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Team:Freiburg/Content/Project/The viral vector
From 2014.igem.org
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| - | + | <h1>Retroviral introduction:</h1> | |
| - | < | + | <p> </p> |
| - | < | + | <p>Retroviruses are ubiquitous viruses, found in fish, avian and mammals. The diameter of the retroviral virion is approximately 80 – 100 nm. The outmost layer of the virus is a lipid bilayer derived from the host cell membrane. It surrounds the spherical capsid in which two identical single stranded RNA strands are located. They are positive-sense and approximately 7 – 11 kB long (5). </p> |
| - | <p> | + | <p>Generally the retroviral life cycle can be divided in two distinct phases: infection and replication. In the following sections we will focus on the Molony Murine Leukemia Virus, which is the origin of the viral vector we are actually using.</p> |
| - | < | + | <h1>Retroviral tropism and infection mechanism:</h1> |
| - | < | + | <p> </p> |
| - | + | <p>Viruses are classified as ecotropic, polytropic or amphotropic depending on the number of different host cells they are able to infect (ref). The Molony Murine Leukemia Virus (MMLV) is an ecotropic gammaretrovirus; it only infects rodent cells (mice and rat)(ref). This tropism of the virus is granted by its high specificity towards the mCAT1 receptor (ref). Once attached to the receptor the MMLV outer membrane fuses with the host cells membrane and the single stranded viral RNA genome is transferred into the cytoplasm. Here it is reverse transcribed into ds DNA by the viral reverse transcriptase. After the conversion the dsDNA is further processed by the viral Integrase protein. Integrase is recessing the 3’ ends of the ds DNA by 2 bp (1). When the cell is going through mitosis the ds DNA can reach the host cells genome due to the breakdown of the nuclear membrane, there the target sequence is cleaved and the 5’ ends are recessed. After integration the dsDNA is called provirus. The provirus starts expression of its genes.</p> | |
| - | + | <h1>The retoviral genome:</h1> | |
| - | + | <p> </p> | |
| - | + | <p>MMLV is a simple retrovirus naturally encoding only three genes called gag, pol and env. Besides this trans-gene elements there are also regulatory cis-gene elements. The trans-gene elements are flanked by long terminal repeats (LTR). The 5’ LTR harbors a promoter region, which initiates transcription of the provirus, whereas the 3’ LTR is needed for polyadenylation of the proviral mRNA. The gag (group antigenes) gene is precursor polyprotein which is further processed after translation. After procession it forms the major proteins building up the core particle, RNA binding proteins and the nucleoprotein core particle. The pol genes codes for the reverse transcriptase, RNase H and the Integrase. It is mandatory for proper processing of the viral RNA genome. The env gene codes for the envelope protein. This protein is responsible for the viral tropism and is located in the viral lipid bilayer which is derived from the host cell membrane. Further the genomes codes for a psi-packaging sequence. This sequence forms a secondary RNA structure and is needed for correct packaging of the viral genome into the virion (3). After proper packaging the virus is leaving the cell and can start the next round of transfection.</p> | |
| - | + | <h1>Retroviral vectors:</h1> | |
| - | + | <p> </p> | |
| - | + | <p>Retroviral vectors are used for the delivery of variable gene cargo into cells, a process termed transduction. To ensure safe handling the cis- and trans-acting gene elements are separated from each other. Helper viruses or packaging cell lines, carrying the trans-acting gene-elements are needed for producing the functional viral vector. Besides safety issues this approach also has the advantage that, due to the deletion, a larger gene cargo (~ 8 kB) can be integrated into the vector (4). Our MLV vector needs the Phoenix cell line in order to replicate. They carry the trans-acting gene elements under non-MLV promoters in order to minimize the probability of a recombination event that would lead to the production of replication efficient viruses. The advantages of retroviral vectors are especially the stable integration of gene cargo and their high specificity but also their flexible genome.</p> | |
| - | + | <p> </p> | |
| - | + | <p>Ref:</p> | |
| - | + | <p align="left">1 - The IN Protein of Moloney Murine Leukemia Virus Processes the Viral NA Ends and Accomplishes Their Integration In Vitro</p> | |
| + | <p> </p> | ||
| + | <p align="left">2- Integration of murine leukemia virus DNA depends on mitosis</p> | ||
| + | <p> </p> | ||
| + | <h1>3- NMR Structure of the 101-nucleotide Core Encapsidation Signal of the Moloney Murine Leukemia Virus</h1> | ||
| + | <p> </p> | ||
| + | <h1>4 - The use of retroviral vectors for gene therapy-what are the risks? A review of retroviral pathogenesis and its relevance to retroviral vector-mediated gene delivery.</h1> | ||
| + | <p> </p> | ||
| + | <h2>5 - Viral vectors for gene therapy: the art of turning infectious agents into vehicles of therapeutics</h2> | ||
| + | <p> </p> | ||
| + | <p>6 – Retroviral integration:</p> | ||
| + | <p align="left">Structure of the initial covalent product andn its</p> | ||
| + | <p align="left">precursor, and a role for the viral IN protein</p> | ||
| + | <p> </p> | ||
</body> | </body> | ||
Revision as of 14:35, 1 October 2014
Retroviral introduction:
Retroviruses are ubiquitous viruses, found in fish, avian and mammals. The diameter of the retroviral virion is approximately 80 – 100 nm. The outmost layer of the virus is a lipid bilayer derived from the host cell membrane. It surrounds the spherical capsid in which two identical single stranded RNA strands are located. They are positive-sense and approximately 7 – 11 kB long (5).
Generally the retroviral life cycle can be divided in two distinct phases: infection and replication. In the following sections we will focus on the Molony Murine Leukemia Virus, which is the origin of the viral vector we are actually using.
Retroviral tropism and infection mechanism:
Viruses are classified as ecotropic, polytropic or amphotropic depending on the number of different host cells they are able to infect (ref). The Molony Murine Leukemia Virus (MMLV) is an ecotropic gammaretrovirus; it only infects rodent cells (mice and rat)(ref). This tropism of the virus is granted by its high specificity towards the mCAT1 receptor (ref). Once attached to the receptor the MMLV outer membrane fuses with the host cells membrane and the single stranded viral RNA genome is transferred into the cytoplasm. Here it is reverse transcribed into ds DNA by the viral reverse transcriptase. After the conversion the dsDNA is further processed by the viral Integrase protein. Integrase is recessing the 3’ ends of the ds DNA by 2 bp (1). When the cell is going through mitosis the ds DNA can reach the host cells genome due to the breakdown of the nuclear membrane, there the target sequence is cleaved and the 5’ ends are recessed. After integration the dsDNA is called provirus. The provirus starts expression of its genes.
The retoviral genome:
MMLV is a simple retrovirus naturally encoding only three genes called gag, pol and env. Besides this trans-gene elements there are also regulatory cis-gene elements. The trans-gene elements are flanked by long terminal repeats (LTR). The 5’ LTR harbors a promoter region, which initiates transcription of the provirus, whereas the 3’ LTR is needed for polyadenylation of the proviral mRNA. The gag (group antigenes) gene is precursor polyprotein which is further processed after translation. After procession it forms the major proteins building up the core particle, RNA binding proteins and the nucleoprotein core particle. The pol genes codes for the reverse transcriptase, RNase H and the Integrase. It is mandatory for proper processing of the viral RNA genome. The env gene codes for the envelope protein. This protein is responsible for the viral tropism and is located in the viral lipid bilayer which is derived from the host cell membrane. Further the genomes codes for a psi-packaging sequence. This sequence forms a secondary RNA structure and is needed for correct packaging of the viral genome into the virion (3). After proper packaging the virus is leaving the cell and can start the next round of transfection.
Retroviral vectors:
Retroviral vectors are used for the delivery of variable gene cargo into cells, a process termed transduction. To ensure safe handling the cis- and trans-acting gene elements are separated from each other. Helper viruses or packaging cell lines, carrying the trans-acting gene-elements are needed for producing the functional viral vector. Besides safety issues this approach also has the advantage that, due to the deletion, a larger gene cargo (~ 8 kB) can be integrated into the vector (4). Our MLV vector needs the Phoenix cell line in order to replicate. They carry the trans-acting gene elements under non-MLV promoters in order to minimize the probability of a recombination event that would lead to the production of replication efficient viruses. The advantages of retroviral vectors are especially the stable integration of gene cargo and their high specificity but also their flexible genome.
Ref:
1 - The IN Protein of Moloney Murine Leukemia Virus Processes the Viral NA Ends and Accomplishes Their Integration In Vitro
2- Integration of murine leukemia virus DNA depends on mitosis
3- NMR Structure of the 101-nucleotide Core Encapsidation Signal of the Moloney Murine Leukemia Virus
4 - The use of retroviral vectors for gene therapy-what are the risks? A review of retroviral pathogenesis and its relevance to retroviral vector-mediated gene delivery.
5 - Viral vectors for gene therapy: the art of turning infectious agents into vehicles of therapeutics
6 – Retroviral integration:
Structure of the initial covalent product andn its
precursor, and a role for the viral IN protein
