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PubMed2005

Construction and characterization of a fowlpox virus field isolate whose genome lacks reticuloendotheliosis provirus nucleotide sequences.

Fowlpox virus (FWPV) has been isolated from vaccinated chicken flocks during subsequent fowlpox outbreaks that were characterized by a high degree of mortality and significant economic losses. This inability of current vaccines to induce adequate immunity in poultry could be reflective of an antigenic and/or biologic distinctiveness of FWPV field isolates. In this regard, whereas an infectious reticuloendotheliosis virus (REV) provirus is present in the majority of the field viruses' genomes, only remnants of REV long terminal repeats (LTR) have been retained in the DNAs of each vaccine strain. Although it has not been demonstrated whether the partial LTRs can provide an avenue for FWPV to reacquire the REV provirus by homologous recombination, utilizing viruses of which genomes lack any known integrated retroviral sequences could resolve concern over this issue. Therefore, such an entity was created by genetically modifying a recently isolated field strain of FWPV. This selection, in lieu of a commercial vaccine virus, as the progenitor was based on the probability that a virus circulating in the environment would be more antigenically similar to others in this locale and thus might be a better candidate for vaccine development. A comparison in vivo of the pathogenic traits of the parental wild-type field isolate, its genetically modified progeny, and a rescue mutant in whose genome the REV provirus was inserted at its previous location, indicated that elimination of the provirus sequence correlated with reduced virulence. However, even with elimination of the parasitic REV, the modified FWPV was still slightly more invasive than a commercial vaccine virus. Interestingly, both types of attenuated FWPV elicited a similar degree of antibody production in inoculated chickens and afforded them protection against a subsequent challenge by a field virus, the origin of which was temporally and geographically distinct from that of the progenitor strain. Due to its antigenicity being retained despite a decrease in virulence, this REV-less FWPV could potentially be developed as a vaccine against fowlpox.

Animals↗

PubMed2004

In vivo analysis of the 3' untranslated region of GB virus B after in vitro mutagenesis of an infectious cDNA clone: persistent infection in a transfected tamarin.

GB virus B (GBV-B), the virus most closely related to hepatitis C virus (HCV), infects tamarins and causes acute hepatitis. The 3' untranslated region (UTR) of an infectious GBV-B clone (pGBB) has a proximal short sequence followed by a poly(U) tract and a 3' terminal sequence. Our investigators previously demonstrated that the 3' terminal sequence was critical for in vivo infectivity. Here, we tested the effect of deleting the short sequence and/or the poly(U) tract from pGBB; infectivity of each mutant was tested by intrahepatic transfection of two tamarins with transcribed RNA. A mutant lacking both regions was not viable. However, mutants lacking either the short sequence or the poly(U) tract were viable. All four tamarins had a wild-type-like acute infection and developed acute hepatitis. Whereas we found that five tamarins transfected with the wild-type clone pGBB had acute resolving infection, one tamarin transfected with the poly(U) deletion mutant became persistently infected. This animal had viremia and hepatitis until its death at week 90. The genomes recovered at weeks 2, 7, 15, 20, 60, and 90 lacked the poly(U) stretch. Eight amino acid changes were identified at week 90. One change, in the putative p7 protein, was dominant at week 15. Thus, persistence of GBV-B, like persistence of HCV, was associated with the emergence of virus variants. Four tamarins inoculated with serum collected at weeks 2 and 90 from the tamarin with persistent infection had an acute resolving infection. Nonetheless, the demonstration that GBV-B can persist in tamarins strengthens its relevance as a surrogate model for the study of HCV.

3' Untranslated Regions↗

PubMed2006

A leuC mutation leading to increased L-lysine production and rel-independent global expression changes in Corynebacterium glutamicum.

We previously found by transcriptome analysis that global induction of amino acid biosynthetic genes occurs in a classically derived industrial L-lysine producer, Corynebacterium glutamicum B-6. Based on this stringent-like transcriptional profile in strain B-6, we analyzed the relevant mutations from among those identified in the genome of the strain, with special attention to the genes that are involved in amino acid biosynthesis and metabolism. Among these mutations, a Gly-456-->Asp mutation in the 3-isopropylmalate dehydratase large subunit gene (leuC) was defined as a useful mutation. Introduction of the leuC mutation into a defined L-lysine producer, AHD-2 (hom59 and lysC311), by allelic replacement led to the phenotype of a partial requirement for L-leucine and approximately 14% increased L-lysine production. Transcriptome analysis revealed that many amino acid biosynthetic genes, including lysC-asd operon, were significantly upregulated in the leuC mutant in a rel-independent manner.

Corynebacterium glutamicum↗

PubMed2005

Deciphering the genes involved in pathogenesis of Mycobacterium tuberculosis.

We had previously reported identification of the virS gene of Mycobacterium tuberculosis (Rv3082c) belonging to the AraC family of transcriptional regulators. The 7 genes (Rv3083-Rv3089) which are present divergently to virS (Rv3082c) constitute an operon designated as the mymA operon. Further investigation on the regulation of this operon showed that transcription of the mymA operon is dependent on the presence of VirS protein. A 4-fold induction of the mymA operon promoter occurs specifically in the wild type M. tuberculosis and not in the virS mutant of M. tuberculosis (M.tbDeltavirS) when exposed to acidic pH. Expression of the mymA operon was also induced in infected macrophages by ten-fold over a six-day period. Bioinformatic analysis suggested the involvement of these proteins in the modification of fatty acids required for cell envelope. This was supported by altered colony morphology and cell envelope structure and increased accumulation of C24/C26 fatty acids by M.tbDeltavirS in comparison to the wild type strain. Thus, induction of mymA operon can play an important role in remodeling the envelope of intracellular M. tuberculosis under acidic conditions. Genomic analysis of M. tuberculosis revealed the presence of two tyrosine phosphatase genes--mptpA (Rv2234) and mptpB (Rv0153c). We have characterized both the tyrosine phosphatases of M. tuberculosis. To evaluate the role of MptpB in the pathogenesis of M. tuberculosis we have disrupted mptpB in the genome of M. tuberculosis. The wild type as well as mptpB mutant strain were comparable in their ability to infect and survive in the resting macrophages. However, the mptpB mutant strain was more sensitive to killing as compared to the wild type strain by IFN-gamma activated macrophages. In guinea pig model of tuberculosis an approximately 70-fold reduced bacillary load was observed in the spleen of the animals infected with mptpB mutant strain as compared to the bacillary load in animals infected with the wild type strain at 6 weeks post-infection. These results suggest that mymA operon as well as mptpB gene of M. tuberculosis play an important role in the survival of the pathogen in the host.

Animals↗

PubMed2004

Sub-genomic replicons of Tick-borne encephalitis virus.

We constructed three sub-genomic replicons of Tick-borne encephalitis virus (TBEV) (Oshima REP, Oshima REP-GFP and Oshima REP-Neo) by deleting genes coding for structural proteins without or with insertion of green fluorescent protein (GFP) or Neo genes, respectively. BHK cells transfected with Oshima REP expressed the viral non-structural antigens in immunofluorescent and western blot analyses. GFP and viral antigens were co-expressed in the transfected cells with Oshima REP-GFP. G418-resistant cells harboring Oshima REP-Neo consistently expressed the antigens without showing any apparent CPE. These replicons constructed in this study will be useful in studies on the replication, assembly and packaging of TBEV, and to develop vaccines and gene-delivering systems.

Animals↗

PubMed2006

Rolling-circle replication of an animal circovirus genome in a theta-replicating bacterial plasmid in Escherichia coli.

A bacterial plasmid containing 1.75 copies of double-stranded porcine circovirus (PCV) DNA in tandem (0.8 copy of PCV type 1 [PCV1], 0.95 copy of PCV2) with two origins of DNA replication (Ori) yielded three different DNA species when transformed into Escherichia coli: the input construct, a unit-length chimeric PCV1(Rep)/PCV2(Cap) genome with a composite Ori but lacking the plasmid vector, and a molecule consisting of the remaining 0.75 copy PCV1(Cap)/PCV2(Rep) genome with a different composite Ori together with the bacterial plasmid. Replication of the input construct was presumably via the theta replication mechanism utilizing the ColE(1) Ori, while characteristics of the other two DNA species, including a requirement of two PCV Oris and the virus-encoded replication initiator Rep protein, suggest they were generated via the rolling-circle copy-release mechanism. Interestingly, the PCV-encoded Rep' protein essential for PCV DNA replication in mammalian cells was not required in bacteria. The fact that the Rep' protein function(s) can be compensated by the bacterial replication machinery to support the PCV DNA replication process echoes previous suggestions that circular single-stranded DNA animal circoviruses, plant geminiviruses, and nanoviruses may have evolved from prokaryotic episomal replicons.

Animals↗

PubMed2002

Targets and assays for discovering novel antibacterial agents.

The increasing frequency of nosocomial infections due to multi-resistant pathogens exerts a significant toll and calls for novel and better antibiotics. Different approaches can be used in the search for novel antibiotics acting on drug-resistant bacterial pathogens. We present some considerations on valid bacterial targets to be used for searching new antibiotics, and how the information from bacterial genome sequences can assist in choosing the appropriate targets. Other factors to be considered in target selection are the chemical diversity available for screening and its uniqueness. We will conclude discussing our strategy for searching novel antibacterials. This is based on a large collection of microbial extracts as a source of chemical diversity and on the use of specific targets essential for the viability of bacterial pathogens. Two assay strategies have been implemented: a pathway-based assay, where a series of essential bacterial targets is screened in a single assay; and a binding assay, where many targets can be screened individually in the same format.

Anti-Bacterial Agents↗

PubMed2007

Conditional somatic mutagenesis in the mouse using site-specific recombinases.

In the last decade, site-specific recombinases (SSRs), such as Cre and Flp, have emerged as indispensable tools for the precise in vivo manipulation of the mouse genome. It is now feasible to control, in space and time, the onset of gene knockouts in almost any tissue of the mouse, thus greatly facilitating the creation of sophisticated animal models for human disease and drug development. This review describes the basic principles and current status of the SSR technology, with a focus on strategies for conditional somatic mutagenesis using the Cre/lox system and ligand-activated Cre recombinases. Practical hints for generating and analysing conditional mouse mutants will be given and exciting novel applications of the SSR technology will be discussed, such as cell fate mapping and the combined use of Cre, Flp and other biotechnological tools. It will be shown how genetic manipulation of the mouse by site-specific recombination can provide new solutions to old problems in the analysis of human physiology and pathophysiology and how it can be employed for drug discovery and development.

Animals↗

PubMed2006

Expression of avian reovirus sigmaC protein in transgenic plants.

Avian reovirus (ARV) structural protein, sigmaC encoded by S1 genome segment, is the prime candidate to become a vaccine against ARV infection. Two plant nuclear expression vectors with expression of sigmaC-encoding gene driven by CaMV 35S promoter and rice actin promoter were constructed, respectively. Agrobacterium containing the S1 expression constructs were used to transform alfalfa, and transformants were selected using hygromysin. The integration of S1 transgene in alfalfa chromosome was confirmed by PCR and histochemical GUS staining. Western blot analysis using antiserum against sigmaC was carried out to determine the expression of sigmaC protein in transgenic alfalfa cells. The highest expression levels of sigmaC protein in the cellular extracts of selected p35S-S1 and pAct1-S1 transgenic alfalfa lines were 0.008% and 0.007% of the total soluble protein, respectively. The transgenic alfalfa cells with expression of sigmaC protein pave the way for the development of edible vaccine.

Capsid Proteins↗

PubMed2001

Construction and in vitro properties of a series of attenuated simian immunodeficiency viruses with all accessory genes deleted.

We have generated simplified simian immunodeficiency virus (SIV) constructs lacking the nef, vpr, vpx, vif, tat, and rev genes (Delta6 viruses). To accomplish this, we began with an infectious molecular clone of SIV, i.e. SIVmac239, and replaced the deleted segments with three alternate elements: (i) a constitutive transport element (CTE) derived from simian retrovirus type 1 to replace the Rev/Rev-responsive element (RRE) posttranscriptional regulation system, (ii) a chimeric SIV long terminal repeat (LTR) containing a cytomegalovirus (CMV) promoter to augment transcription and virus production, and (iii) an internal ribosome entry site (IRES) upstream of the env gene to ensure expression of envelope proteins. This simplified construct (Delta6CCI) efficiently produced all viral structural proteins, and mature virions possessed morphology typical of wild-type virus. It was also observed that deletion of the six accessory genes dramatically affected both the specificity and efficiency of packaging of SIV genomic RNA into virions. However, the presence of both the CTE and the chimeric CMV promoter increased the specificity of viral genomic RNA packaging, while the presence of the IRES augmented packaging efficiency. The Delta6CCI virus was extremely attenuated in replication capacity yet retained infectiousness for CEMx174 and MT4 cells. We also generated constructs that retained either the rev gene or both the rev and vif genes and showed that these viruses, when complemented by the CMV promoter, i.e., Delta5-CMV and Delta4-CMV, were able to replicate in MT4 cells with moderate and high-level efficiency, respectively. Long-term culture of each of these constructs over 6 months revealed no potential for reversion. We hope to shortly evaluate these simplified constructs in rhesus macaques to determine their long-term safety as well as ability to induce protective immune responsiveness as proviral DNA vaccines.

Animals↗

PubMed1998

A DNA clone encoding the full-length infectious genome of odontoglossum ringspot tobamovirus and mutagenesis of its coat protein gene.

A full-length DNA clone encoding the genome of odontoglossum ringspot tobamovirus (ORSV) was synthesized and placed adjacent to a bacteriophage T7 RNA polymerase promoter. Capped-RNA transcripts produced in vitro were highly infectious when mechanically inoculated onto seedlings of Nicotiana benthamiana and Oncidium Gower Ramsey. A representative clone, designated pOT2, caused a disease phenotype identical to that produced by parental viral RNA. ELISA, Western blot analysis, Northern blot hybridization and electron microscopy verified the infectivity of pOT2. A coat protein deficient mutant of the clone, pO delta CP1, was produced with the initiation codon of the coat protein cistron of ORSV abolished. Transcripts from pO delta CP1 were infective, able to move in N. benthamiana but produced no coat protein. This demonstrates that the coat protein was dispensable for RNA replication and for movement. This is believed to be the first report of an ORSV infectious clone driven by a T7 RNA polymerase promoter.

Blotting, Northern↗

PubMed2007

Genome-based identification and characterization of a putative mucin-binding protein from the surface of Streptococcus pneumoniae.

Streptococcus pneumoniae open reading frame SP1492 encodes a surface protein that contains a novel conserved domain similar to the repeated fragments of mucin-binding proteins from lactobacilli and lactococci. To investigate the functional role(s) of this protein and its potential adhesive properties, the surface-exposed region of SP1492 was expressed in Escherichia coli, purified to homogeneity, and partially characterized by biophysical and immunological methods. Circular dichroism and sedimentation measurements confirmed that SP1492 is an all-beta protein that exists in solution as a monomer. The SP1492 protein has been shown to be expressed by S. pneumoniae and was experimentally localized to its surface. The protein functional domain binds to mucins II and III from porcine stomach and to purified submaxillary bovine gland mucin. It appears to be one of the very few unambiguous pneumococcal adhesin molecules known to date. A hypothetical model constructed by ab initio techniques predicts a novel beta-sandwich protein structure.

Amino Acid Sequence↗

PubMed2004

The vaccinia virus K1L gene product inhibits host NF-kappaB activation by preventing IkappaBalpha degradation.

Vaccinia virus wild-type strains such as Ankara and WR synthesize proteins capable of inhibiting the activation of host NF-kappaB, a family of transcription factors that regulate the expression of inflammatory genes. In contrast, an infection by the attenuated MVA strain, whose genome lacks many immunoregulatory genes present in the DNA of its Ankara parent, induces NF-kappaB activation. Insertion of NF-kappaB inhibitory genes into the MVA DNA, then, would alter the MVA phenotype. By this method, a 5.2-kb region of Ankara DNA containing the K1L gene and two other genes that are absent in the MVA genome that was identified as NF-kappaB was inhibited in cells infected with the MVA/5.2kb virus. To determine if K1L was responsible, the relevant biological properties of both a recombinant MVA containing a copy of the WR strain's K1L (MVA/K1L) and a WR deletion mutant lacking the K1L gene (DeltaK1L) were examined. Indeed, unlike its progenitor, the altered MVA halted degradation of the host regulatory protein IkappaBalpha-a key event in the pathway of transcriptional activation by NF-kappaB factors. Moreover, MVA/K1L gained the ability to repress artificially contrived and natural NF-kappaB-regulated expression of a transfected luciferase and the cellular tumor necrosis factor gene, respectively. In contrast, although these functions could also be performed by WR, the DeltaK1L virus lost these abilities. Thus, one apparent molecular function of K1L is to prevent IkappaBalpha degradation. This impediment to NF-kappaB-induced host proinflammatory gene expression, in turn, might enhance virus survival.

Animals↗

PubMed1993

Enhanced in vivo monooxygenase activities of mammalian P450s in engineered yeast cells producing high levels of NADPH-P450 reductase and human cytochrome b5.

We have engineered yeast genomic DNA to construct a set of strains producing various relative amounts of yeast NADPH-P450 reductase (Yred) and human cytochrome b5 (Hb5). Expression of cDNAs encoding human P450 1A1, 1A2, 3A4, 19A and mouse P450 1A1 in the different oxido-reduction backgrounds thus constituted were achieved after strain transformation by plasmid-based P450-encoding expression cassettes. The results indicate that the level of Yred strongly affects all activities tested. In contrast, the amount of Hb5 affects activities in a manner that is dependent both on the P450 isoform considered and the Yred level. In a strain containing optimized amounts of Hb5 and Yred, human P450 3A4-specific testosterone-6 beta-hydroxylase activity can be enhanced as much as 73-fold in comparison with the activity observed in a wild-type strain. Bioconversion of sterols or xenobiotics was easily achieved in vivo using this new co-expression system.

Animals↗

PubMed2002

Increased production of nutriments by genetically engineered crops.

Plants are the basis of human nutrition and have been selected and improved to assure this purpose. Nowadays, new technologies such as genetic engineering and genomics approaches allow further improvement of plants. We describe here three examples for which these techniques have been employed. We introduced the first enzyme involved in fructan synthesis, the sucrose sucrose fructosyltransferase (isolated from Jerusalem artichoke), into sugar beet. The transgenic sugar beet showed a dramatic change in the nature of the accumulated sugar, 90% of the sucrose being converted into fructan. The use of transgenic sugar beet for the production and isolation of fructans will result in a more efficient plant production system of fructans and should promote their use in human food. The second example shows how the over-expression of the key enzyme of flavonoid biosynthesis could increase anti-oxidant levels in tomato. Introduction of a highly expressed chalcone isomerase led to a seventyfold increase of the amount of quercetin glucoside, which is a strong anti-oxidant in tomato. We were also able to modify the essential amino acid content of potato in order to increase its nutritional value. The introduction of a feedback insensitive bacterial gene involved in biosynthesis of aspartate family amino acids led to a sixfold increase of the lysine content. Because the use of a bacterial gene could appear to be controversial, we also introduced a mutated form of the plant key enzyme of lysine biosynthesis (dihydrodipicolinate synthase) in potato. This modification led to a 15 times increase of the lysine content of potato. This increase of the essential amino acid lysine influences the nutritional value of potato, which normally has low levels of several essential amino acids. These three examples show how the metabolism of primary constituents of the plant cell such as sugar or amino acids, but also of secondary metabolites such as flavonoids, can be modified by genetic engineering. Producing fructan, a soluble fiber, increasing the level of flavonoids, an antioxidant, in tomato or increasing the level of essential amino acids in potato are all clear examples of plant genetic modifications with possible positive effects on human nutrition.

Crops, Agricultural↗

PubMed2002

A gene capable of blocking apoptosis can substitute for the herpes simplex virus type 1 latency-associated transcript gene and restore wild-type reactivation levels.

After ocular herpes simplex virus type 1 (HSV-1) infection, the virus travels up axons and establishes a lifelong latent infection in neurons of the trigeminal ganglia. LAT (latency-associated transcript), the only known viral gene abundantly transcribed during HSV-1 neuronal latency, is required for high levels of reactivation. The LAT function responsible for this reactivation phenotype is not known. Recently, we showed that LAT can block programmed cell death (apoptosis) in neurons of the trigeminal ganglion in vivo and in tissue culture cells in vitro (G.-C. Perng et al., Science 287:1500-1503, 2000; M. Inman et al., J. Virol. 75:3636-3646, 2001). Consequently, we proposed that this antiapoptosis function may be a key to the mechanism by which LAT enhances reactivation. To study this further, we constructed a recombinant HSV-1 virus in which the HSV-1 LAT gene was replaced by an alternate antiapoptosis gene. We used the bovine herpes virus 1 (BHV-1) latency-related (LR) gene, which was previously shown to have antiapoptosis activity, for this purpose. The resulting chimeric virus, designated CJLAT, contains two complete copies of the BHV-1 LR gene (one in each viral long repeat) in place of the normal two copies of the HSV-1 LAT, on an otherwise wild-type HSV-1 strain McKrae genomic background. We report here that in both rabbits and mice reactivation of CJLAT was significantly greater than the LAT null mutant dLAT2903 (P < 0.0004 and P = 0.001, respectively) and was at least as efficient as wild-type McKrae. This strongly suggests that a BHV-1 LR gene function was able to efficiently substitute for an HSV-1 LAT gene function involved in reactivation. Although replication of CJLAT in rabbits and mice was similar to that of wild-type McKrae, CJLAT killed more mice during acute infection and caused more corneal scarring in latently infected rabbits. This suggested that the BHV-1 LR gene and the HSV-1 LAT gene are not functionally identical. However, LR and LAT both have antiapoptosis activity. These studies therefore strongly support the hypothesis that replacing LAT with an antiapoptosis gene restores the wild-type reactivation phenotype to a LAT null mutant of HSV-1 McKrae.

Animals↗

PubMed2005

A novel gnd mutation leading to increased L-lysine production in Corynebacterium glutamicum.

Toward more efficient L-lysine production, we have been challenging genome-based strain breeding by the approach of assembling only relevant mutations in a single wild-type background. Following the creation of a new L-lysine producer Corynebacterium glutamicum AHP-3 that carried three useful mutations (lysC311, hom59, and pyc458) on the relevant downstream pathways, we shifted our target to the pentose phosphate pathway. Comparative genomic analysis for the pathway between a classically derived L-lysine producer and its parental wild-type identified several mutations. Among these mutations, a Ser-361-->Phe mutation in the 6-phosphogluconate dehydrogenase gene (gnd) was defined as a useful mutation for L-lysine production. Introduction of the gnd mutation into strain AHP-3 by allelic replacement led to approximately 15% increased L-lysine production. Enzymatic analysis revealed that the mutant enzyme was less sensitive than the wild-type enzyme to allosteric inhibition by intracellular metabolites, such as fructose 1,6-bisphosphate, D-glyceraldehyde 3-phosphate, phosphoribosyl pyrophosphate, ATP, and NADPH, which were known to inhibit this enzyme. Isotope-based metabolic flux analysis demonstrated that the gnd mutation resulted in 8% increased carbon flux through the pentose phosphate pathway during L-lysine production. These results indicate that the gnd mutation is responsible for diminished allosteric regulation and contributes to redirection of more carbon to the pentose phosphate pathway that was identified as the primary source for NADPH essential for L-lysine biosynthesis, thereby leading to improved product formation.

Corynebacterium↗

PubMed1996

Pathogen-derived resistance to dengue type 2 virus in mosquito cells by expression of the premembrane coding region of the viral genome.

The full-length premembrane (prM) coding region of the dengue virus type 2 (DEN-2; Jamaica) genome was expressed in C6/36 (Aedes albopictus) cells in either the sense or the antisense orientation from a double subgenomic Sindbis (dsSIN) virus. Northern (RNA) blot analysis confirmed the expression of sense or antisense DEN-2 prM RNA in infected C6/36 cells. PrM protein was demonstrated in cells infected with dsSIN virus expressing DEN-2 sense RNAs by an immunofluorescence assay. C6/36 cells were infected with each dsSIN virus at a multiplicity of infection (MOI) of 50 and challenged 48 h later with DEN-2 virus at an MOI of 0.1. Whereas C6/36 cells infected with a control of dsSIN virus supported high levels of DEN-2 replication, C6/36 cells infected with the dsSIN virus expressing prM antisense RNA were completely resistant to DEN-2 challenge. Cells expressing prM protein or untranslatable prM sense RNA also were resistant to DEN-2 challenge. Cells expressing prM protein demonstrated some breakthrough of DEN-2 virus when challenged at an MOI of 10. However, expressed untranslatable sense prM RNA conferred complete protection to challenge at the high MOI.

Aedes↗