Post by skyship on Jan 13, 2010 4:22:00 GMT -5
I will try to take this down to its beginning if I can.
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T1
Novel alphavirus vectors for gene delivery and transgene expression
Kenneth Lundstrom
F. Hoffmann-La Roche, Research Laboratories, Basel Switzerland
Expression vectors based on the replicons of Semliki Forest virus (SFV) and Sindbis virus, two common alphaviruses, have been engineered for efficient gene expression both in vitro and in vivo. Attractive features of alphavirus vectors are the rapid generation of high-titer recombinant virus particles, the broad host range, cytoplasmic RNA replication and extreme levels of recombinant protein expression. Although efficient infection and recombinant protein expression has been established, the high cytotoxicity of alphavirus vectors has restricted their use. Novel less cytotoxic vectors based on site-directed mutagenesis of the SFV nonstructural genes have therefore been engineered.
Earlier studies have demonstrated that SFV-based expression of recombinant G protein-coupled receptors and ligand-gated ion channels produces high specific binding activity and functional responses measured by coupling to G proteins (intracellular Ca2+-release, inositol phosphate accumulation, cAMP stimulation and GTPg S binding) and by electrophysiological recordings. Establishment of SFV-based large-scale expression in serum-free suspension cultures of various mammalian cell lines has allowed production of hundreds of milligrams of recombinant receptors for purification and biostructural studies. Recently, alphavirus vectors have been demonstrated to generate efficient neuron-specific transgene expression in organotypic hippocampal slice cultures as well as in rodent brain.
To further increase the application range of SFV vectors, novel mutants have been engineered to reduce host cell cytotoxicity and to prolong the survival of host cells. An amino acid substitution in the nuclear localization signal sequence of the nsP2 gene was earlier described to lead to a less virulent phenotype of a replication-competent SFV vector in mice. Introduction of an additional point mutation at position 259 in the nsP2 gene generated an expression vector (SFV-PD) with a further reduced cytotoxicity and prolonged survival of host cells. Point mutations at various other sites of the nonstructural genes nsP2 and nsP4 resulted in novel SFV vector demonstrating a temperature-sensitive phenotype. A triple mutant vector, SFV-PDP, with three point mutations in the nsP2 gene showed a significantly prolonged expression pattern (>20 days) in BHK-21 cells and primary cortical neurons. Because these less cytotoxic SFV vectors do not cause the rapid termination of host cell protein synthesis typically observed shortly after infection with the conventional SFV vector, it is possible to apply antisense, ribozyme and RNA interference technologies. Preliminary results indicated that efficient gene-specific inhibition could be obtained for several target genes in various mammalian host cells. For instance, the human dopamine D2 receptor stably expressed in a CHO cell line could be down-regulated after co-infection with SFV-PD vectors containing 500 bp sense and antisense fragments, respectively, from the coding region of the human dopamine D2 receptor gene.
www.boku.ac.at/IAM/5gdcpe/abstract_book.htm
===============================
replicons of Semliki Forest virus (SFV) and Sindbis virus, two common alphaviruses,
Alphavirus:
en.wikipedia.org/wiki/Alphavirus
semliki Forest Virus
The Semliki Forest virus was first isolated from mosquitoes in the Semliki Forest, Uganda by the Uganda Virus Research Institute in 1942. It is known to cause disease in both animals and man. It is an Alphavirus found in central, eastern, and southern Africa.
The Semliki Forest virus is a positive-stranded RNA virus with icosahedral capsid which is enveloped by a lipid bilayer, derived from the host cell. The outermost surface of the virus is almost entirely covered by heterodimers of glycoproteins E1 and E2, arranged in interconnective trimers, which form an outer shell. Trimers are anchored in the membrane by an E2 cytoplasmic domain that associates with the nucleocapsid.
The size of the virus genome is approximately 13,000 base pairs. The 5’ two thirds of the genome encode non-structural proteins and the structural proteins are encoded in the 3’ third. Replication occurs via a negative strand intermediate giving rise to a full length genomic RNA for export in new virions and a subgenomic message that is translated into the structural proteins.
Semliki Forest virus is spread mainly by mosquito bites. It is not able to infect mammals through inhalation or gastrointestinal exposure although rodents in the laboratory can be infected by intranasal instillation. The virus is able to cause a lethal encephalitis in rodents, but only one lethal human infection has been reported. Even in this one case, the patient was immunodeficient and had been exposed to large amounts of virus in the laboratory.
Semliki Forest virus has been used extensively in biological research as a model of the viral life cycle and of viral neuropathy. Due to its broad host range and efficient replication, it has also been developed as a vector for genes encoding vaccines and anti-cancer agents, and as a tool in gene therapy.
en.wikipedia.org/wiki/Semliki_forest_virus
Sindbis virus
Sindbis Virus (SINV) is a member of the Togaviridae family, in the alphavirus subfamily. The virus was first isolated in 1952 in Cairo, Egypt. The virus is transmitted by mosquitoes (Culex spp.) SINV causes sindbis fever in humans and the symptoms include arthralgia, rash and malaise. Sindbis fever is most common in South and East Africa, Egypt, Israel, Philippines and parts of Australia. Sindbis virus is an "arbovirus" (arthropod-borne) and is maintained in nature by transmission between vertebrate (bird) hosts and invertebrate (mosquito) vectors. Humans are infected with Sindbis virus when bitten by an infected mosquito. Recently SINV has been linked to Pogosta disease in Finland.[1]
Virus structure, genome and replication
Sindbis viruses are enveloped particles with an icosahedral capsid. Its genome is a single stranded RNA approximately 11.7kb long. It has a 5' cap and 3' polyadenylated tail therefore serves directly as messenger RNA (mRNA) in a host cell. The genome encodes four non-structural proteins at the 5' end and the capsid and two envelope proteins at the 3' end. This is characteristic of all Togaviruses. Replication is cytoplasmic and rapid. The genomic RNA is partially translated at the 5’ end to produce the non-structural proteins which are then involved in genome replication and the production of new genomic RNA and a shorter sub-genomic RNA strand. This sub-genomic strand is translated into the structural proteins. The viruses assemble at the host cell surfaces and acquire their envelope through budding"
en.wikipedia.org/wiki/Sindbis_virus
skyship
================================
T1
Novel alphavirus vectors for gene delivery and transgene expression
Kenneth Lundstrom
F. Hoffmann-La Roche, Research Laboratories, Basel Switzerland
Expression vectors based on the replicons of Semliki Forest virus (SFV) and Sindbis virus, two common alphaviruses, have been engineered for efficient gene expression both in vitro and in vivo. Attractive features of alphavirus vectors are the rapid generation of high-titer recombinant virus particles, the broad host range, cytoplasmic RNA replication and extreme levels of recombinant protein expression. Although efficient infection and recombinant protein expression has been established, the high cytotoxicity of alphavirus vectors has restricted their use. Novel less cytotoxic vectors based on site-directed mutagenesis of the SFV nonstructural genes have therefore been engineered.
Earlier studies have demonstrated that SFV-based expression of recombinant G protein-coupled receptors and ligand-gated ion channels produces high specific binding activity and functional responses measured by coupling to G proteins (intracellular Ca2+-release, inositol phosphate accumulation, cAMP stimulation and GTPg S binding) and by electrophysiological recordings. Establishment of SFV-based large-scale expression in serum-free suspension cultures of various mammalian cell lines has allowed production of hundreds of milligrams of recombinant receptors for purification and biostructural studies. Recently, alphavirus vectors have been demonstrated to generate efficient neuron-specific transgene expression in organotypic hippocampal slice cultures as well as in rodent brain.
To further increase the application range of SFV vectors, novel mutants have been engineered to reduce host cell cytotoxicity and to prolong the survival of host cells. An amino acid substitution in the nuclear localization signal sequence of the nsP2 gene was earlier described to lead to a less virulent phenotype of a replication-competent SFV vector in mice. Introduction of an additional point mutation at position 259 in the nsP2 gene generated an expression vector (SFV-PD) with a further reduced cytotoxicity and prolonged survival of host cells. Point mutations at various other sites of the nonstructural genes nsP2 and nsP4 resulted in novel SFV vector demonstrating a temperature-sensitive phenotype. A triple mutant vector, SFV-PDP, with three point mutations in the nsP2 gene showed a significantly prolonged expression pattern (>20 days) in BHK-21 cells and primary cortical neurons. Because these less cytotoxic SFV vectors do not cause the rapid termination of host cell protein synthesis typically observed shortly after infection with the conventional SFV vector, it is possible to apply antisense, ribozyme and RNA interference technologies. Preliminary results indicated that efficient gene-specific inhibition could be obtained for several target genes in various mammalian host cells. For instance, the human dopamine D2 receptor stably expressed in a CHO cell line could be down-regulated after co-infection with SFV-PD vectors containing 500 bp sense and antisense fragments, respectively, from the coding region of the human dopamine D2 receptor gene.
www.boku.ac.at/IAM/5gdcpe/abstract_book.htm
===============================
replicons of Semliki Forest virus (SFV) and Sindbis virus, two common alphaviruses,
Alphavirus:
en.wikipedia.org/wiki/Alphavirus
semliki Forest Virus
The Semliki Forest virus was first isolated from mosquitoes in the Semliki Forest, Uganda by the Uganda Virus Research Institute in 1942. It is known to cause disease in both animals and man. It is an Alphavirus found in central, eastern, and southern Africa.
The Semliki Forest virus is a positive-stranded RNA virus with icosahedral capsid which is enveloped by a lipid bilayer, derived from the host cell. The outermost surface of the virus is almost entirely covered by heterodimers of glycoproteins E1 and E2, arranged in interconnective trimers, which form an outer shell. Trimers are anchored in the membrane by an E2 cytoplasmic domain that associates with the nucleocapsid.
The size of the virus genome is approximately 13,000 base pairs. The 5’ two thirds of the genome encode non-structural proteins and the structural proteins are encoded in the 3’ third. Replication occurs via a negative strand intermediate giving rise to a full length genomic RNA for export in new virions and a subgenomic message that is translated into the structural proteins.
Semliki Forest virus is spread mainly by mosquito bites. It is not able to infect mammals through inhalation or gastrointestinal exposure although rodents in the laboratory can be infected by intranasal instillation. The virus is able to cause a lethal encephalitis in rodents, but only one lethal human infection has been reported. Even in this one case, the patient was immunodeficient and had been exposed to large amounts of virus in the laboratory.
Semliki Forest virus has been used extensively in biological research as a model of the viral life cycle and of viral neuropathy. Due to its broad host range and efficient replication, it has also been developed as a vector for genes encoding vaccines and anti-cancer agents, and as a tool in gene therapy.
en.wikipedia.org/wiki/Semliki_forest_virus
Sindbis virus
Sindbis Virus (SINV) is a member of the Togaviridae family, in the alphavirus subfamily. The virus was first isolated in 1952 in Cairo, Egypt. The virus is transmitted by mosquitoes (Culex spp.) SINV causes sindbis fever in humans and the symptoms include arthralgia, rash and malaise. Sindbis fever is most common in South and East Africa, Egypt, Israel, Philippines and parts of Australia. Sindbis virus is an "arbovirus" (arthropod-borne) and is maintained in nature by transmission between vertebrate (bird) hosts and invertebrate (mosquito) vectors. Humans are infected with Sindbis virus when bitten by an infected mosquito. Recently SINV has been linked to Pogosta disease in Finland.[1]
Virus structure, genome and replication
Sindbis viruses are enveloped particles with an icosahedral capsid. Its genome is a single stranded RNA approximately 11.7kb long. It has a 5' cap and 3' polyadenylated tail therefore serves directly as messenger RNA (mRNA) in a host cell. The genome encodes four non-structural proteins at the 5' end and the capsid and two envelope proteins at the 3' end. This is characteristic of all Togaviruses. Replication is cytoplasmic and rapid. The genomic RNA is partially translated at the 5’ end to produce the non-structural proteins which are then involved in genome replication and the production of new genomic RNA and a shorter sub-genomic RNA strand. This sub-genomic strand is translated into the structural proteins. The viruses assemble at the host cell surfaces and acquire their envelope through budding"
en.wikipedia.org/wiki/Sindbis_virus
skyship