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Team:Potsdam Bioware/Project/Part Virus
From 2012.igem.org
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.
