Semliki forest virus replicon particles production in. In order to generate a semliki forest virus sfv noncytopathic vector we. In order to package the replicon rna into particles, a helper vector has been used to provide structural proteins rheme et al. An sfv vector expressing luciferase was constructed. Semliki forest virus vectors for in vitro and in vivo. The expression of the fusion tumor antigen survivin and hcg. Pdf mobilization of fulllength semliki forest virus replicon by.
Typically, these vectors are constructed in a format known as a replicon, due to the selfamplifying nature of the vector rna. Conciliating biosafety with efficient gene transfer remains a constant concern in the development of retroviral vectors. We expressed the n protein nucleoprotein of puumala virus via the semliki forest virus sfv replicon in mammalian cells and compared its antigenic properties with those of the native antigen derived from puumala virus infected cells. It was recently shown that infection of ise6 tick cells by a recombinant semliki forest virus sfv expressing a heterologous gene induced small interfering rnas sirnas and silencing of the gene. Semliki forest virus rarely causes disease in humans. Control of the rescue and replication of semliki forest virus recombinants by. In the context of an siv vaccine model, the replicon was constructed to encode the sfv replicase machinery, an siv immunogen, and the vesicular stomatitis virus vsv. Enhancement of antitumor immunity using a dnabased. These are the sources and citations used to research autophagy in mosquito cells infected with semliki forest virus. Membrane fusion of semliki forest virus involves homotrimers of the fusion protein. Four positive clones were analyzed, and the orientation of the insert was determined by sequencing. Semliki forest virus replicon dna immune response goatpox, caused by goatpox virus gtpv, is an acute feverish and contagious disease in goats often associated with high morbidity and high mortality.
A new generation of animal cell expression vectors based. Abstract we have developed a novel dna expression system, based on the semliki forest virus sfv replicon, which combines a wide choice of animal cell hosts, high efficiency and ease of use. Following intramuscular injection, dispersal of the rna occurs to the local lymph node only, persisting only for 7 days. In turn, replicons derived from alphaviruses like semliki forest virus sfv are potent protein expression and immunization vectors but stocks are difficult to produce and maintain. This is especially the case for sindbis virus sinv, semliki forest virus sfv, and venezuelan equine encephalitis virus veev 35. Immunogenicity and protective efficacy of semliki forest virus replicon based dna vaccines encoding goatpox virus structural proteins. Alphavirus vaccines such as those based on the semliki forest virus sfv replicon have been widely studied as candidate vaccines. Replicon rnas can also be efficiently packed into virusreplicon particles. Alphaviruses, like all other group iv viruses, have a positive sense, singlestranded rna genome. Polo jm, belli ba, driver da, frolov i, sherril s, hariharan mj, et al. Its genome has been introduced into two plasmid vectors as cdna copies.
Stable alphavirus packaging cell lines for sindbis virus. The expression vector contains the sfv nonstructural genes nsp14. Mobilization of fulllength semliki forest virus replicon by. Semliki forest virus sfv vectors can be produced faster, and have a wider host range, than baculovirus vectors. The ligandgated ion channels showed high specific binding activity and highamplitude electrophysiol. Alphavirus replicon vaccines animal health research. A dnabased replicon vaccine derived from semliki forest virus, psvkshfcggmb7. Semliki forest virus and kunjin virus rna replicons elicit. Alphaviruses are enveloped viruses containing a positivestrand rna genome and belonging to the togaviridae family. Semliki forest virus biodistribution in tumorfree and 4t1. Production of sfvn virus and characterization of the recombinant n protein. Autophagy in mosquito cells infected with semliki forest. Nairovirus rna sequences expressed by a semliki forest.
We have generated a system that is wholly dnabased. Institute of technology, faculty of science and technology, university of. High affinity binding and functional coupling to g proteins were obtained for the receptors. A sindbis virus replicon based dna vaccine encoding the rabies virus glycoprotein elicits immune responses and complete protection in mice from lethal challenge. However, the original sfv system requires in vitro manipulation of rna. Semliki forest virus biodistribution in tumorfree and 4t1 mammary.
The j b c 1998 by the american society for biochemistry. Expression of the nucleoprotein of the puumala virus from. Potent therapeutic efficacy of an alphavirus replicon dna. As with any viral vector, they possess a limited cloning capacity, so infection with different sfv vectors may be required to introduce multiple transgenes into individual cells. To resolve potential safety risks and vaccination side effects of existing live attenuated goatpox vaccine av41, two semliki forest virus sfv. In biology and immunology, an alphavirus belongs to group iv of the baltimore classification of the togaviridae family of viruses, according to the system of classification based on viral genome composition introduced by david baltimore in 1971. Development of a new noncytopathic semliki forest virus vector. Highlevel expression of g protein coupled receptors has allowed specific binding and functional studies in a variety of mammalian cell lines.
To gain information on rna interference rnai in ticks, three known viral inhibitors that act in different ways, the ns1 protein of influenza virus, nss of tospovirus tomato spotted wilt virus and. Alphavirus vectors, derived principally from sindbis virus sin, semliki forest virus sfv, and venezuelan equine encephalitis virus, are widely used for gene expression studies in vitro and are being developed for both vaccine and gene therapy applications. Among them, semliki forest virus is commonly employed for this purpose. In an attempt to demonstrate the use of a poxvirus as a means for the delivery of small vaccine vectors, we have constructed and characterized vvsfv hybrid vectors. Both the replicon vector, encoding sfv polymerase and the protein of. In this context, production of virus replicon particles vrps has been described, which allows the expression of foreign genes via so called single round infectious particles. In the present study, sfv dna vectors dna vectors based on th. Semliki forest virus vesicular stomatitis virus hiv1 r na replicons from alphaviruses including semliki forest virus sfv have been developed and used for transient expression of foreign proteins in mammalian cells and also as experimental vaccine vectors. Plasmid psfvnpuu was constructed by inserting the entire open reading frame of the puumala virus n protein into psfv1 at the unique bamhi site. However, it has been widely adopted as a research tool because of its efficient replication and broad host range. In psfv1enhanced green fluorescent protein egfp vector viral structural genes have been substituted by the egfp marker gene. Studies on the semliki forest virus replicase protein nsp1. Twohelper rna system for production of recombinant semliki forest virus particles.
To study g glycoprotein function we have cloned two variants of the rsv g gene into a semliki forest virus sfv expression vector, a full length rg and soluble srg g glycoprotein variant. Replicon vectors derived from sindbis virus and semliki forest virus that establish persistent replication in host cells. Semliki forest virus sfv replicons allow important retroviral vector. Alphavirus replicon vectors are well suited for applications where transient, high level expression of a heterologous gene is required. Sindbis virus, semliki forest virus sfv, and venezuelan equine enceph. A semliki forest virus vector engineered to express ifn. Semliki forest virus sfv vectors are widely used in neurobiological studies because they efficiently infect neurons. The use of dna vectors based on the sfv semliki forest virus replicon have not been reported in the modality of dna prime virus boost. Replicon vectors derived from sindbis virus and semliki. Ljungberg k, whitmore ac, fluet me, moran tp, shabman rs, collier ml, kraus aa, thompson jm, montefiori dc, beard c and johnston re 2007.
Alphavirus vectors are usually based on rna replicons in which the viral. A new generation of animal cell expression vectors based on the semliki forest virus replicon. Semliki forest virus replicon particles production in serumfree medium bhk21 cell cultures and their use to express different proteins sandra fernanda suarezpatino, thaissa consoni bernardino, eutimio gustavo fernandez nunez, renato mancini astray, carlos augusto pereira, hugo r. Replicon vectors derived from sindbis virus and semliki forest. Semliki forest virus replicon particles production in serumfree medium bhk21 cell cultures and their use to express different proteins. Semliki forest virus sfv vectors are promising tools for cancer gene therapy because they ensure a high level of transgene expression and a rapid and strong cytopathic effect. Rapid virus generation, broad host range, efficient rna replication in the cytoplasm, and high expression levels are features that have made the use of semliki forest virus sfv vectors attractive. Giles, uk and quantitated with imagequant tl software ge healthcare.
The respiratory syncytial virus rsv g glycoprotein mediates attachment of rsv to cells via an unknown receptor. We have developed a novel dna expression system, based on the semliki forest virus sfv replicon, which combines a wide choice of animal cell hosts, high efficiency and ease of use. Construction and characterisation of a semliki forest. An sfv vector expressing luciferase was constructed by replacing the.
Utr untranslated region veev venezuelan equine encephalitis virus vrp virus replicon particle wt wild type. Ctp37 and adjuvant molecular protein granulocytemacrophage colonystimulating factor gmcsfb7. Using a recombinant semliki forest alphavirus replicon that replicates in these cells, we were able to inhibit replication of hazv, and we showed that this blockage is mediated by the replication of the semliki forest alphavirus replicon. It is known to cause disease in animals including humans. The first part 5end of genome encodes the nonstructural proteins, while the second part 3end encodes the structural proteins dudek and knipe 2006. A vaccinia virus recombinant transcribing an alphavirus.
B transmobilization of fulllength sfv replicon by retrovirus particles. Replicon vectors derived from sindbis virus and semliki forest virus. It is an alphavirus found in central, eastern, and southern africa. The semliki forest virus sfv expression system utilises the efficient replication of sfv to transiently express foreign proteins in eukaryotic cells. Dna of interest is cloned into sfv plasmid vectors that serve as templates for in vitro synthesis of recombinant rna. Respiratory syncytial virus g glycoprotein expressed using. Vector replicon rna can be transcribed in vitroand used directly, or the replicon rna can be packaged into infectious vector particles by cotransfection of cultured cells with a complementing defective helper rna, which provides the virion structural proteins in trans. The classical semliki forest virus sfv replicon vector is generated in such. Immunogenicity and protective efficacy of semliki forest.
Semliki forest virus semliki forest virus sfv rna replicons have been developed to propagate in membrane vesicles. The highly efficient semliki forest virus sfv system has been applied to express several recombinant 7tm receptors and ligandgated ion channels. Semliki forest virus sfv represents a promising gene therapy vector. Simliki has been used in the development of gene therapy and as a vector for vaccines and anticancer agents. Replicon dna templates containing the bacteriophage sp6 rna polymerase promoter were used for rna transcription. Semliki forest virus an overview sciencedirect topics. Reverse genetic system, genetically stable reporter viruses and packaged subgenomic replicon based on a brazilian zika virus. Viral suppressors of rna interference impair rna silencing. Joseph charles glorioso, university of pittsburgh school of. The semliki forest virus replicon plasmid, psfv3lacz, was purchased from gibcobrl and the kunjin virus replicon plasmid, pc20dx bgal2ahdvrep1 has been described previously. In the basic version, cells are infected with a recombinant sfv where the part of the viral ribonucleic acid rna genome that encodes the virus structural proteins is replaced by a gene of interest transgene. Naked rna immunization with replicons derived from. Naked rna immunization with replicons derived from poliovirus and semliki forest virus genomes for the generation of a cytotoxic t cell response against the influenza a virus nucleoprotein member.