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At least 1,207 records · Page 67Linked to original sources
PubMed2001

VP5 and the N terminus of VP2 are not responsible for the different pathotype of serotype I and II infectious bursal disease virus.

Two serotypes have been identified in infectious bursal disease virus (IBDV), a member of the family BIRNAVIRIDAE: A reverse genetics system was used for generation of chimeras in genome segment A of the two serotypes, in which the complete viral VP5 gene and 3' noncoding region (NCR), or parts thereof, were exchanged. The engineered viruses were characterized in vitro and in vivo in comparison to serotype I and II IBDV. Our results show that IBDV chimeras exhibit a different phenotype in cell culture compared to the wild-type viruses. In in vitro-cultivated bursal-derived cells, chimeric viruses infected B lymphocytes, as does serotype I IBDV. Surprisingly, serotype II virus was also able to infect in vitro-cultivated bursal cells, but these were neither B lymphocytes nor macrophages. After infection of susceptible chickens all chimeras replicated in the bursa of Fabricius (BF), and three chimeric viruses caused mild depletion of bursal cells. In contrast, after infection of chickens with a chimeric IBDV containing exchanged VP5 as well as 3'-NCR, no depletion was detectable. The serotype II strain did not replicate in the BF nor did it cause depletion of bursal cells. Thus, the origin of VP5 does not explain the different pathotype of IBDV serotype I and II.

3' Untranslated Regions↗
PubMed2001

Recombinant bovine respiratory syncytial virus with deletions of the G or SH genes: G and F proteins bind heparin.

Bovine respiratory syncytial virus (BRSV) encodes three transmembrane envelope glycoproteins, namely the small hydrophobic (SH) protein, the attachment glycoprotein (G) and the fusion glycoprotein (F). The BRSV reverse genetics system has been used to generate viable recombinant BRSV lacking either the G gene or the SH gene or both genes. The deletion mutants were fully competent for multicycle growth in cell culture, proving that, of the BRSV glycoprotein genes, the SH and G genes are non-essential. Virus morphogenesis was not impaired by either of the deletions. The deletion mutants were used to study the role of the F glycoprotein and the contributions of SH and G with respect to virus attachment. Attachment mediated by the F protein alone could be blocked by soluble heparin, but not by chondroitin sulphate. Heparin affinity chromatography revealed that both the BRSV G and F glycoproteins have heparin-binding activity, with the affinity of the F glycoprotein being significantly lower than that of G. Therefore, the roles of the BRSV glycoproteins in virus attachment and receptor binding have to be reconsidered.

Animals↗
PubMed2001

Expression of VP5 of infectious pancreatic necrosis virus strain VR299 is initiated at the second in-frame start codon.

Infectious pancreatic necrosis virus (IPNV), a member of the BIRNAVIRIDAE: with two double-stranded RNA genome segments, encodes five proteins designated VP1 to VP5. To study the function of the 17 kDa nonstructural protein VP5 during virus replication several mutated IPNV genome segments A were constructed and included in a reverse genetics system for IPNV to obtain recombinant virus. Mutations between nt 68 and 85 or nt 94 and 103 in the noncoding region failed to yield viable virus. Only mutations located between nt 86 and 92 and downstream of nt 104 were tolerated, and viable virus could be generated. All IPNV generated showed no difference in replication compared with the wild-type IPNV, indicating that the absence of expression of VP5 did not influence virus growth in vitro. Furthermore, the results presented here indicate that initiation of translation of VP5 occurs at position 113, the second in-frame start codon.

Amino Acid Sequence↗
PubMed2001

Infectious RNA transcribed in vitro from a cDNA copy of the human coronavirus genome cloned in vaccinia virus.

The coronavirus genome is a positive-strand RNA of extraordinary size and complexity. It is composed of approximately 30000 nucleotides and it is the largest known autonomously replicating RNA. It is also remarkable in that more than two-thirds of the genome is devoted to encoding proteins involved in the replication and transcription of viral RNA. Here, a reverse-genetic system is described for the generation of recombinant coronaviruses. This system is based upon the in vitro transcription of infectious RNA from a cDNA copy of the human coronavirus 229E genome that has been cloned and propagated in vaccinia virus. This system is expected to provide new insights into the molecular biology and pathogenesis of coronaviruses and to serve as a paradigm for the genetic analysis of large RNA virus genomes. It also provides a starting point for the development of a new class of eukaryotic, multi-gene RNA vectors that are able to express several proteins simultaneously.

Animals↗
PubMed2002

Alteration of amino acids in VP2 of very virulent infectious bursal disease virus results in tissue culture adaptation and attenuation in chickens.

Reverse genetics technology offers the possibility to study the influence of particular amino acids of infectious bursal disease virus (IBDV) on adaptation to tissue culture. Genomic segments A and B of the very virulent (vv) IBDV field strain UK661 were completely cloned and sequenced, and the strain was rescued from full-length cDNA copies of both segments (UK661rev). Using site-directed mutagenesis, alteration of a single amino acid in the segment A-encoded VP2 (A284T) resulted in a limited capacity of UK661 to replicate in tissue culture. Additional alteration of a second amino acid (Q253H) increased replication efficiency in tissue culture. The second mutant (UK661-Q253H-A284T) was used to infect chickens and results were compared with UK661 and UK661rev. Whereas UK661 and UK661rev induced 100% morbidity and 50-80% mortality, UK661-Q253H-A284T proved to be strikingly attenuated, producing neither morbidity nor mortality. Moreover, UK661-Q253H-A284T-infected animals were protected from challenge infection. Thus, alteration of two specific amino acids in the VP2 region of IBDV resulted in tissue culture adaptation and attenuation in chickens of vvIBDV. The data demonstrate that VP2 plays a decisive role in pathogenicity of IBDV.

Adaptation, Physiological↗
PubMed1997

XylUW, two genes at the start of the upper pathway operon of TOL plasmid pWW0, appear to play no essential part in determining its catabolic phenotype.

The upper pathway operon of the toluene catabolic pathway of TOL plasmid pWW0 was shown to carry two open reading frames between the start of transcription and xylC (encoding benzaldehyde dehydrogenase), the first previously reported gene of the operon. These were designated xylUW: xylU encoded a protein of 131 amino acid residues (M(r) 14,244) which bore no relationship with any protein in the databases, and xylW encoded a protein of 348 residues (M(r) 36,992) which was strongly homologous to other long-chain Zn-containing alcohol dehydrogenases. Extracts of Escherichia coli carrying xylUW in expression vector pTrc99A contained a novel protein corresponding to XylW, but no NAD(+)-dependent dehydrogenase activity against benzyl alcohol, mandelate or bezylamine. A mini-Tn5 transposon carrying the meta pathway operon was constructed and from it two strains of Pseudomonas putida were constructed with the normally plasmid-encoded catabolic operons integrated into the chromosome. Three derivatives of plasmid pKNG101 containing modified xylUW genes were constructed, two of which had frameshifts in xylU and xylW, respectively, and a third with a deletion from the 3' end of xylU into the 5' end of xylW. The wild-type genes of the two Pseudomonas strains were substituted by the mutant alleles by reverse genetics. The ability of the constructed mutant strains to utilize the aromatic substrates of the TOL pathway was not significantly affected.

Amino Acid Sequence↗
PubMed2000

Expression and purification of four different rhizobial acyl carrier proteins.

In rhizobia, besides the constitutive acyl carrier protein (AcpP) involved in the biosynthesis and transfer of common fatty acids, there are at least three specialized acyl carrier proteins (ACPs): (1) the flavonoid-inducible nodulation protein NodF; (2) the RkpF protein, which is required for the biosynthesis of rhizobial capsular polysaccharides; and (3) AcpXL, which transfers 27-hydroxyoctacosanoic acid to a sugar backbone during lipid A biosynthesis. Whereas the nucleotide sequences encoding the three specialized ACPs are known, only the amino acid sequence of the AcpP of Sinorhizobium meliloti was available. In this study, using reverse genetics, the genes for the constitutive AcpPs of S. meliloti and of Rhizobium leguminosarum were cloned and sequenced. Previously, it had been shown that NodF and RkpF can be overproduced in Escherichia coli using the T7 polymerase expression system. Using the same system, the constitutive AcpPs of S. meliloti and of R. leguminosarum, together with the specialized ACP AcpXL, were overproduced and purified. All the known ACPs of rhizobia can be labelled in vivo during expression in E. coli with radioactive beta-alanine added to the growth medium due to their modification with a 4'-phosphopantetheine prosthetic group. The availability of all functionally different ACPs should help to unravel how different fatty acids are targeted towards different biosynthetic pathways in one organism.

Acyl Carrier Protein↗
PubMed2001

A Bacillus amyloliquefaciens ChbB protein binds beta- and alpha-chitin and has homologues in related strains.

A small (19.8 kDa) protein was identified in Bacillus amyloliquefaciens ALKO 2718 cultures during growth in the presence of yeast extract and chitin, but not with glucose. The protein targets beta-chitin best, then alpha-chitin, but barely any other polysaccharide. This described chitin-binding protein (ChbB) is the first of its type from a Bacillus strain and cross-reacts with antibodies raised against the Streptomyces alpha-chitin-binding protein CHB1. Using reverse genetics, the chromosomal chbB gene of strain ALKO 2718 was identified, cloned and sequenced. ChbB shares several motifs with the alpha-chitin-binding proteins CHB1 and CHB2 of Streptomyces and CBP21 of Serratia marcescens predominantly targeting beta-chitin. Synthesis was repressed by glucose and the presence of cre boxes suggests catabolite control. Using PCR, Southern hybridization and anti-ChbB antibodies, the presence of a chbB gene, as well as of a ChbB protein homologue, was ascertained in several tested B. amyloliquefaciens strains, but not in Bacillus subtilis 168. Contrary to B. subtilis 168, all B. amyloliquefaciens strains secreted varying amounts of enzymic activity, degrading carboxymethyl chitin coupled with Remazol brilliant violet.

Amino Acid Sequence↗
PubMed2002

Bacillus amyloliquefaciens orthologue of Bacillus subtilis ywrO encodes a nitroreductase enzyme which activates the prodrug CB 1954.

A nitroreductase with distinct properties that can activate the prodrug 5-aziridinyl-2,4-dinitrobenzamide (CB 1954) was isolated from Bacillus amyloliquefaciens. The encoding gene was identified as a homologue of the ywrO of Bacillus subtilis, and was obtained as a PCR product by reverse genetics, cloned and the entire nucleotide sequence determined. The gene was found to reside between homologues of the B. subtilis alsD and yswB genes; however, the ywrO and yswB genes of B. amyloliquefaciens were not separated by a fourth gene, ywsA. The B. amyloliquefaciens ywrO gene was overexpressed, the recombinant protein purified and its properties were compared with those of two CB 1954-activating enzymes, Escherichia coli B nitroreductase (NTR) and Walker DT-diaphorase (DTD). In common with these enzymes menadione was an electron acceptor (K(m) 3 microM) and activity with this substrate was inhibited by the presence of dicoumarol (K(i) 1.0 microM). In contrast, YwrO showed a marked preference for NADPH as a cofactor (K(m) 40 microM) and therefore could not be classified as a DTD (EC 1.6.99.2). The flavin FMN was an acceptor with high affinity. B. amyloliquefaciens YwrO was shown to be a flavoprotein with a monomeric molecular mass of 21.5 kDa by calculation and SDS-PAGE. The cytotoxic 4-hydroxylamine derivative was the single CB 1954 reduction product, but B. amyloliquefaciens YwrO was inactive with the bischloroethyl analogue of CB 1954, SN 23862. In both of these properties B. amyloliquefaciens YwrO more closely resembles DTD than NTR. Its K(m) for CB 1954 was lower than that of NTR (617 microM compared to 862 microM). Enhanced in vitro cytotoxicity of CB 1954 was demonstrated on incubation of V79 cells with prodrug, NADPH and B. amyloliquefaciens YwrO. The work has led to the identification of a previously unknown nitroreductase, B. amyloliquefaciens YwrO, with distinct properties which will aid the rational selection of appropriate genes for applications in directed enzyme prodrug therapy (DEPT).

Amino Acid Sequence↗
PubMed1995

Mesentericin Y105 gene clusters in Leuconostoc mesenteroides Y105.

Because of their potential usefulness as natural food preservatives, increased interest has focused on bacteriocins from lactic acid bacteria. Mesentericin Y105 is a small non-lantibiotic bacteriocin (class II) encoded within a 35 kb plasmid from Leuconostoc mesenteroides Y105 and it is active against Listeria monocytogenes. Using reverse genetic methodologies, an 8 kb DraII fragment has been cloned that contains the mesentericin Y105 structural gene, mesY, which encodes a precursor of the bacteriocin with a 24 amino acid N-terminal extension ending with a Gly-Gly motif upstream of the cleavage site, which is typical of class II bacteriocins. Four other putative genes are associated with mesY within two divergent putative operons. In addition to mesY, the first putative operon is predicted to encode a protein, similar to that encoded by ORF2 in the leucocin A operon, whose function remains to be elucidated. The second putative operon contains three ORFs, two of which, mesD and mesE, encode proteins that resemble ATP-dependent transporters and accessory factors, respectively. For three other class II bacteriocin systems (lactococcin A, pediocin PA-1, colicin V), these proteins have been shown to be involved in bacteriocin secretion independently of the general sec-dependent secretion pathway. The last putative gene (mesC) does not resemble any previously characterized gene. Results concerning the heterologous expression of the cloned mesY in Lactobacillus johnsonii NCK64 suggest that the maturation and secretion functions dedicated to lactacin F (another class II bacteriocin) are efficient for mesentericin Y105 as well. This characteristic may be of great interest in the development of industrial fermentation starters producing multiple bactericidal activities.

ATP-Binding Cassette Transporters↗
PubMed2005

Aminopeptidases and dipeptidyl-peptidases secreted by the dermatophyte Trichophyton rubrum.

The nature of secreted aminopeptidases in Trichophyton rubrum was investigated by using a reverse genetic approach. T. rubrum genomic and cDNA libraries were screened with Aspergillus spp. and Saccharomyces cerevisiae aminopeptidase genes as the probes. Two leucine aminopeptidases, ruLap1 and ruLap2, and two dipeptidyl-peptidases, ruDppIV and ruDppV, were characterized and compared to orthologues secreted by Aspergillus fumigatus using a recombinant protein from Pichia pastoris. RuLap1 is a 33 kDa nonglycosylated protein, while ruLap2 is a 58-65 kDa glycoprotein. The hydrolytic activity of ruLap1, ruLap2 and A. fumigatus orthologues showed various preferences for different aminoacyl-7-amido-4-methylcoumarin substrates, and various sensitivities to inhibitors and cations. ruDppIV and ruDppV showed similar activities to A. fumigatus orthologues. In addition to endopeptidases, the four aminopeptidases ruLap1, ruLap2, ruDppIV and ruDppV were produced by T. rubrum in a medium containing keratin as the sole nitrogen source. Synergism between endo- and exopeptidases is likely to be essential for dermatophyte virulence, since these fungi grow only in keratinized tissues.

Amino Acid Sequence↗
PubMed2003

Functional L polymerase of La Crosse virus allows in vivo reconstitution of recombinant nucleocapsids.

La Crosse virus (LACV), a member of the family Bunyaviridae, is the primary cause of paediatric encephalitis in the United States. In this study, a functional RNA polymerase (L) gene of LACV was cloned and a reverse genetics system established. A reporter minireplicon mimicking the viral genome was constructed by flanking the Renilla luciferase gene with the 3' and 5' noncoding regions of the genomic M segment. These noncoding regions serve as promoters for the viral polymerase. Both L and nucleocapsid (N) genes were expressed by means of T7 RNA polymerase, which was provided by the recombinant T7-expressing modified vaccinia virus Ankara. Renilla reporter activity in transfected cells reflected reconstitution of recombinant nucleocapsids by functional L and N gene products. Time-course experiments revealed a rapid increase in minireplicon activity from 10 to 18 h after the onset of L and N expression. Minireplicon activity was found to be dependent on the correct ratio of L to N plasmids, with too much of either construct resulting in downregulation. Furthermore, a specific inhibitory effect of LACV NSs protein on minireplicon activity was found. In passaging experiments using parental helper virions, it was demonstrated that the recombinant nucleocapsids are a useful model for transcription, replication and packaging of LACV.

Animals↗
PubMed2003

Recombinant Newcastle disease virus as a viral vector: effect of genomic location of foreign gene on gene expression and virus replication.

Newcastle disease virus (NDV) was examined for its suitability as a vector for the expression and delivery of foreign genes for vaccination and gene therapy. A reporter gene encoding human secreted alkaline phosphatase (SEAP) was inserted as an additional transcription unit at four different positions in the NDV genome, between the NP and P, M and F, and HN and L genes and behind the L gene. Eight infectious recombinant NDV (rNDV) viruses, four in the non-virulent strain NDFL and four in the virulent derivative NDFLtag, were generated by reverse genetics. SEAP expression levels, replication kinetics and virus yield were examined. Replication kinetics of the rNDV viruses in primary chicken embryo fibroblasts showed that the insertion of an additional gene resulted in a delay in the onset of replication. This effect was most prominent when the gene was inserted between the NP and P genes. With the exception of the strain that carried the SEAP gene behind the L gene, all recombinant strains expressed high levels of SEAP, both in cell culture and in embryonated chicken eggs. In embryonated eggs, the rNDV viruses showed a 2.6- to 5.6-fold (NDFL) or 2.1- to 8.1-fold (NDFLtag) reduction in yield compared with the parent strains. These results show that foreign genes can be inserted at different positions in the NDV genome without severely affecting replication efficiency or virus yield.

Alkaline Phosphatase↗
PubMed2003

Conserved cysteine and histidine residues in the putative zinc finger motif of the influenza A virus M1 protein are not critical for influenza virus replication.

The influenza virus matrix protein (M1) possesses a cysteine and histidine (CCHH) motif in the helix 9 (H9) and adjacent region ((148)CATCEQIADSQHRSH(162)). The CCHH motif has been proposed as a putative zinc finger motif and zinc-binding activity has been implicated in virus uncoating as well as transcription inhibition and mRNA regulation. The function of the CCHH motif in the influenza virus life cycle was investigated by site-directed mutagenesis (alanine replacement) and by rescuing mutant viruses by reverse genetics. Mutant viruses containing an alanine replacement of the cysteine and histidine residues, either individually or in combination, were seen to exhibit wt phenotype in multiple virus growth cycles and plaque morphology. In addition, synthetic peptides containing the putative zinc finger motif did not inhibit virus replication in MDCK cells. However, mutation of Ala(155) in H9 was lethal for rescuing infectious virus. These data show that the CCHH motif does not provide a critical function in the influenza virus life cycle in cell culture and that the zinc-binding function may not be involved in virus biology. However, the lethal phenotype of the Ala(155) mutation shows that the H9 region of M1 provides some other critical function(s) in virus replication.

Amino Acid Motifs↗
PubMed2003

Virions of Pariacoto virus contain a minor protein translated from the second AUG codon of the capsid protein open reading frame.

Virions of the alphanodavirus Pariacoto virus (PaV) have T=3 icosahedral symmetry and are assembled from multiple copies of a precursor protein that is cleaved into two mature capsid proteins after assembly. The crystal structure of PaV shows that the N-terminal approximately 30 amino acid residues of the subunits surrounding the 5-fold axes interact extensively with icosahedrally ordered regions of the encapsidated positive-sense genomic RNAs. We found that wild-type PaV particles also contain a minor capsid protein that is truncated by 24 residues at its N terminus. Reverse genetic experiments showed that translation of this protein initiated at the second AUG of the capsid protein open reading frame. When either the longer or shorter version of the capsid protein was expressed independently of the other, it assembled into virus particles and underwent maturational cleavage. Virions that lacked the shorter capsid protein retained infectivity for cultured insect cells and Galleria mellonella larvae.

Animals↗
PubMed2005

Genome sequence of the non-pathogenic strain 15 of pneumonia virus of mice and comparison with the genome of the pathogenic strain J3666.

Pneumonia virus of mice (PVM) is a member of the subfamily Pneumovirinae and is the closest known relative of respiratory syncytial virus. Both viruses cause pneumonia in their respective hosts. Here, the genome sequences of two strains of PVM, non-pathogenic strain 15 and pathogenic strain J3666, are reported. Comparison of the genome sequences revealed 59 nucleotide differences between the two strains, 37 of which were coding. The nucleotide differences were spread throughout the genome, affecting cis-acting regulatory regions and seven of the ten genes. Development of a reverse-genetics system for PVM should allow further elucidation of the functional importance of the genetic differences between the two strains identified here.

Amino Acid Sequence↗
PubMed2005

Characterization of Potato virus Y (PVY) molecular determinants involved in the vein necrosis symptom induced by PVYN isolates in infected Nicotiana tabacum cv. Xanthi.

Viral molecular determinant(s) involved in the tobacco vein necrosis (TVN) symptom induced by necrotic isolates of Potato virus Y (PVY) on Nicotiana tabacum cv. Xanthi leaves remain undetermined. Reference isolates belonging to PVY(N) (infectious PVY(N)-605 clone) and PVY(O) (PVY(O)-139) were used to produce PVY chimeric genomes by using reverse-genetic techniques. These chimeric clones were inoculated biolistically onto Nicotiana clevelandii plants to establish the clone, prior to being tested on N. tabacum for their ability to induce TVN symptoms. Comparison between sequence data and symptoms observed for each mutated PVY construct shows that the C-terminal part of the multifunctional HC-Pro protein includes two residues (K(400) and E(419)) that are involved in TVN induced by PVY(N) isolates. Site-directed mutagenesis was used to confirm that these two HC-Pro residues are involved in the TVN phenotype.

Amino Acid Sequence↗
PubMed2006

Proteolytic maturation of replicase polyprotein pp1a by the nsp4 main proteinase is essential for equine arteritis virus replication and includes internal cleavage of nsp7.

The positive-stranded RNA genome of the arterivirus Equine arteritis virus (order Nidovirales) encodes the partially overlapping replicase polyproteins pp1a (1727 aa) and pp1ab (3175 aa). Previously, three viral proteinases were reported to cleave these large polyproteins into 12 non-structural proteins (nsps). The chymotrypsin-like viral main proteinase residing in nsp4 is responsible for eight of these cleavages. Processing of the C-terminal half of pp1a (the nsp3-8 region) was postulated to occur following either of two alternative proteolytic pathways (the 'major' and 'minor' pathways). Here, the importance of these two pathways was investigated by using a reverse-genetics system and inactivating each of the cleavage sites by site-directed mutagenesis. For all of these pp1a cleavage sites, mutations that prevented cleavage by the nsp4 proteinase were found to block or severely inhibit EAV RNA synthesis. Furthermore, our studies identified a novel nsp4 cleavage site (Glu-1575/Ala-1576) that is located within nsp7 and is conserved in arteriviruses. The N-terminal nsp7 fragment (nsp7alpha) derived from this cleavage was detected in lysates of both EAV-infected cells and cells transiently expressing pp1a. Mutagenesis of the novel cleavage site in the context of an EAV full-length cDNA clone proved to be lethal, underlining the fact that the highly regulated, nsp4-mediated processing of the C-terminal half of pp1a is a crucial event in the arterivirus life cycle.

Animals↗