Creation of Rift Valley Fever Viruses with four-segmented Genomes reveals flexibility in Bunyavirus Genome Packaging.
Wichgers Schreur, P.J. ; Oreshkova, N. ; Moormann, R.J.M. ; Kortekaas, J.A. - \ 2014
Journal of Virology 88 (2014)18. - ISSN 0022-538X - p. 10883 - 10893.
defective interfering particles - noncoding regions - immune-response - golgi retention - messenger-rna - nsm protein - replication - segment - glycoprotein - expression
Bunyavirus genomes comprise a small (S), medium (M) and a large (L) RNA segment of negative polarity. Although the untranslated regions (UTRs) have been shown to comprise signals required for transcription, replication and encapsidation, the mechanisms that drive the packaging of at least one S, M and L segment into a single virion to generate infectious virus are largely unknown. One of the most important members of the Bunyaviridae family that causes devastating disease in ruminants and occasionally humans is the Rift Valley fever virus (RVFV). Here we studied the flexibility of RVFV genome packaging by splitting the glycoprotein precursor gene, encoding the (NSm)GnGc polyprotein, into two individual genes encoding either (NSm)Gn or Gc. Using reverse-genetics, six viruses with a segmented glycoprotein precursor gene were rescued, varying from a virus comprising 2 S-type segments in the absence of an M-type segment to a virus consisting of 4 segments (RVFV-4s) of which 3 are M-type. Despite that all virus variants were able to grow in mammalian cell lines, they were unable to spread efficiently in cells of mosquito origin. Moreover, in vivo studies demonstrated that RVFV-4s is unable to cause disseminated infection and disease in mice, even in the presence of the main virulence factor NSs, but induced a protective immune response against a lethal challenge with wild-type virus. In summary, splitting bunyavirus glycoprotein precursor genes provides new opportunities to study bunyavirus genome packaging and offers new methods to develop next-generation live-attenuated bunyavirus vaccines.
Enhanced protein secretion from insect cells by co-expression of the chaperone calreticulin and translation initiation factor eIF4E
Teng, C.Y. ; Chang, S.L. ; Oers, M.M. van; Wu, T.Y. - \ 2013
Molecular Biotechnology 54 (2013)1. - ISSN 1073-6085 - p. 68 - 78.
baculovirus expression system - ribosome entry site - endoplasmic-reticulum - quality-control - messenger-rna - calnexin - infection - pathway - er - glycoprotein
Host protein synthesis is shut down in the lytic baculovirus expression vector system (BEVS). This also affects host proteins involved in routing secretory proteins through the endoplasmic reticulum (ER)-Golgi system. It has been demonstrated that a secretory alkaline phosphatase–EGFP fusion protein (SEFP) can act as a traceable and sensitive secretory reporter protein in BEVS. In this study, a chaperone, calreticulin (CALR), and the translation initiation factor eIF4E were co-expressed with SEFP using a bicistronic baculovirus expression vector. We observed that the intracellular distribution of SEFP in cells co-expressing CALR was different from co-expressing eIF4E. The increased green fluorescence emitted by cells co-expressing CALR had a good correlation with the abundance of intracellular SEFP protein and an unconventional ER expansion. Cells co-expressing eIF4E, on the other hand, showed an increase in extracellular SEAP activity compared to the control. Utilization of these baculovirus expression constructs containing either eIF4E or CALR offers a significant advantage for producing secreted proteins for various biotechnological and therapeutic applications.
The major autolysin Acm2 from Lactobacillus plantarum undergoes cytoplasmic O-glycosylation
Fredriksen, L. ; Mathiesen, G. ; Moen, A. ; Bron, P.A. ; Kleerebezem, M. ; Eijsink, V.G.H. ; Egge-Jacobsen, W. - \ 2012
Journal of Bacteriology 194 (2012)2. - ISSN 0021-9193 - p. 325 - 333.
binding-protein gspb - n-acetylglucosaminidase - signal peptides - gene-expression - system - peptidoglycan - endopeptidase - glycoprotein - specificity - diversity
The major autolysin Acm2 from the probiotic strain Lactobacillus plantarum WCFS1 contains high proportions of alanine, serine, and threonine in its N-terminal so-called AST domain. It has been suggested that this extracellular protein might be glycosylated, but this has not been experimentally verified. We used high-resolution liquid chromatography-tandem mass spectrometry (LC-MS/MS) to study the possible occurrence of glycans on peptides generated from lactobacillary surface proteins by protease treatment. This approach yielded five glycopeptides in various glycoforms, all derived from the AST domain of Acm2. All five glycopeptides contained the hydroxy-amino acids serine and threonine, suggesting that Acm2 is O-glycosylated. By using lectin blotting with succinylated wheat germ agglutinin, and by comparing the wild-type strain with an Acm2-negative derivative (NZ3557), we found that the attached N-acetylhexosamines are most likely N-acetylglucosamines (GlcNAc). NZ3557 was further used as a genetic background to express an Acm2 variant lacking its secretion signal, resulting in intracellular expression of Acm2. We show that this intracellular version of Acm2 is also glycosylated, indicating that the GlcNAc modification is an intracellular process.
Glycan-dependent immunogenicity of recombinant soluble trimeric hemagglutinin
Vries, R.P. de; Smit, C.H. ; Bruin, E. de; Rigter, A. ; Vries, E. de; Cornelissen, A.H.M. ; Eggink, D. ; Chung, N.P.Y. ; Moore, J.P. ; Sanders, R.W. ; Hokke, C.H. ; Koopmans, M.P.G. ; Rottier, P.J.M. ; Haan, C.A.M. de - \ 2012
Journal of Virology 86 (2012)21. - ISSN 0022-538X - p. 11735 - 11744.
influenza-virus hemagglutinin - receptor-binding - carbohydrate moiety - dc-sign - glycosylation - glycoprotein - proteins - antibody - recognition - specificity
Recombinant soluble trimeric influenza A virus (IAV) hemagglutinin (sHA3) has proven an effective vaccine antigen against IAV. Here, we investigate to what extent the glycosylation status of the sHA3 glycoprotein affects its immunogenicity. Different glycosylation forms of subtype H5 trimeric HA protein (sH53) were produced by expression in insect cells and different mammalian cells in the absence and presence of inhibitors of N-glycan-modifying enzymes or by enzymatic removal of the oligosaccharides. The following sH53 preparations were evaluated: (i) HA proteins carrying complex glycans produced in HEK293T cells; (ii) HA proteins carrying Man9GlcNAc2 moieties, expressed in HEK293T cells treated with kifunensine; (iii) HA proteins containing Man5GlcNAc2 moieties derived from HEK293S GnTI(-) cells; (iv) insect cell-produced HA proteins carrying paucimannosidic N-glycans; and (v) HEK293S GnTI(-) cell-produced HA proteins treated with endoglycosidase H, thus carrying side chains composed of only a single N-acetylglucosamine each. The different HA glycosylation states were confirmed by comparative electrophoretic analysis and by mass spectrometric analysis of released glycans. The immunogenicity of the HA preparations was studied in chickens and mice. The results demonstrate that HA proteins carrying terminal mannose moieties induce significantly lower hemagglutination inhibition antibody titers than HA proteins carrying complex glycans or single N-acetylglucosamine side chains. However, the glycosylation state of the HA proteins did not affect the breadth of the antibody response as measured by an HA1 antigen microarray. We conclude that the glycosylation state of recombinant antigens is a factor of significant importance when developing glycoprotein-based vaccines, such as recombinant HA proteins.
Heparan sulfate facilitates Rift Valley fever virus entry into the cell
Boer, S.M. de; Kortekaas, J.A. ; Haan, C.A. de; Rottier, P.J.M. ; Moormann, R.J.M. ; Bosch, B.J. - \ 2012
Journal of Virology 86 (2012)24. - ISSN 0022-538X - p. 13767 - 13771.
hamster ovary cells - protein - receptor - binding - infection - biosynthesis - glycoprotein - replication - phlebovirus - expression
Rift Valley fever virus (RVFV), an emerging arthropod-borne pathogen, has a broad host and cell tropism. Here we report that the glycosaminoglycan heparan sulfate, abundantly present on the surface of most animal cells, is required for efficient entry of RVFV. Entry was significantly reduced by preincubating the virus inoculum with highly-sulfated heparin, by enzymatic removal of heparan sulfate from cells and in cells genetically deficient in heparan sulfate synthesis.
The F-like protein Ac23 enhances the infectivity of the budded virus of gp64-null Autographa california multinucleocapsid nucleopolyhedrovirus pseudotyped with baculovirus envelope fusion protein F
Wang, M. ; Tan, Y. ; Yin, F. ; Deng, F. ; Vlak, J.M. ; Hu, Z.H. ; Wang, H. - \ 2008
Journal of Virology 82 (2008)19. - ISSN 0022-538X - p. 9800 - 9804.
multicapsid nucleopolyhedrovirus - multiple nucleopolyhedrovirus - gp64 - identification - glycoprotein - homolog - virions - gene
The GP64 and F proteins were previously identified as the sole functional envelope fusion proteins in Baculoviridae. F-like proteins, present only in group I nucleopolyhedroviruses (NPVs), are remnant, nonfunctional F proteins. In this report, we describe the effect of the presence or absence of the F-like protein Ac23 in a gp64-null Autographa californica multinucleocapsid NPV pseudotyped with the F protein from Spodoptera exigua multicapsid NPV (SeF). We found that the presence of Ac23 elevates the infectivity of the pseudotyped virus. This is in contrast to the results of Lung et al. (J. Virol. 76:5729-5736, 2002), who found no such effect. The possible reasons for the differing results are discussed
Presence of indigestible peptide aggregates of soybean meal in pig ileal digesta residue
Fischer, M. ; Voragen, A.G.J. ; Piersma, S.R. ; Kofod, L.V. ; Joergensen, C.I. ; Guggenbuhl, P. ; Nunes, C.S. ; Gruppen, H. - \ 2007
Journal of the Science of Food and Agriculture 87 (2007)12. - ISSN 0022-5142 - p. 2229 - 2238.
n-15-isotope dilution technique - small-intestinal mucus - beta-lactoglobulin - enzymatic extractability - soy proteins - growing pigs - glycine-max - hydrolysis - proteolysis - glycoprotein
With the purpose of analysing the molecular size and composition, proteinaceous material was extracted from the insoluble components of a digesta sample obtained from pigs fed a feed consisting of only soybean meal. Gel permeation chromatography indicated that the alkali-extractable fraction of the proteinaceous material from the residue was of relatively high apparent molecular weight. However, the combined results from gel electrophoresis, RPLC-MS, and MALDI-ToF MS showed that the extracted protein material was in fact, to a high extent, composed of aggregated peptides. To our knowledge this has not previously been described. Aggregates extracted by dilute alkali were fully degraded upon subsequent proteolytic treatment. N-terminal sequencing of selected protein bands from SDS-PAGE gels indicated the presence of partly degraded -conglycinin alpha subunits in the residue
Hydrodynamic Flow-Mediated Protein Sorting on the Cell Surface of Trypanosomes
Engstler, M. ; Pfohl, T. ; Herminghaus, S. ; Boshart, M. ; Wiegertjes, G.F. ; Heddergott, N. ; Overath, P. - \ 2007
Cell 131 (2007). - ISSN 0092-8674 - p. 505 - 515.
blood-stream forms - antigenic variation - dna-molecules - brucei - glycoprotein - endocytosis - membrane - movement - motility - antibody
The unicellular parasite Trypanosoma brucei rapidly removes host-derived immunoglobulin (Ig) from its cell surface, which is dominated by a single type of glycosylphosphatidylinositol-anchored variant surface glycoprotein (VSG). We have determined the mechanism of antibody clearance and found that Ig-VSG immune complexes are passively sorted to the posterior cell pole, where they are endocytosed. The backward movement of immune complexes requires forward cellular motility but is independent of endocytosis and of actin function. We suggest that the hydrodynamic flow acting on swimming trypanosomes causes directional movement of Ig-VSG immune complexes in the plane of the plasma membrane, that is, immunoglobulins attached to VSG function as molecular sails. Protein sorting by hydrodynamic forces helps to protect trypanosomes against complement-mediated immune destruction in culture and possibly in infected mammals but likewise may be of functional significance at the surface of other cell types such as epithelial cells lining blood vessels.
Baculovirus envelope fusion proteins F and GP64 exploit distinct receptors to gain entry into cultured insect cells
Westenberg, M. ; Uijtdewilligen, P. ; Vlak, J.M. - \ 2007
Journal of General Virology 88 (2007). - ISSN 0022-1317 - p. 3302 - 3306.
nuclear polyhedrosis-virus - californica multicapsid nucleopolyhedrovirus - membrane-fusion - recombinant baculovirus - lymantria-dispar - mammalian-cells - genome sequence - lines - glycoprotein - mechanism
Group II nucleopolyhedroviruses (NPVs), e.g. Helicoverpa armigera (Hear) NPV and Spodoptera exigua (Se) MNPV (multiple NPV), lack a GP64-like protein that is present in group I NPVs, e.g. Autographa californica (Ac)MNPV, but have an unrelated envelope fusion protein named F. Three AcMNPV viruses were constructed by introducing AcMNPV gp64, HearNPV f or SeMNPV f genes, respectively, into a gp64-negative AcMNPV bacmid. Sf21 cells were incubated with different amounts of inactivated budded virus to occupy receptors and were subsequently infected with a fixed amount of infectious virus to compete for attachment. The results suggest that GP64 and F act on their own and use different receptors, while the two different F proteins exploit the same receptor. Additionally, gp64-null AcMNPV pseudotyped with baculovirus F was, in contrast to GP64, unable to transduce mammalian cells, indicating that mammalian cells do not possess baculovirus F protein receptors despite the structural similarity of baculovirus F to vertebrate viral fusion proteins.
Aggregation of -Lactoglobulin Regulated by Glucosylation
Broersen, K. ; Elshof, E. ; Groot, J. de; Voragen, A.G.J. ; Hamer, R.J. ; Jongh, H.H.J. de - \ 2007
Journal of Agricultural and Food Chemistry 55 (2007)6. - ISSN 0021-8561 - p. 2431 - 2437.
heat-induced aggregation - amyloid formation - fibril formation - protein - glycoprotein - stability - hydrophobicity - glycosylation - calreticulin - denaturation
A large number of proteins are glycosylated, either in vivo or as a result of industrial processing. Even though the effect of glycosylation on the aggregation of proteins has been studied extensively in the past, some reports show that the aggregation process is accelerated, whereas others found that the process is inhibited by glycosylation. This paper investigates the reasons behind these controversial results as well as the potential mechanism of the effect of glucosylation on aggregation using bovine -lactoglobulin as a model. Glucosylation was found to inhibit denaturant-induced aggregation, whereas heat-induced aggregation was accelerated. It was also found that the kinetic partitioning from an unfolded state was driven toward refolding for glucosylated protein, whereas aggregation was the preferred route for the nonglucosylated protein. Keywords: Aggregation; glucosylation; -lactoglobulin; hydrophobicity; electrostatic repulsion; unfolding/refolding
The effect on pathogenesis of Newcastle Disease Virus LaSota strain from a mutation of the fusion cleavage site to a virulent sequence.
Wakamatsu, N. ; King, D. ; Seal, B.S. ; Peeters, B.P.H. ; Brown, C.C. - \ 2006
Avian Diseases 50 (2006)4. - ISSN 0005-2086 - p. 483 - 488.
domestic chickens - exotic birds - pathogenicity - activation - protein - glycoprotein - determinant - passage
The principal molecular determinant of virulence of Newcastle disease virus (NDV) is the amino acid sequence at the fusion cleavage activation site. To extend the understanding of the role of the fusion cleavage activation site in NDV virulence, the pathogenesis in chickens of a lentogenic LaSota isolate and two infectious clones, NDFL and NDFLtag, were compared. NDFL is an infectious clone of a lentogenic NDV strain (LaSota E13-1), and NDFLtag is the infectious clone with the fusion cleavage site sequence mutated to the virulent motif. NDFL and NDFLtag were described by Peeters et al. The viruses were inoculated intraconjunctivally into groups of 4-wk-old white leghorn chickens and compared in a pathogenesis study for determination of disease causation (clinical signs of disease, gross lesions, histology, virus isolation, and serology) and viral distribution (presence of viral nucleoprotein and mRNA was detected by immunohistochemistry and in situ hybridization, respectively). The modification of the fusion cleavage activation site to the virulent motif in the infectious clone only slightly increased disease severity and viral distribution in the pathogenesis assessment, even though dramatically increased pathogenicity of NDFLtag was confirmed by standard pathogenicity index tests. The result, that the mutated fusion cleavage site of NDV¿NDFLtag had only a small influence on pathogenesis in chickens compared to either E13-1 or NDFL, suggests that the pathogenic effects of NDV are not dependent on the fusion cleavage site alone.
Functional role of the cytoplasmic tail domain of the major envelope fusion protein of group II baculoviruses
Long, G. ; Pan, M. ; Westenberg, M. ; Vlak, J.M. - \ 2006
Journal of Virology 80 (2006)22. - ISSN 0022-538X - p. 11226 - 11234.
californica multicapsid nucleopolyhedrovirus - virus env protein - membrane-fusion - transmembrane protein - cell-surface - r-peptide - glycoprotein - identification - truncation - cleavage
F proteins from baculovirus nucleopolyhedrovirus (NPV) group II members are the major budded virus (BV) viral envelope fusion proteins. They undergo furin-like proteolysis processing in order to be functional. F proteins from different baculovirus species have a long cytoplasmic tail domain (CTD), ranging from 48 (Spodoptera litura multicapsid NPV [MNPV]) to 78 (Adoxophyes honmai NPV) amino acid (aa) residues, with a nonassigned function. This CTD is much longer than the CTD of GP64-like envelope fusion proteins (7 aa), which appear to be nonessential for BV infectivity. Here we have investigated the functional role of the CTD of Helicoverpa armigera single-capsid NPV (HearNPV), a group II NPV. We combined a newly constructed HearNPV f-null bacmid knockout-repair system and an Autographa californica MNPV (AcMNPV) gp64-null bacmid knockout-pseudotype system with mutation and rescue experiments to study the functional role of the baculovirus F protein CTD. We show that except for the 16 C-terminal aa, the HearNPV F CTD is essential for virus spread from cell to cell. In addition, the CTD of HearNPV F is involved in BV production in a length-dependent manner and is essential for BV infectivity. The tyrosine residue Y658, located 16 aa from the C terminus, seems to be critical. However, HearNPV F without a CTD still rescues the infectivity of gp64-null AcMNPV BV, indicating that the CTD is not involved in processing and fusogenicity. Altogether, our results indicate that the F protein is essential for baculovirus BV infectivity and that the CTD is important for F protein incorporation into BV.
A DNA vaccine coding for gB and gD of pseudorabies virus (suid herpes type 1) primes the immune system in the presence of maternal immunity more efficiently than conventional vaccines
Rooij, E.M.A. van; Moonen-Leusen, H.W.M. ; Visser-Hendriksen, Y.E. de; Middel, W.G. ; Boersma, W.J.A. ; Bianchi, A.T.J. - \ 2006
Vaccine 24 (2006)9. - ISSN 0264-410X - p. 1264 - 1273.
aujeszkys disease virus - cell-mediated-immunity - intranasal vaccination - antibody-titers - pigs - responses - protection - infection - glycoprotein - immunization
DNA vaccines are capable of priming the immune system of neonates in the presence of maternal antibodies. However, it is still not clear whether the extent of priming and protection against challenge infections induced by a DNA vaccine in maternally immune newborns is better than that induced by conventional vaccines. To study this, we used the pseudorabies virus (PRV) infection model in the natural host, the pig. We compared the efficacy of a DNA vaccine with the efficacy of a conventional modified live vaccine (MLV) and an inactivated vaccine (IV) in maternally immune newborn piglets. We measured the priming of the immune response and the degree of protection against challenge infection for all vaccine types. We vaccinated piglets with or without maternal immunity twice, at the age of 5 and 9 weeks, and we assessed protection by challenge infection with virulent PRV at the age of 15 weeks. Vaccination with DNA or conventional vaccines induced both humoral and cell-mediated immune responses in maternally immune animals. DNA vaccination seemed not to suffer from suppression by maternal immunity and resulted in similar or stronger immune responses in maternally immune piglets as compared in naïve piglets. In contrast, vaccination with conventional vaccines resulted in weaker immune responses in maternally immune piglets than in naïve piglets. Moreover, DNA vaccination provided better protection against challenge infection in maternally immune piglets than in naive piglets, whereas vaccination with conventional vaccines did not.
Novel rabies virus-neutralizing epitope recognized by human monoclonal antibody: Fine mapping and escape mutant analysis
Marissen, W.E. ; Kramer, R.A. ; Rice, A. ; Weldon, W.C. ; Niezgoda, M. ; Faber, M. ; Slootstra, J.W. ; Meloen, R.H. ; Clijsters-van der Horst, M. ; Visser, T.J. ; Jongeneelen, M. ; Thijsse, S. ; Throsby, M. ; Kruif, J. de; Rupprecht, C.E. ; Dietzschold, B. ; Goudsmit, J. ; Bakker, A.B.H. - \ 2005
Journal of Virology 79 (2005)8. - ISSN 0022-538X - p. 4672 - 4678.
high-level expression - postexposure prophylaxis - glycoprotein - pathogenicity - determinant - virulence - mice - igg
Anti-rabies virus immunoglobulin combined with rabies vaccine protects humans from lethal rabies infections. For cost and safety reasons, replacement of the human or equine polyclonal immunoglobulin is advocated, and the use of rabies virus-specific monoclonal antibodies (MAbs) is recommended. We produced two previously described potent rabies virus-neutralizing human MAbs, CR57 and CRJB, in human PER.C6 cells. The two MAbs competed for binding to rabies virus glycoprotein. Using CR57 and a set of 15-mer overlapping peptides covering the glycoprotein ectodomain, a neutralization domain was identified between amino acids (aa) 218 and 240. The minimal binding region was identified as KLCGVL (aa 226 to 231), with key residues K-CGV- identified by alanine replacement scanning. The critical binding region of this novel nonconformational rabies virus epitope is highly conserved within rabies viruses of genotype 1. Subsequently, we generated six rabies virus variants escaping neutralization by CR57 and six variants escaping CRJB. The CR57 escape mutants were only partially covered by CRJB, and all CRJB-resistant variants completely escaped neutralization by CR57. Without exception, the CR57-resistant variants showed a mutation at key residues within the defined minimal binding region, while the CRJB escape viruses showed a single mutation distant from the CR57 epitope (N182D) combined with mutations in the CR57 epitope. The competition between CR57 and CRJB, the in vitro escape profile, and the apparent overlap between the recognized epitopes argues against including both CR57 and CRJB in a MAb cocktail aimed at replacing classical immunoglobulin preparations.
Envelope protein requirements for the assembly of infectious virions of porcine reproductive and respiratory syndrome virus
Wissink, E.H.J. ; Kroese, M.V. ; Wijk, H.A. van; Rijsewijk, F.A.M. ; Meulenberg, J.J. ; Rottier, P.J.M. - \ 2005
Journal of Virology 79 (2005)19. - ISSN 0022-538X - p. 12495 - 12506.
equine-arteritis-virus - dehydrogenase-elevating virus - lelystad-virus - structural protein - membrane-fusion - alveolar macrophages - culture-medium - fever virus - glycoprotein - identification
Virions of porcine reproductive and respiratory syndrome virus (PRRSV) contain six membrane proteins: the major proteins GP5 and M and the minor proteins GP2a, E, GP3, and GP4. Here, we studied the envelope protein requirements for PRRSV particle formation and infectivity using full-length cDNA clones in which the genes encoding the membrane proteins were disrupted by site-directed mutagenesis. By transfection of RNAs transcribed from these cDNAs into BHK-21 cells and analysis of the culture medium using ultracentrifugation, radioimmunoprecipitation, and real-time reverse transcription-PCR, we observed that the production of viral particles is dependent on both major envelope proteins; no particles were released when either the GP5 or the M protein was absent. In contrast, particle production was not dependent on the minor envelope proteins. Remarkably, in the absence of any one of the latter proteins, the incorporation of all other minor envelope proteins was affected, indicating that these proteins interact with each other and are assembled into virions as a multimeric complex. Independent evidence for such complexes was obtained by coexpression of the minor envelope proteins in BHK-21 cells using a Semliki Forest virus expression system. By analyzing the maturation of their N-linked oligosaccharides, we found that the glycoproteins were each retained in the endoplasmic reticulum unless expressed together, in which case they were collectively transported through the Golgi complex to the plasma membrane and were even detected in the extracellular medium. As the PRRSV particles lacking the minor envelope proteins are not infectious, we hypothesize that the virion surface structures formed by these proteins function in viral entry by mediating receptor binding and/or virus-cell fusion
Differential gene expression in rat colon by dietary heme and calcium
Meer - van Kraaij, C. van der; Kramer, E.H.M. ; Jonker - Termont, D. ; Katan, M.B. ; Meer, R. van der; Keijzer, J. - \ 2005
Carcinogenesis 26 (2005)1. - ISSN 0143-3334 - p. 73 - 79.
carbonic-anhydrase-i - amyloid-p component - red meat - colorectal-cancer - protein - cells - glycoprotein - pentraxins - colitis - cloning
Dietary heme and calcium are alleged modulators of colon cancer risk. Little is known about the molecular and cellular changes in the colon epithelium that are induced by consumption of these unabsorbed nutrients. In this nutrigenomics study, we fed rats high- and low-calcium diets with or without heme. In agreement with previous studies, we found that dietary heme increased the cytotoxicity of fecal water in the colon and elevated epithelial proliferation, a risk factor in colon carcinogenesis. Calcium reduced cytotoxicity and inhibits heme-induced effects. Among 365 colon-expressed genes, we could identify 10 diet-modulated genes that show >2-fold altered expression, of which several are related to colon cell turnover and disease. Mucosal pentraxin (Mptx) was the strongest differentially expressed gene, similar to10-fold down-regulated by dietary heme and 3-fold up-regulated by calcium. cDNA microarray and quantitative PCR analysis show that calcium significantly inhibits the effects of heme, which correlates with the physiological effects. Our results indicate that Mptx expression is related to colonic cell turnover, and that Mptx might be a marker for diet-modulated mucosal integrity. We also show that Mptx expression is restricted to the intestine, and occurs predominantly in the colon.
Glutathione peroxidases of the potato cyst nematode Globodera Rostochiensis
Jones, J.T. ; Reavy, B. ; Smant, G. ; Prior, A.E. - \ 2004
Gene 324 (2004). - ISSN 0378-1119 - p. 47 - 54.
heterodera-glycines - messenger-rna - retinol-binding - brugia-pahangi - expression - gene - selenium - identification - transcription - glycoprotein
We report the cloning and characterisation of full-length DNAs complementary to RNA (cDNAs) encoding two glutathione peroxidases (GpXs) from a plant parasitic nematode, the potato cyst nematode (PCN) Globodera rostochiensis. One protein has a functional signal peptide that targets the protein for secretion from animal cells while the other is predicted to be intracellular. Both genes are expressed in all parasite stages tested. The mRNA encoding the intracellular GpX is present throughout the nematode second stage juvenile and is particularly abundant in metabolically active tissues including the genital primordia. The mRNA encoding the secreted GpX is restricted to the hypodermis, the outermost cellular layer of the nematode, a location from which it is likely to be secreted to the parasite surface. Biochemical studies confirmed the secreted protein as a functional GpX and showed that, like secreted GpXs of other parasitic nematodes, it does not metabolise hydrogen peroxide but has a preference for larger hydroperoxide substrates. The intracellular protein is likely to have a role in metabolism of active oxygen species derived from internal body metabolism while the secreted protein may protect the parasite from host defences. Other functional roles for this protein are discussed.
Functional analysis of the putative fusion domain of the Baculovirus envelope fusion protein F
Westenberg, M. ; Veenman, F. ; Roode, E.C. ; Goldbach, R.W. ; Vlak, J.M. - \ 2004
Journal of Virology 78 (2004)13. - ISSN 0022-538X - p. 6946 - 6954.
californica multicapsid nucleopolyhedrovirus - membrane-fusion - genome sequence - cell-fusion - glycoprotein - virus - gp64 - peptide - site - mutagenesis
Group II nucleopolyhedroviruses (NPVs), e.g., Spodoptera exigua MNPV, lack a GP64-like protein that is present in group I NPVs but have an unrelated envelope fusion protein named F. In contrast to GP64, the F protein has to be activated by a posttranslational cleavage mechanism to become fusogenic. In several vertebrate viral fusion proteins, the cleavage activation generates a new N terminus which forms the so-called fusion peptide. This fusion peptide inserts in the cellular membrane, thereby facilitating apposition of the viral and cellular membrane upon sequential conformational changes of the fusion protein. A similar peptide has been identified in NPV F proteins at the N terminus of the large membrane-anchored subunit F-1. The role of individual amino acids in this putative fusion peptide on viral infectivity and propagation was studied by mutagenesis. Mutant F proteins with single amino acid changes as well as an F protein with a deleted putative fusion peptide were introduced in gp64-null Autographa californica MNPV budded viruses (BVs). None of the mutations analyzed had an major effect on the processing and incorporation of F proteins in the envelope of BVs. Only two mutants, one with a substitution for a hydrophobic residue (F152R) and one with a deleted putative fusion peptide, were completely unable to rescue the gp64-null mutant. Several nonconservative substitutions for other hydrophobic residues and the conserved lysine residue had only an effect on viral infectivity. In contrast to what was expected from vertebrate virus fusion peptides, alanine substitutions for glycines did not show any effect.
Evaluation of baculovirus expression vectors with enhanced stability in continuous cascaded insect-cell bioreactors
Pijlman, G.P. ; Vrij, J. de; End, E.J. van den; Vlak, J.M. ; Martens, D.E. - \ 2004
Biotechnology and Bioengineering 87 (2004)6. - ISSN 0006-3592 - p. 743 - 753.
nuclear polyhedrosis-virus - beta-galactosidase production - non-hr origin - spodoptera-exigua - recombinant baculoviruses - dna-replication - genome sequence - infection - gene - glycoprotein
Continuous protein production with baculovirus expression vectors in insect-cell bioreactors is characterized by a dramatic drop in heterologous protein production within a few weeks. This is mainly due to the spontaneous deletion of the heterologous gene(s) from the baculovirus genome and/or to the rapid accumulation of defective interfering baculoviruses (DIs). Cell culture experiments with bacmid-derived baculoviruses showed that spontaneous deletions in the foreign bacterial artificial chromosome (BAC) sequences readily occurred, These deletions correlated with a low density of baculovirus homologous (repeat) regions (hrs), which are located dispersed throughout the baculovirus genome and are believed to act as origins of viral DNA replication (oris). To test the hypothesis that deletions are more likely to occur in regions with a low ori density, the properties of bacmidderived baculoviruses with an additional hr in the unstable BAC sequences were compared to the standard bacmidderived baculovirus in a continuous cascaded insect-cell bioreactor configuration. All viruses were equipped with a green fluorescent protein (GFP) gene and a gene encoding the classical swine fever virus E2 glycoprotein (CSFV-E2). The insertion of an extra hr in the BAC vector led to improved genetic stability of adjacent sequences, resulting in prolonged protein expression. The maintenance of the BAC sequences appeared to be dependent on the orientation of the inserted hr. The advantages of the utilization of hrs to improve the stability of baculovirus expression vectors for the large-scale protein production in insect-cell bioreactors are discussed. (C) 2004 Wiley Periodicals, Inc.
Effect of fusion protein cleavage site mutations on virulence of Newcastle disease virus: non-virulent cleavage site mutants revert to virulence after one passage in chicken brain.
Leeuw, O.S. de; Hartog, L. ; Koch, G. ; Peeters, B.P.H. - \ 2003
Journal of General Virology 84 (2003)2. - ISSN 0022-1317 - p. 475 - 484.
phylogenetic-relationships - proteolytic cleavage - nucleotide-sequence - molecular evolution - avirulent strains - mammalian-cells - activation site - factor-x - glycoprotein - pathogenicity
Virulence of Newcastle disease virus (NDV) is mainly determined by the amino acid sequence of the fusion (F0) protein cleavage site. Full-length NDV cDNA clone pNDFL was used to generate infectious NDV with defined mutations in the F0 cleavage site (RRQRR downward arrow L, GRQGR downward arrow F, RRQGR downward arrow F, RGQRR downward arrow F and RKQKR downward arrow F). All the mutants were viable and the mutations were maintained after virus propagation in embryonated eggs. The mutants showed single-cell infections on chicken embryo fibroblasts, which suggested that they were non-virulent. However, virulence tests in 1-day-old chickens resulted in an intracerebral pathogenicity index (ICPI) between 0 and 1.3. Moreover, virulent virus was isolated from chickens that had died in the virulence tests. Subsequent sequence analysis showed that the mutants RRQRR downward arrow L, RRQGR downward arrow F, RGQRR downward arrow F and RKQKR downward arrow F gave rise to the appearance of revertants containing the virulent cleavage site RRQ(K/R)R downward arrow F and an ICPI of 1.4 or higher. This indicated that reversion to virulence was caused by alteration of the amino acid sequence of the F0 cleavage site from a non-virulent to a virulent type. Furthermore, the ICPI of the revertants was higher than that of cDNA-derived strain NDFLtag, which has the same cleavage site, RRQRR downward arrow F (ICPI=1.3). NDFLtag(Pass), which was isolated from dead chickens after intracerebral inoculation of NDFLtag, also showed an increase in the ICPI from 1.3 to 1.5. This study proves that reversion to virulence occurs within non-virulent NDV populations and that the virulence may increase after one passage in chicken brain