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Team:Potsdam Bioware/Project/Part Virus
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| + | CHO-cells with GFP-nanobody on the surface generated by our group bind YFP. Our task was to produce recombinant virus with YFP on the surface. Such a ligand-receptor complex should provide a targeted and efficient transport of the virus through the cell membrane and also allow the release of the viral DNA in the cell nucleus. To modify the surface exposed proteins of the recombinant virus we fused the desired genes to the N-terminus of VP2 cap-gene. VP2 cap-gene is not essential for viral infectivity, what makes it an ideal candidate for the insertions of sequences to retarget the particle. Therefor to reduce and to define the AAV tropism, the primary receptor motif, heparan sulfat proteoglycan motif, was koncked-out. | ||
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| + | The transgene flanked by the ITRs we used in our system was CFP reporter gene. When the cells were infected with the recombinant AAV, the reporter gene was first trascribed in the nucleous and translated to the mature protein in the cytoplasm. The CFP-detection was under UV-light, where the cells should exhibit bright cyan fluorescence. But no cyan fluorescence was detected. The yellow fluorescence was detectible, pointing out that the virus was completly assembled. To check whether the viral assembly worked we infected the AAV-HT1080 cells with the recombinant virus, like it was described in the AAV Helper-Free System Instruction Manual. The next day several HT1080 cells were gleaming cyan by the excitation. | ||
Revision as of 11:10, 24 September 2012
Part Virus
Contents |
Introduction
In our project AAV-virus acts as a selectionsystem for CHO-cells. The CHO-cells express antibodies presenting them on their surface. To check the Antibodies being right expressed we focused on two different selectionsystems with two different antigens presented from recombinant AAV-Virus. After a stable formation of antigen-antibody-komplex the virus infect the cells with a gene necessery for selection.
AAV2 as a selectionsystem
The first Selectionsystem is based on the expression of several fluorescence proteins. The CHO-cells present on their surface GFP-nanobody, which binds both GFP and YFP with a high affinity and high specifity. The recombinant AAV-Virus carries on its surface YFP to target specifically the CHO-cells with GFP-nanobodies and to infect them.
To examine the effect of the enzyme AID stably trasfected in the CHO-cells to induce the hypersomatic mutation, we designed a recombinant virus with CFP on its surface. GFP-nanobody expressed on CHO-cells binds YFP, but not the closely realted CFP. Our goal was to show the AID mutates the GFP-nanobody to an Antibody with a higher affinity to the CFP. Formation of a stable antigen-antibody complex enables the virus binding to the cell and infect it with a mcherry-signal. By excitation with the UV-light the infected cells could be visualised by shining with red colour.
The second selection system is based on the Virus giving the cells a survival signal by infecting them. Virus with the same CFP-antigen on the surface couldn't bind to the GFP-nanobody. Altered by the AID GFP-nanobody binds CFP linked to the virus following by the infection with Kanamycinresistance genecassette. The Kanamycin/Neomycin gene codes for the aminoglycoside phosphotransferase enzyme, which inactivates by phosphorylation the aminoglycoside antibiotics such as geneticin (G418). Cultured in the correct Geneticin concentration, which was determined by titration against nontransfected controls, selection for stable transfectants is possible.
AAV2 Genome
Adeno-associated virus (AAV) is a DNA virus with single-stranded linear genome of 4.7 kb. The genome consists of two open reading frames rep and cap which are flanked by two inverted terminal repeats (ITR).
The Rep gene located on the 5'-end of the genome encodes four overlapping multifunctional regulatory proteins. The transcription of the Rep proteins begins at the promoters p5 (Rep 78/Rep 68) and p19 (Rep 52/Rep 40). The two larger Rep proteins Rep78 and Rep68 are required for genome replication, the regulation of gene expression, and for site specific integration. The smaller Rep proteins Rep52 and Rep40 are involved in the encapsidation of the AAV-Genome.
The open reading frame cap on the 3'-end of the AAV-genome encodes the three viral capsid proteins VP1, VP2, and VP3. The transcription of the mRNA is regulated by the p40 promoter. Alternative splicing results in two mRNA the one encodes VP1 and the other VP2/ VP3. Appropriation (Verwendung) of an alternate splice acceptor removes the first AUG start codon for VP1. Thereby mainly formed mRNA encodes the VP2 and VP3 capsid proteins. The first AUG codon in the transcript is the initiation codon for VP3. The translation of VP2 begins at an upstream ACG non-methionin start codon resulting in an approximately 10-fold lower translation of VP2. The stop codon of the three capsid proteins is identical. The stoichiometric ratio of the three capsid proteins VP1, VP2 and VP3 forming the icosahedral form is 1:1:10.
Infection by AAV2
AAV2 viruses kill cancer cells without harming healthy ones. Viruses cross the membrane by a process known as penetration.
The virus particals present in the cell media come closer to the cells by diffusion. After several attachments to the cell membrane the virus binds to the cell receptors and enter the cell by receptor mediated endocytosis. There are three cell receptors playing a major role in infectivity. Ubiquitously expressed cell surface Heparan sulfate proteoglycan (HSPG) functions as a primary receptor account for the broad host range of AAV . aVß5 integrin and fibroblast growth factor 1 (FGFR-1) have a co-receptor activity for successful viral entry into the host cell. Bound AAV particles enter the cell very rapidly through clathrin mediated endocytosis. Each endosome carries a single AAV particle. After internalization the virus requires acidic environment for sufficient realease into the cytosol. AAv accumulates in the perinuclear and entry through the nuclear pore complex (NPC) into the nucleus.There uncoating and gene expression take place.
Lytic and lysogenic pathways
After entry the nucleous, AAV follows either lytic or lysogenic life cycles. In the presence of a helper virus, Adenovirus or Herpesvirus, the AAV is directed in a lytic life cycle. AAV undergoes a productive infection, which includes rapid replication in the nucleous and the afterward release of the new particles into the environment by bursting cells.
If there is no helper virus, AAV establish latency by integration into the host genome. The Rep expression is limited. When AAV infects a human cell, it integrates in a site-specific manner into the chromosome 19, called AAVS1. Essential components are the ITRs and the AAVS1 in cis and Rep proteins 78/68 in trans. Rep78/68 binds to the Rep-binding site (RBS) on ITR and unwinds the ITR at the terminal resolution site (trs).
The productive cycle is initiated by infecting with the helper virus, what affects stronger Rep expression and episomal AAV DNA replication. Rep proteins excise the AAV genes out of the host cell’s chromosome. The excised genes are replicated in the nucleous and the capsid proteins package the replicated genes in the cell. When a large number of these particles have assembled, the helper virus induce cell lysis and release the newly assembled partikles into the environment.
recombinant AAV2
Adeno-associated viruses are replication-deficient parvoviruses, although AAV can replicate in the absence of a helper virus, they require a coinfection of adenovirus for an efficient replication. To bypass the co-infection with adenoviruses, which is associated with disadvantages as the contamination of AAV preparation, all of the required genes of adenovirus were encoded on a helperplasmid. The Helperplasmid consiting of adenovirus E2A, VA und E4 genes, is transfected in the AAV-293-cells, which stably express the adenovirus E1 gene. The production of infectious adeno-associated virus particles is able by cotransfecting the AAV-293 cells with three plasmids: as even said the plasmid that carries the helper genes taken from adenovirus, a vectorplasmid that contains the AAV2 Rep-Cap and an AAV2 ITR-providing plasmid carrying the gene of interest. The transgenecassette flanked by the ITRs is wrapped as a single strand in the virus capsid. The replication of the Plasmid with transgenecassette is implemented with rep-proteins and enveloped in the viral capsid formed by cap-proteins.
Results
...
CHO-cells with GFP-nanobody on the surface generated by our group bind YFP. Our task was to produce recombinant virus with YFP on the surface. Such a ligand-receptor complex should provide a targeted and efficient transport of the virus through the cell membrane and also allow the release of the viral DNA in the cell nucleus. To modify the surface exposed proteins of the recombinant virus we fused the desired genes to the N-terminus of VP2 cap-gene. VP2 cap-gene is not essential for viral infectivity, what makes it an ideal candidate for the insertions of sequences to retarget the particle. Therefor to reduce and to define the AAV tropism, the primary receptor motif, heparan sulfat proteoglycan motif, was koncked-out.
The transgene flanked by the ITRs we used in our system was CFP reporter gene. When the cells were infected with the recombinant AAV, the reporter gene was first trascribed in the nucleous and translated to the mature protein in the cytoplasm. The CFP-detection was under UV-light, where the cells should exhibit bright cyan fluorescence. But no cyan fluorescence was detected. The yellow fluorescence was detectible, pointing out that the virus was completly assembled. To check whether the viral assembly worked we infected the AAV-HT1080 cells with the recombinant virus, like it was described in the AAV Helper-Free System Instruction Manual. The next day several HT1080 cells were gleaming cyan by the excitation.
