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At least 127 records · Page 7Linked to original sources
PubMed2002

Reverse genetics demonstrates that proteolytic processing of the Ebola virus glycoprotein is not essential for replication in cell culture.

Ebola virus, a prime example of an emerging pathogen, causes fatal hemorrhagic fever in humans and in nonhuman primates. Identification of major determinants of Ebola virus pathogenicity has been hampered by the lack of effective strategies for experimental mutagenesis. Here we exploit a reverse genetics system that allows the generation of Ebola virus from cloned cDNA to engineer a mutant Ebola virus with an altered furin recognition motif in the glycoprotein (GP). When expressed in cells, the GP of the wild type, but not of the mutant, virus was cleaved into GP1 and GP2. Although posttranslational furin-mediated cleavage of GP was thought to be an essential step in Ebola virus infection, generation of a viable mutant Ebola virus lacking a furin recognition motif in the GP cleavage site demonstrates that GP cleavage is not essential for replication of Ebola virus in cell culture.

Animals↗
PubMed1992

Tn9 CAT gene contains a promoter for vaccinia virus transcription: implications for reverse-genetic techniques.

Vaccinia virus-dependent CAT expression was observed in virus-infected cells cotransfected with a promoterless CAT gene. Restriction endonuclease resection of the CAT plasmid indicated that expression was due to recognition by vaccinia virus RNA polymerase of sequences within the CAT gene itself, probably located within the 5' untranslated region of the gene. This observation is relevant to the design of reverse-genetic systems which use CAT as a reporter gene to detect replication of negative-strand RNA virus pseudogenomes.

Base Sequence↗
PubMed2006

Live vaccines for human metapneumovirus designed by reverse genetics.

Human metapneumovirus (HMPV) was first described in 2001 and has quickly become recognized as an important cause of respiratory tract disease worldwide, especially in the pediatric population. A vaccine against HMPV is required to prevent severe disease associated with infection in infancy. The primary strategy is to develop a live-attenuated virus for intranasal immunization, which is particularly well suited against a respiratory virus. Reverse genetics provides a means of developing highly characterized 'designer' attenuated vaccine candidates. To date, several promising vaccine candidates have been developed, each using a different mode of attenuation. One candidate involves deletion of the G glycoprotein, providing attenuation that is probably based on reduced efficiency of attachment. A second candidate involves deletion of the M2-2 protein, which participates in regulating RNA synthesis and whose deletion has the advantageous property of upregulating transcription and increasing antigen synthesis. A third candidate involves replacing the P protein gene of HMPV with its counterpart from the related avian metapneumovirus, thereby introducing attenuation owing to its chimeric nature and host range restriction. Another live vaccine strategy involves using an attenuated parainfluenza virus as a vector to express HMPV protective antigens, providing a bivalent pediatric vaccine. Additional modifications to provide improved vaccines will also be discussed.

Animals↗
PubMed2003

Development and application of a reverse genetics system for Japanese encephalitis virus.

Japanese encephalitis virus (JEV) is a common agent of viral encephalitis that causes high mortality and morbidity among children. Molecular genetic studies of JEV are hampered by the lack of a genetically stable full-length infectious JEV cDNA clone. We describe here the development of such a clone. A JEV isolate was fully sequenced, and then its full-length cDNA was cloned into a bacterial artificial chromosome. This was then further engineered so that transcription of the cDNA in vitro would generate synthetic RNAs with authentic 5' and 3' ends. The synthetic RNAs thus produced were highly infectious in susceptible cells (>10(6) PFU/ micro g), and these cells rapidly generated a high titer of synthetic viruses (>5 x 10(6) PFU/ml). The recovered viruses were indistinguishable from the parental virus in terms of plaque morphology, growth kinetics, RNA accumulation, protein expression, and cytopathogenicity. Significantly, the structural and functional integrity of the cDNA was maintained even after 180 generations of growth in Escherichia coli. A single point mutation acting as a genetic marker was introduced into the cDNA and was found in the genome of the recovered virus, indicating that the cDNA can be manipulated. Furthermore, we showed that JEV is an attractive vector for the expression of heterologous genes in a wide variety of cell types. This novel reverse genetics system for JEV will greatly facilitate research into JEV biology. It will also be useful as a heterologous gene expression vector and will aid the development of a vaccine against JEV.

Animals↗
PubMed1997

Characterization of DNA damage inflicted by free radicals from a mutagenic sunscreen ingredient and its location using an in vitro genetic reversion assay.

We describe an in vitro approach to assessing the potential genotoxicity of illuminated sunscreens. The photomutagenic sunscreen Padimate-O attacks DNA on illumination with simulated sunlight, producing strand breaks and lesions that are labile to N,N'-dimethylethylenediamine but few, if any, cyclobutane dimers or other direct photoproducts. The damage can be completely suppressed by the free radical quenchers Tris, ethanol, mannitol and dimethylsulfoxide, which is commonly used as a solvent in conventional photomutagenicity assays. Using a genetic reversion assay that depends on regenerating beta-galactosidase activity in photodamaged plasmids we find that GC base pairs are particularly susceptible to attack by Padimate-O.

4-Aminobenzoic Acid↗
PubMed2005

Reverse genetic approaches for functional genomics of rice.

T-DNA and transposable elements e.g., Ds and Tos17, are used to generate a large number of insertional mutant lines in rice. Some carry the GUS or GFP reporter for gene trap or enhancer trap. These reporter systems are valuable for identifying tissue- or organ-preferential genes. Activation tagging lines have also been generated for screening mutants and isolating mutagenized genes. To utilize these resources more efficiently, tagged lines have been produced for reverse genetic approaches. DNA pools of the T-DNA tagged lines and Tos17 lines have been prepared for PCR screening of insertional mutants in a given gene. Tag end sequences (TES) of the inserts have also been produced. TES databases are beneficial for analyzing the function of a large number of rice genes.

Binding Sites↗
PubMed2005

Introducing point and deletion mutations into the P/C gene of human parainfluenza virus type 1 (HPIV1) by reverse genetics generates attenuated and efficacious vaccine candidates.

The P/C gene of human parainfluenza virus type 1 (HPIV1) encodes a nested set of related accessory C proteins, C'/C/Y1/Y2, which have been shown in other paramyxoviruses to have a role in evasion of the type I interferon (IFN) response following virus infection. We previously demonstrated that a set of two amino acid substitutions, CR84G/HNT553A, and a separate amino acid substitution, CF170S, are independently attenuating for HPIV1 in African green monkeys (AGMs). However, in each case the attenuation (att) phenotype is vulnerable to reversion by a single nucleotide change back to wild type. Using reverse genetics, recombinant HPIV1 (rHPIV1) vaccine candidates were generated that were designed for increased genetic and phenotypic stability by: (i) creating a two-amino acid deletion and substitution at the site of the CF170S mutation, yielding CDelta170; (ii) introducing a six amino acid deletion in the N-terminal region of C, CDelta10-15; and (iii) combining these stable deletion mutations with the att CR84G/HNT553A mutation. The resulting rHPIV1 vaccine candidates were evaluated for attenuation in hamsters and AGMs and for immunogenicity and protective efficacy in AGMs. The CDelta10-15 mutation was attenuating in hamsters but not in AGMs, and likely will be of limited value for an HPIV1 vaccine. Conversely, the CR84G/HNT553A mutation set was attenuating in AGMs but not in hamsters. Thus, these two mutations demonstrated reciprocal host range phenotypes involving different regions of C. The CDelta170 mutation conferred a significant level of attenuation in hamsters and AGMs that closely resembled that of CF170S and will be of particular utility for vaccine development because it involves a deletion of six nucleotides rendering it highly refractory to reversion. The combination of the CR84G/HNT553A mutation set and the CDelta170 deletion mutation yielded a virus, rCR84G/Delta170 HNT553A, that exhibited a satisfactory level of attenuation in hamsters and AGMs and was immunogenic and highly protective against HPIV1 wt challenge. This virus will be evaluated clinically as a live intranasal HPIV1 vaccine, one that can be further attenuated as necessary by the introduction of additional stabilized att mutations previously developed in the L protein.

Animals↗
PubMed2006

[Development of attenuated H5N1 avian influenza vaccines using reverse genetic technology].

Outbreaks caused by highly pathogenic H5N1 avian influenza viruses have spread worldwide and the containment is impossible no longer. During the outbreaks, over 200 human infection cases with 55% fatality are confirmed at the moment and some human-to -human transmission in family clusters have been observed in Vietnam, Thailand and Indonesia. The potency of pandemic caused by highly pathogenic H5N1 avian influenza is increasing gradually. Consequently, development of effective H5N1 vaccine and its licensing for vaccination to human are urgent countermeasures for pandemic preparedness. In the present article, the author reviews the development of H5N1 attenuated vaccine by reverse genetic technology and discuss the problems on the vaccine production to be dissolved before pandemic occur.

Animals↗
PubMed2004

Reverse genetics studies of attenuation of the ca A/AA/6/60 influenza virus: the role of the matrix gene.

The matrix (M) gene of influenza virus has been implicated in the attenuation phenotype of the cold adapted (ca) A/AA/6/60 vaccine. Previous studies have evaluated the ca M from A/AA/6/60 in different wild type (wt) virus backgrounds with varying results. In experiments described here, the ca M gene was transfected into the background of its own wt A/AA/6/60 to eliminate the possibility of confounding gene constellation effects. Comparison of the sequence of the wt and the ca A/AA/6/60 revealed one substitution in the nucleotide sequence of M. The molecular techniques of reverse genetics were used to rescue the ca M gene into the virulent wt A/AA/6/60 virus. The selection system used to identify the desired transfectant virus was amantadine resistance, which was introduced into the M2 gene using mutagenesis. The ca A/AA/6/60, the wt A/AA/6/60, a virus which contained wt M and was wt in the remaining seven genes and amantadine resistant (wt/969), a virus which contained the ca M but wt in the other seven genes (ca/969) were all evaluated in mice determine the effect of the ca M. The ca/969 virus was not attenuated in the mouse model when compared to the wt/969 virus, indicating that the ca A/AA/6/60 M does not independently contribute to the attenuation phenotype attributed to the ca A/AA/6/60 vaccine virus.

Animals↗
PubMed2001

Analysis of allosteric effector binding sites of potato ADP-glucose pyrophosphorylase through reverse genetics.

ADP-glucose pyrophosphorylase (AGPase) is a key regulatory enzyme of bacterial glycogen and plant starch synthesis as it controls carbon flux via its allosteric regulatory behavior. Unlike the bacterial enzyme that is composed of a single subunit type, the plant AGPase is a heterotetrameric enzyme (alpha2beta2) with distinct roles for each subunit type. The large subunit (LS) is involved mainly in allosteric regulation through its interaction with the catalytic small subunit (SS). The LS modulates the catalytic activity of the SS by increasing the allosteric regulatory response of the hetero-oligomeric enzyme. To identify regions of the LS involved in binding of effector molecules, a reverse genetics approach was employed. A potato (Solanum tuberosum L.) AGPase LS down-regulatory mutant (E38A) was subjected to random mutagenesis using error-prone polymerase chain reaction and screened for the capacity to form an enzyme capable of restoring glycogen production in glgC(-) Escherichia coli. Dominant mutations were identified by their capacity to restore glycogen production when the LS containing only the second site mutations was co-expressed with the wild-type SS. Sequence analysis showed that most of the mutations were decidedly nonrandom and were clustered at conserved N- and C-terminal regions. Kinetic analysis of the dominant mutant enzymes indicated that the K(m) values for cofactor and substrates were comparable with the wild-type AGPase, whereas the affinities for activator and inhibitor were altered appreciably. These AGPase variants displayed increased resistance to P(i) inhibition and/or greater sensitivity toward 3-phosphoglyceric acid activation. Further studies of Lys-197, Pro-261, and Lys-420, residues conserved in AGPase sequences, by site-directed mutagenesis suggested that the effectors 3-phosphoglyceric acid and P(i) interact at two closely located binding sites.

Allosteric Site↗
PubMed2000

Interdependence of hemagglutinin glycosylation and neuraminidase as regulators of influenza virus growth: a study by reverse genetics.

The hemagglutinin (HA) of fowl plague virus A/FPV/Rostock/34 (H7N1) carries two N-linked oligosaccharides attached to Asn123 and Asn149 in close vicinity to the receptor-binding pocket. In previous studies in which HA mutants lacking either one (mutants G1 and G2) or both (mutant G1,2) glycosylation sites had been expressed from a simian virus 40 vector, we showed that these glycans regulate receptor binding affinity (M. Ohuchi, R. Ohuchi, A. Feldmann, and H. D. Klenk, J. Virol. 71:8377-8384, 1997). We have now investigated the effect of these mutations on virus growth using recombinant viruses generated by an RNA polymerase I-based reverse genetics system. Two reassortants of influenza virus strain A/WSN/33 were used as helper viruses to obtain two series of HA mutant viruses differing only in the neuraminidase (NA). Studies using N1 NA viruses revealed that loss of the oligosaccharide from Asn149 (mutant G2) or loss of both oligosaccharides (mutant G1,2) has a pronounced effect on virus growth in MDCK cells. Growth of virus lacking both oligosaccharides from infected cells was retarded, and virus yields in the medium were decreased about 20-fold. Likewise, there was a reduction in plaque size that was distinct with G1,2 and less pronounced with G2. These effects could be attributed to a highly impaired release of mutant progeny viruses from host cells. In contrast, with recombinant viruses containing N2 NA, these restrictions were much less apparent. N1 recombinants showed lower neuraminidase activity than N2 recombinants, indicating that N2 NA is able to partly overrule the high-affinity binding of mutant HA to the receptor. These results demonstrate that N-glycans flanking the receptor-binding site of the HA molecule are potent regulators of influenza virus growth, with the glycan at Asn149 being dominant and that at Asn123 being less effective. In addition, we show here that HA and NA activities need to be highly balanced in order to allow productive influenza virus infection.

Animals↗
PubMed1993

Two complementary approaches to study peroxisome biogenesis in Saccharomyces cerevisiae: forward and reversed genetics.

In order to investigate the mechanisms of peroxisome biogenesis and to identify components of the peroxisomal import machinery we studied these processes in the yeast Saccharomyces cerevisiae. The forward genetic approach has led to pas-mutants (peroxisomal assembly) which fall into 12 complementation groups and allowed to identify 10 of the corresponding wild-type PAS genes (PAS 1-7, 9, 11 and 12). Recent sequence analysis data of some of these genes are beginning to provide first hints as to the possible function of their gene products. The PAS genes and their corresponding mutants are presently used to address some important questions of peroxisomal biogenesis. Reversed genetics has been started as a complementary approach to characterize especially the function of peroxisomal membrane proteins. For this purpose we describe a technique to isolate highly purified peroxisomes. This led to the identification of 21 polypeptides as constituents of this organelle. Some of them are presently sequenced.

Adenosine Triphosphatases↗
PubMed1991

Reverse genetics in the mouse and its application to the study of deafness.

Genetic variants of the laboratory mouse can serve as useful models for hereditary deafness syndromes in humans. Recessive mutations at the shaker-1 (sh-1) and whirler (wi) loci, in chromosomes 7 and 4, respectively, both result in circling behavior and a deafness syndrome. In sh-1 homozygotes this deafness is associated with neurophysiological abnormalities that may be accompanied by structural abnormalities of the inner ear. Radiation-induced deletion mutations are being used in a strategy of reverse genetics to identify the genes defined by these mutations. Genetic analyses have refined the position of sh-1 to a chromosomal interval between break points of deletions involving the closely linked albino (c) locus. A cDNA encoding olfactory marker protein (OMP) and the anonymous locus D7OR1 have also been mapped to this interval. These clones contribute to the physical map of the sh-1 region and could be important for accessing the sh-1 gene itself. Similarly, we have identified a radiation-induced deletion of the brown (b) locus that covers the wi locus and two that do not. Thus, the wi locus has been located within a chromosome 4 interval defined by structural rearrangements, which should likewise aid in identifying closely linked molecular clones.

Animals↗
PubMed2006

Reverse genetic generation of recombinant Zaire Ebola viruses containing disrupted IRF-3 inhibitory domains results in attenuated virus growth in vitro and higher levels of IRF-3 activation without inhibiting viral transcription or replication.

The VP35 protein of Zaire Ebola virus is an essential component of the viral RNA polymerase complex and also functions to antagonize the cellular type I interferon (IFN) response by blocking activation of the transcription factor IRF-3. We previously mapped the IRF-3 inhibitory domain within the C terminus of VP35. In the present study, we show that mutations that disrupt the IRF-3 inhibitory function of VP35 do not disrupt viral transcription/replication, suggesting that the two functions of VP35 are separable. Second, using reverse genetics, we successfully recovered recombinant Ebola viruses containing mutations within the IRF-3 inhibitory domain. Importantly, we show that the recombinant viruses were attenuated for growth in cell culture and that they activated IRF-3 and IRF-3-inducible gene expression at levels higher than that for Ebola virus containing wild-type VP35. In the context of Ebola virus pathogenesis, VP35 may function to limit early IFN-beta production and other antiviral signals generated from cells at the primary site of infection, thereby slowing down the host's ability to curb virus replication and induce adaptive immunity.

Animals↗
PubMed2005

Reverse genetics on fish rhabdoviruses: tools to study the pathogenesis of fish rhabdoviruses.

Rhabdoviruses, mainly in rainbow trout, are among the most devastating viruses for worldwide aquaculture. To date no effective treatments to fight against these viruses are available. During the past years, several approaches to develop efficient vaccines have been undertaken such as the use of immunogenic recombinant viral proteins, naked DNA or inactivated viruses. However, although these vaccines have been proven to be very effective on a small scale, they have never been used in the field because the vaccines would have to be injected into thousands of yearling trouts. The only alternative to injection consists of the development of attenuated live vaccines that can be administrated to trouts by bath immersion. Reverse genetics on trout rhabdoviruses offer the possibility of recovering a series of live recombinant viruses in which the viral genome has been irreversibly modified to generate cost-effective live, safe vaccines.

Animals↗
PubMed2004

Accessory genes of the paramyxoviridae, a large family of nonsegmented negative-strand RNA viruses, as a focus of active investigation by reverse genetics.

The Paramyxoviridae, a large family of nonsegmented negative-strand RNA viruses, comprises several genera each containing important human and animal pathogens. They possess in common six basal genes essential for viral replication and, in addition, a subset of accessory genes that are largely unique to each genus. These accessory genes are either encoded in one or more alternative overlapping frames of a basal gene, which are accessed transcriptionally or translationally, or inserted before or between the basal genes as one or more extra genes. However, the question of how the individual accessory genes contribute to actual viral replication and pathogenesis remained unanswered. It was not even established whether they are dispensable or indispensable for the viral life cycle. The plasmid-based reverse genetics of the full-length viral genome has now come into wide use to demonstrate that most, if not all, of these putative accessory genes can be disrupted without destroying viral infectivity, conclusively defining them as indeed dispensable accessory genes. Studies on the phenotypes of the resulting gene knockout viruses have revealed that the individual accessory genes greatly contribute specifically and additively to the overall viral fitness both in vitro and in vivo.

Animals↗
PubMed1999

Function search in a large transcription factor gene family in Arabidopsis: assessing the potential of reverse genetics to identify insertional mutations in R2R3 MYB genes.

More than 92 genes encoding MYB transcription factors of the R2R3 class have been described in Arabidopsis. The functions of a few members of this large gene family have been described, indicating important roles for R2R3 MYB transcription factors in the regulation of secondary metabolism, cell shape, and disease resistance, and in responses to growth regulators and stresses. For the majority of the genes in this family, however, little functional information is available. As the first step to characterizing these genes functionally, the sequences of >90 family members, and the map positions and expression profiles of >60 members, have been determined previously. An important second step in the functional analysis of the MYB family, through a process of reverse genetics that entails the isolation of insertion mutants, is described here. For this purpose, a variety of gene disruption resources has been used, including T-DNA-insertion populations and three distinct populations that harbor transposon insertions. We report the isolation of 47 insertions into 36 distinct MYB genes by screening a total of 73 genes. These defined insertion lines will provide the foundation for subsequent detailed functional analyses for the assignment of specific functions to individual members of the R2R3 MYB gene family.

Arabidopsis↗
PubMed2006

Development of a live attenuated dengue virus vaccine using reverse genetics.

There are four serotypes of dengue (DEN1-DEN4) virus that are endemic in most areas of Southeast Asia, Central and South America, and other subtropical regions. The number of cases of severe disease associated with DEN virus infection is growing because of the continued spread of the mosquito vector, Aedes aegypti, which transmits the virus to humans. Infection with DEN virus can result in an asymptomatic infection, a febrile illness called dengue fever (DF), and the very severe disease called dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). Currently, a licensed vaccine is not available. However, a tetravalent vaccine is urgently needed to prevent DF and DHF/DSS, the latter of which occurs predominantly in partially immune individuals. A live attenuated, tetravalent DEN virus vaccine candidate has been generated using reverse genetics that is able to provide immunity to each of the four serotypes of DEN. Attenuation has been achieved by generating recombinant DEN (rDEN) viruses which are modified by deletion or, alternatively, by antigenic chimerization between two related DEN viruses using the following two strategies: 1) introduction of an attenuating 30 nucleotide deletion (Delta30) mutation into the 3' untranslated region of DEN1 and DEN4; and 2) replacement of structural proteins of the attenuated rDEN4Delta30 vaccine candidate with those from DEN2 or DEN3. Attenuation of the four monovalent vaccine candidates has been achieved for rhesus monkeys or humans and an immunogenic tetravalent vaccine candidate has been formulated. The level of attenuation of each dengue vaccine component can be increased, if needed, by introduction of additional attenuating mutations that have been well characterized.

Dengue↗