Team:Wageningen UR/ObtainingthePoleroVLP

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(→Isolation and BioBricking of the potato leaf roll virus (PLRV) coat protein.)
(→Potato Leaf Roll Virus(PLRS) and Turnip Yellows Virus(TuYV))
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== Potato Leaf Roll Virus(PLRS) and Turnip Yellows Virus(TuYV) ==
== Potato Leaf Roll Virus(PLRS) and Turnip Yellows Virus(TuYV) ==
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'''Why work on PLRV?'''
 
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Different viruses can be used to form VLPs. In our team we used both Hepatitis B and Cowpea Chlorotic Mottle Virus. But one can imagine that for different purposes of the VLP's, different characteristics of the VLP are desired. For example: the VLP should be stable at pH 7.4 when used for medical applications. For other applications the pH at which the VLP should be stable can be different. One could imagine that for various applications the desired size of the VLP can be different. In order to show that the production of VLP's can be done with a virus of choice. We worked on Potato Leaf Roll Virus (PLRV) because this virus shows a very peculiar read trough event  at expression, more about this is explained below . We isolated the Potato Leaf Roll Virus from infected potato plants and BioBricked the gene that encodes the viral coat protein. By creating a PLRV Coat Protein BioBrick we showed that it is possible to isolate a virus of your own choice, express the coat proteins in ''E.coli'' and form VLP's. This makes the PLRV CP BioBricks our favorite Natural BioBricks.
 
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'''Aim''': Our primary aim is to obtain self-assembling VLP’s from PLRV in E. coli. If we succeed, we can attempt to modify the spike on the outside.
 
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'''More about PLRV'''
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Potato Leaf Roll Virus (PLRV) and Turnip Yellows Virus (TuYV) both are members of the genus Polerovirus and family Luteoviridae, which are both positive sense RNA virus as well. (PLRV and TuYV will be called Polerovirus all together). Moreover, they both distribute all over the world and cause great yield loss for crops yearly. However, the host for PLVR and TuYV are different: PLRV mostly infect potatoes and other plants in family Solanaceae; TuTV mainly infect rapeseed (Brassica. napus) and cabbage. [1] [2]
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The Polero (potato leaf roll virus, PLRV) virus coat proteins can be used as building blocks to form VLP’s. PLRV is a positive sense RNA virus (group IV). Viruses from this group have their genome directly utilized as if it were mRNA. Ribosomes from the host cell translate their genome into a protein, with RNA-dependent RNA polymerase as one of it. This means that to isolate the coat protein gene we need to isolate the RNA of infected potato plants which would include the viral RNA.
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In nature, the stopcodon of the coat protein is sometimes ‘’missed’’, which results in a 70kDa read through product. This is considerably larger than the 23kDa coat protein on its own. When the coat protein as well as the coat protein plus read through are assembled together to form the virus capsid, spikes are formed on the capsid. These spikes might have a function in the transmission by aphids, which are the vectors which spread the virus. The green peach aphid, ''Myzus persicae'' is the most important vector for spreading the virus.  We expect that these so-called ‘’spikes’’ can be changed without having a large effect on VLP formation. This is based on findings by Lamb et al. which show that PLRV VLP’s can be formed with and without the read trough [1]. The fact that PLRV VLP’s can be formed either with or without the read trough led to the idea that addition of the P’NAS (described in P’NAS section) would be allowable to still form VLP’s. The expression of coat protein monomers, needed for VLP formation, has never been done in E.coli. If this step is successful, further modifications on the VLP’s will be done.
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[[File:PLRV infected potato plants.jpg]]
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Figure 1: PLRV infected potato plants. [3]
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The genome of PLRV and TuYV is similar to each other: both of them have a leaky coat protein stop codon. Consequently, sometimes the 23kDa coat protein will be extended to 70kDa with an extra readthrough part. It has been reported that either coat protein or coat protein with readthrough can form VLPs.[4]
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[[File:orfs.jpg|center]]
 
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[[File:models.jpg|center]]
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[[File:The PLRV genome.jpg]]
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Figure 2:The PLRV genome.
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'''Work done on PLRV'''
 
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After obtaining the coat protein gene of PLRV, the PLRV coat protein was sent to be sequenced. The sequence of our isolate together with other isolates described in literature gave us information about the conserved regions in the gene. Alignment experiments were performed, also together with data of other members of the Luteoviridae.
 
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The PLRV coat protein was thereafter ‘’BioBricked’’. This brick was expressed in Escherichia coli and produced monomers were isolated and purified.
 
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'''Results''':
 
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*RNA isolation of potato leaf tissue
 
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*Reverse transcriptase reaction to obtain cDNA
 
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*PCR on cDNA using coat protein primers   
 
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*Addition of Prefix and Suffix to CP genes                         
 
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*Transformation of the gene, inserted into iGEM pSB1C3 backbone, into E.coli
 
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*Sequencing of coat protein gene
 
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*Submission to Registry
 
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*Luteoviridae coat protein/read trough analogy experiments
 
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[[File:PCR.jpg|center]]
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Compared with CCMV and Hepatitis B, Polerovirus has its own unique advantage: the spike on the C terminus makes it much easier to be modified on the outside. CCMV and Hepatitis B have a loop on the outside, in order to modify them, extra extension or deletion are needed, while the C terminus of Polerovirus will be stuck out and form a spike after VLP assembly. The spike is not involved in the VLP formation, so the natural characteristics of the VLP will not be changed after modification, in this case, adding the PnAS. What’s more, the PLRV VLP has only been produced in the eukaryotic cells, more specifically, insect cells. We would like to explore the possibility to produce PLRV in prokaryotic cells, such as E.coli, which will make producing PLRV VLP less laborious and cheaper. Based on two reasons above, we choose Polerovirus.
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[[File:Structure of PLRV coat protein.jpg]]
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Figure 3: Structure of PLRV coat protein.
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Four PLRV parts were sequenced: three PLRV coat proteins from different potato cultivars, one PLRV coat protein with a Histidine-tag added to the N-terminal. The three bare PLRV coat proteins were confirmed to be positioned nicely within the iGEM prefix and suffix. Unfortunately the sequence results showed the absence of the PstI restriction site in the suffix.
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In order to show the possibility of obtaining of biobrick from nature, we asked some PLRV infected potato plant leaves from ‘Dutch General Inspection Service for Agricultural seed and seed potatoes’ (www.nak.nl) and they were sent to us by normal service. Later, we isolated the RNA from the infected leaves and synthesized cDNA from the RNA template. With designed primers, the coat protein gene of the PLRV was isolated and bricked with iGEM prefix and suffix. Because we obtained the biobrick totally from nature, this makes the PLRV coat protein biobrick is our favorite natural biobricks.
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The three different PLRV coat protein BioBrick parts were submitted to the Registry with the following accession numbers: BBa_K883402, BBa_K883403 and BBa_K883404.
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[[File:cps.jpg|center]]
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The viral gene encoding the Coat Protein for TuYV (GenBank: NC_003743.1 (3483..5495)) was obtained from a plasmid encoding the entire viral genome (GenBank: X13063.1). This plasmid was provided to us, via Dr. Kormelink of Wageningen UR’s Virology faculty, by Véronique Brault of the UMR SVQV in Strasbourg. With designed primers, the TuYV coat protein and coat protein with partial readthrough, both with or without his-tag, were bricked into iGEM standard backbone as well.  
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Alignment of the different PLRV isolates shows 9 SNP’s but not all SNP’s result in a different amino acid (only 3). The position of these amino acid changes if shown in the figure below.
 
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[[File:aapositions.jpg|center]]
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References:  
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1. Potato leafroll virus. Available from: http://en.wikipedia.org/wiki/Potato_leafroll_virus.
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'''Future work'''
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2. Juergens, M., et al., Genetic analyses of the host-pathogen system Turnip yellows virus (TuYV)-rapeseed (Brassica napus L.) and development of molecular markers for TuYV-resistance. Theor Appl Genet, 2010. 120(4): p. 735-44.
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3. diseases: Potato leafroll luteovirus - Potato Leaf Roll Virus (PLRV) Available from: http://www.agroatlas.ru/en/content/diseases/Solani/Solani_Potato_leafroll_luteovirus/.
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*Isolation of monomers and formation of VLP’s
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4. Lamb, J.W., et al., Assembly of virus-like particles in insect cells infected with a baculovirus containing a modified coat protein gene of potato leafroll luteovirus. J Gen Virol, 1996. 77(Pt 7): p. 1349-58.
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*VLP analysis/characterization
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We inserted an IPTG inducible promotor upstream the PLRV CP gene. We performed 2 attempts to express the PLRV coat protein in E.coli but these were not successful. We obtained PLRV antibodies to use with Western blot to check for PLRV CP expression. Right now, we focus on getting the PLRV CP expressed. After successful expression we will try to isolate formed VLP's.  
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''Further progress will be posted asap;)''
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[1]: Journal of General Virology (1996), 77, 1349-1358
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Assembly of virus-like particles in insect cells infected with a baculovirus containing a modified coat protein gene of potato leafroll luteovirus
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J. W. Lamb, G. H. Duncan, B. Reavy, F. E. Gildow, M. A. Mayo and R. T. Hay
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Revision as of 20:38, 20 September 2012


Potato Leaf Roll Virus(PLRS) and Turnip Yellows Virus(TuYV)

Potato Leaf Roll Virus (PLRV) and Turnip Yellows Virus (TuYV) both are members of the genus Polerovirus and family Luteoviridae, which are both positive sense RNA virus as well. (PLRV and TuYV will be called Polerovirus all together). Moreover, they both distribute all over the world and cause great yield loss for crops yearly. However, the host for PLVR and TuYV are different: PLRV mostly infect potatoes and other plants in family Solanaceae; TuTV mainly infect rapeseed (Brassica. napus) and cabbage. [1] [2] PLRV infected potato plants.jpg Figure 1: PLRV infected potato plants. [3] The genome of PLRV and TuYV is similar to each other: both of them have a leaky coat protein stop codon. Consequently, sometimes the 23kDa coat protein will be extended to 70kDa with an extra readthrough part. It has been reported that either coat protein or coat protein with readthrough can form VLPs.[4] The PLRV genome.jpg Figure 2:The PLRV genome. Compared with CCMV and Hepatitis B, Polerovirus has its own unique advantage: the spike on the C terminus makes it much easier to be modified on the outside. CCMV and Hepatitis B have a loop on the outside, in order to modify them, extra extension or deletion are needed, while the C terminus of Polerovirus will be stuck out and form a spike after VLP assembly. The spike is not involved in the VLP formation, so the natural characteristics of the VLP will not be changed after modification, in this case, adding the PnAS. What’s more, the PLRV VLP has only been produced in the eukaryotic cells, more specifically, insect cells. We would like to explore the possibility to produce PLRV in prokaryotic cells, such as E.coli, which will make producing PLRV VLP less laborious and cheaper. Based on two reasons above, we choose Polerovirus. Structure of PLRV coat protein.jpg Figure 3: Structure of PLRV coat protein. In order to show the possibility of obtaining of biobrick from nature, we asked some PLRV infected potato plant leaves from ‘Dutch General Inspection Service for Agricultural seed and seed potatoes’ (www.nak.nl) and they were sent to us by normal service. Later, we isolated the RNA from the infected leaves and synthesized cDNA from the RNA template. With designed primers, the coat protein gene of the PLRV was isolated and bricked with iGEM prefix and suffix. Because we obtained the biobrick totally from nature, this makes the PLRV coat protein biobrick is our favorite natural biobricks. The viral gene encoding the Coat Protein for TuYV (GenBank: NC_003743.1 (3483..5495)) was obtained from a plasmid encoding the entire viral genome (GenBank: X13063.1). This plasmid was provided to us, via Dr. Kormelink of Wageningen UR’s Virology faculty, by Véronique Brault of the UMR SVQV in Strasbourg. With designed primers, the TuYV coat protein and coat protein with partial readthrough, both with or without his-tag, were bricked into iGEM standard backbone as well. References: 1. Potato leafroll virus. Available from: http://en.wikipedia.org/wiki/Potato_leafroll_virus. 2. Juergens, M., et al., Genetic analyses of the host-pathogen system Turnip yellows virus (TuYV)-rapeseed (Brassica napus L.) and development of molecular markers for TuYV-resistance. Theor Appl Genet, 2010. 120(4): p. 735-44. 3. diseases: Potato leafroll luteovirus - Potato Leaf Roll Virus (PLRV) Available from: http://www.agroatlas.ru/en/content/diseases/Solani/Solani_Potato_leafroll_luteovirus/. 4. Lamb, J.W., et al., Assembly of virus-like particles in insect cells infected with a baculovirus containing a modified coat protein gene of potato leafroll luteovirus. J Gen Virol, 1996. 77(Pt 7): p. 1349-58.

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