present throughout the life cycle of the insect; eggs, larvae, pupae, and adults. We failed to detect the presence of a latent virus in a second culture of M. brassicae insects, obtained from the environment and only recently adapted to growth in laboratory conditions, and thus these insects acted as an effective negative control in all the PCR and activation experiments. Using PCR analysis of DNA isolated from dissected tissues of fourth instar MbLC larvae, latent virus sequences were only detected in the fat body. Cell lines established from the isolated MbLC fat body tissue were also shown to harbor the latent virus sequences and should prove useful in further studies to elucidate the mechanisms of latency and virus activation.

Animals↗
PubMed1994

Expression of the Sendai virus fusion protein in insect cells and characterization of its post-translational modifications.

The fusion (F) protein of Sendai virus was expressed with a baculovirus system in insect cells. The F protein is synthesized in its uncleaved form F0, which appears in SDS-containing gels as two bands with M(r) values of 66K and 64K. Digestion of the F protein with endoglycosidase H revealed that the 66K species contains high mannose-type carbohydrates, whereas partially processed oligosaccharides are attached to the 64K species. Pulse-chase experiments showed that the F protein is initially synthesized as its 66K form. After 1 h of chase this precursor glycoprotein is partially converted to the 64K species and exposed at the cell surface. Other modifications of the F protein in insect cells are the formation of intramolecular disulphide linkages and oligomerization. However, processing and plasma membrane transport of the F protein in insect cells are incomplete and retarded compared to the F protein synthesized in Sendai virus-infected mammalian cells.

Animals↗
PubMed2004

Novel insect picorna-like virus identified in the brains of aggressive worker honeybees.

To identify candidate genes involved in the aggressive behavior of worker honeybees, we used the differential display method to search for RNAs exclusively detected in the brains of aggressive workers that had attacked a hornet. We identified a novel, 10,152-nucleotide RNA, termed Kakugo RNA. Kakugo RNA encodes a protein of 2,893 amino acid residues that shares structural features and sequence similarities with various picorna-like virus polyproteins, especially those from sacbrood virus, which infects honeybees. The Kakugo protein contains several domains that correspond to the virion protein, helicase, protease, and RNA-dependent RNA polymerase domains of various picorna-like virus polyproteins. When the worker bee tissue lysate was subjected to sucrose density gradient centrifugation, Kakugo RNA, except for the material at the bottom, was separated into two major peaks. One of the peaks corresponded to the position of Kakugo mRNA, and the other corresponded to the position of the poliovirus virion. These results suggest that the Kakugo RNA exists as an mRNA-like free RNA and virion RNA in the honeybee. Furthermore, injection of the lysate supernatant from the attacker heads into the heads of noninfected bees resulted in a marked increase in Kakugo RNA. These results demonstrate that Kakugo RNA is a plus-strand RNA of a novel picorna-like virus and that the brains of aggressive workers are infected by this novel virus. Kakugo RNA was detected in aggressive workers but not in nurse bees or foragers. In aggressive workers, Kakugo RNA was detected in the brain but not in the thorax or abdomen, indicating a close relation between viral infection in the brain and aggressive worker behaviors.

Aggression↗
PubMed2002

Tomato glycoalkaloids: role in the plant and in the diet.

Tomatoes, a major food source for humans, accumulate a variety of secondary metabolites including phenolic compounds, phytoalexins, protease inhibitors, and glycoalkaloids. These metabolites protect against adverse effects of hosts of predators including fungi, bacteria, viruses, and insects. Because glycoalkaloids are reported to be involved in host-plant resistance, on the one hand, and to have a variety of pharmacological and nutritional properties in animals and humans, on the other, a need exists to develop a better understanding of the role of these compounds both in the plant and in the diet. To contribute to this effort, this integrated review presents data on the history, composition, and nutrition of tomatoes, with special focus on the assessment of the chemistry, analysis, composition, nutrition, microbiology, and pharmacology of the tomato glycoalkaloids comprising alpha-tomatine and dehydrotomatine; their content in different parts of the tomato plant, in processed tomato products, and in wild and transgenic tomatoes; their biosynthesis, inheritance, metabolism, and catabolism; plant-microbe relationships with fungi, bacteria, viruses, insects, and worms; interactions with ergosterol and cholesterol; disruption of cell membranes; tomatine-induced tomatinases, pantothenate synthetase, steroid hydroxylases, and cytokines; and inhibition of acetylcholinesterase. Also covered are tomato-human pathogen relationships and tomatine-induced lowering of plasma cholesterol and triglycerides and enhancement of the immune system. Further research needs in each of these areas are suggested. The overlapping aspects are discussed in terms of general concepts for a better understanding of the impact of tomato glycoalkaloids in the plant in general and in food in particular. Such an understanding can lead to the creation of improved tomatoes and to improved practices on the farm and in the consumption of tomatoes.

Animals↗