Team:Wageningen UR/ObtainingthePoleroVLP

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== Isolation and BioBricking of the potato leaf roll virus (PLRV) coat protein. ==
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== ''Polerovirus'' ==
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'''Why work on PLRV?'''
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Potato Leaf Roll Virus (PLRV) and Turnip Yellows Virus (TuYV) are both members of the genus ''Polerovirus'' and family Luteoviridae, which are both positive sense RNA viruses as well. PLRV and TuYV will be called ''Polerovirus'' all together. Moreover, they are both distributed all over the world and cause great yield loss for crops yearly. Symptoms include chlorosis, necrosis and leaf curling (Figure 1). However, the hosts for PLVR and TuYV are different: PLRV mostly infects potatoes and other plants in the Solanaceae family; TuYV mainly infects rapeseed (Brassica. napus) and cabbage. [1,2]
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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). 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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<br>
 
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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, according to literature, have a function in the transmission by aphids, primarily the green peach aphid, ''Myzus persicae''. We expect that these spikes can be changed without having a large effect on VLP formation. This, thus, seems a good strategy to build in either E-coils or K-coils, described in the general project introduction.
 
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Virus like particles (VLPs) are being formed either with or without read trough proteins [1]. 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|center|900px|thumb|<p align="justify">''Figure 1: PLRV infected potato plants. [3]''</p>]]
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<br></p>
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[[File:orfs.jpg|center]]
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<p align="justify">
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The genomes of PLRV and TuYV are similar to each other: both of them have a leaky coat protein stop codon which is missed by the polymerase. Consequently, sometimes the 23kDa coat protein will be extended to 70kDa with an extra readthrough part (Figure 2). After capsid formation the C terminus of ''Polerovirus'' readthrough products will stick out and form a spike on the outside of the capsid. Capsids have been reported to form either with or without readthrough spikes. [4]
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[[File:models.jpg|center]]
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[[File: The PLRV genome.jpg|center|900px|thumb|<p align="justify">''Figure 2: PLRV genome.''</p>]]
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'''Work done on PLRV'''
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Compared with [[Team:Wageningen_UR/ModifyingtheCCMV|CCMV]] and [[Team:Wageningen_UR/ModifyingtheHepatitisB|Hepatitis B]], ''Polerovirus'' has its own unique advantage: the spike on the C terminus makes it much easier to modify a VLP on the outside. CCMV and Hepatitis B have a loop on the outside which can be modified, but extra extensions or deletions are needed. The C terminus spike of ''Polerovirus'' is not involved in VLP formation, so the natural characteristics of the VLP will not be changed after modification （Figure 3). Modification on the outside, in this case addition of the [[Team:Wageningen_UR/Coil_system|Plug and Apply System]], can be done more freely and in fewer steps.
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But the readthrough translation system has another advantage. For most VLP modifications, a mixture of wildtype and modified subunits is required to maintain VLP formation stability. The natural Polero system essentially automates and simplifies this procedure without requiring a duplicate gene or separate production strain. The ratio at which a readthrough translation event occurs can even be influenced using genetic architecting. [5]
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What’s more, the PLRV VLP has only been produced in eukaryotic (insect) cells. We would like to explore the possibility to produce ''Polerovirus'' in prokaryotic (''E.coli'') cells, which will make producing ''Polerovirus'' VLPs less laborious and cheaper. These are three of the reasons why we choose ''Polerovirus'' as our favorite [[Team:Wageningen_UR/ObtainingthePoleroVLP#Natural_BioBrick|Natural BioBrick]].
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</p>
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[[File:Structure of PLRV coat protein.jpg|center|900px|thumb|<p align="justify">''Figure 3: Structure of PLRV coat protein''</p>]]
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== Isolation of the PLRV coat protein ==
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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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In order to show the possibility of obtaining a BioBrick from nature, we investigated potato fields in the surroundings of Wageningen. But after some more inquiry we found out that PLRV is almost extinct in the Netherlands. Luckily we could obtain PLRV infected potato plant leaves from the Dutch General Inspection Service for Agricultural seed and seed potatoes (http://www.nak.nl). Later, we isolated the RNA from the infected leaves and synthesized cDNA from the RNA template.  
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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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<br><br>
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'''Progress''':
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The results of the RNA isolation is shown in Figure 4. The 28s rRNA bands appears equal to or more abundant than the 18s rRNA band, thereby indicating that little or no RNA degradation occurred during extraction.
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<br></p>
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[[File:RNApotato.jpg|left|900px|thumb|''Figure 4: RNA isolation from the PLRV infected leafs'']]
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[[File:Races.jpg|right|900px|thumb|''Table 1: Description of lanes in Figure 4'']]
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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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== Results ==
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We isolated the PLRV CP genes from multiple potato cultivars (table 1). Four of these were sequenced and the results were used to find natural variations in the viral coat protein: three PLRV coat proteins from different potato cultivars and 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 between the iGEM prefix and suffix. Unfortunately the sequence results of the PLRV CP with Histidine-tag showed the absence of the PstI restriction site in the suffix.
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The three different PLRV coat protein BioBrick parts were submitted to the Registry with the following accession numbers: [[Team:Wageningen_UR/Parts|BBa_K883402]], [[Team:Wageningen_UR/Parts|BBa_K883403]] and  [[Team:Wageningen_UR/Parts|BBa_K883404]],
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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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Alignment of the obtained sequences showed 9 single nucleotide polymorphisms (SNP's) of which only 3 resulted in a different amino acid. Amino acid replacement positions can be seen in the Figure 5. The PLRV isolated from Solanum tuberosum cv. Desirée (potato cultivar Desirée) showed the substitution of a valine with an alanine, which have very similar hydrophobic properties. Also the two other amino acid variations have more or less similar physical properties. These results show that the structure of the PLRV coat protein is very conserved.  
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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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[[File:aa differences.jpg|center|600px|thumb|''Figure 5: Amino acid substitutions in natural PLRV coat protein isolates'']]
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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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<br>
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== Natural BioBrick ==
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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 and know the potential of VLP coat proteins based on our results with the CCMV and Hepatatis B VLPs, we firmly believe in the significance of the isolated PLRV coat protein. This makes the PLRV coat protein biobrick our favorite natural biobrick.
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== Readthrough ==
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<p align="justify">
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The PLRV readthrough proteins are thought to have a function in the transmission by aphids, which are the vectors that spread the virus. We expect that these so-called ''spikes'' can be changed without having a large effect on VLP formation. It is shown that Polero VLPs can be formed either with or without the readthrough protein [4]. We wanted to know to what extent we could modify this spike for application of the [[Team:Wageningen_UR/Coil_system|Plug and Apply (PnA) System]]. Therefore, we isolated the PLRV coat protein including the readthrough part. PCR with primers specific to the start of the coat protein and to the end of the readthrough protein showed DNA segments of various sizes (Figure 6). This might indicate that there is large natural variation in the size of the readthrough. Unfortunately, all attempts to clone and sequence the PLRV readthrough failed. For the close relative TuYV though, we did manage to [[Team:Wageningen_UR/Parts#Parts_we_made|brick]] part of the readthrough.
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</p>
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[[File:isolation polero natural biobrick from infected potato plant leaves.jpg|center|600px|thumb|''Figure 6: Isolation of PLRV coat protein (CP) and coat protein with readthrough from (CP-RT)infected potato plants'']]
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== Isolation of the TuYV constructs==
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<p align="justify">
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The viral genes encoding the TuYV constructs were 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. Using procedures similar to those for PLRV, we successfully made four TuYV BioBricks (TuYV Coat Protein (CP), TuYV CP with N-terminal His-Tag (HT), TuYV CP with partial readthrough sequence (RT) and TuYV CP with both HT and RT) from the genome plasmid as well.
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</p>
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'''Future work'''
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==References==
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*Isolation of monomers and formation of VLP’s
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1. ''Potato leafroll virus''. Available from: http://en.wikipedia.org/wiki/Potato_leafroll_virus.
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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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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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''Further progress will be posted asap;)''
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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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5. Mao, P.L., et al., ''Predicting the efficiency of UAG translational stop signal through studies of physicochemical properties of its composite mono- and dinucleotides'' Computational Biology and Chemistry, 2004. 28: p. 245–256.
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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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Latest revision as of 03:21, 27 September 2012


Contents

Polerovirus

Potato Leaf Roll Virus (PLRV) and Turnip Yellows Virus (TuYV) are both members of the genus Polerovirus and family Luteoviridae, which are both positive sense RNA viruses as well. PLRV and TuYV will be called Polerovirus all together. Moreover, they are both distributed all over the world and cause great yield loss for crops yearly. Symptoms include chlorosis, necrosis and leaf curling (Figure 1). However, the hosts for PLVR and TuYV are different: PLRV mostly infects potatoes and other plants in the Solanaceae family; TuYV mainly infects rapeseed (Brassica. napus) and cabbage. [1,2]


Figure 1: PLRV infected potato plants. [3]

The genomes of PLRV and TuYV are similar to each other: both of them have a leaky coat protein stop codon which is missed by the polymerase. Consequently, sometimes the 23kDa coat protein will be extended to 70kDa with an extra readthrough part (Figure 2). After capsid formation the C terminus of Polerovirus readthrough products will stick out and form a spike on the outside of the capsid. Capsids have been reported to form either with or without readthrough spikes. [4]

Figure 2: 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 modify a VLP on the outside. CCMV and Hepatitis B have a loop on the outside which can be modified, but extra extensions or deletions are needed. The C terminus spike of Polerovirus is not involved in VLP formation, so the natural characteristics of the VLP will not be changed after modification （Figure 3). Modification on the outside, in this case addition of the Plug and Apply System, can be done more freely and in fewer steps.
But the readthrough translation system has another advantage. For most VLP modifications, a mixture of wildtype and modified subunits is required to maintain VLP formation stability. The natural Polero system essentially automates and simplifies this procedure without requiring a duplicate gene or separate production strain. The ratio at which a readthrough translation event occurs can even be influenced using genetic architecting. [5]
What’s more, the PLRV VLP has only been produced in eukaryotic (insect) cells. We would like to explore the possibility to produce Polerovirus in prokaryotic (E.coli) cells, which will make producing Polerovirus VLPs less laborious and cheaper. These are three of the reasons why we choose Polerovirus as our favorite Natural BioBrick.


Figure 3: Structure of PLRV coat protein

Isolation of the PLRV coat protein

In order to show the possibility of obtaining a BioBrick from nature, we investigated potato fields in the surroundings of Wageningen. But after some more inquiry we found out that PLRV is almost extinct in the Netherlands. Luckily we could obtain PLRV infected potato plant leaves from the Dutch General Inspection Service for Agricultural seed and seed potatoes (http://www.nak.nl). Later, we isolated the RNA from the infected leaves and synthesized cDNA from the RNA template.

The results of the RNA isolation is shown in Figure 4. The 28s rRNA bands appears equal to or more abundant than the 18s rRNA band, thereby indicating that little or no RNA degradation occurred during extraction.

Figure 4: RNA isolation from the PLRV infected leafs
Table 1: Description of lanes in Figure 4











Results

We isolated the PLRV CP genes from multiple potato cultivars (table 1). Four of these were sequenced and the results were used to find natural variations in the viral coat protein: three PLRV coat proteins from different potato cultivars and 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 between the iGEM prefix and suffix. Unfortunately the sequence results of the PLRV CP with Histidine-tag showed the absence of the PstI restriction site in the suffix. 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,

Alignment of the obtained sequences showed 9 single nucleotide polymorphisms (SNP's) of which only 3 resulted in a different amino acid. Amino acid replacement positions can be seen in the Figure 5. The PLRV isolated from Solanum tuberosum cv. Desirée (potato cultivar Desirée) showed the substitution of a valine with an alanine, which have very similar hydrophobic properties. Also the two other amino acid variations have more or less similar physical properties. These results show that the structure of the PLRV coat protein is very conserved.

Figure 5: Amino acid substitutions in natural PLRV coat protein isolates



Natural BioBrick

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 and know the potential of VLP coat proteins based on our results with the CCMV and Hepatatis B VLPs, we firmly believe in the significance of the isolated PLRV coat protein. This makes the PLRV coat protein biobrick our favorite natural biobrick.

Readthrough

The PLRV readthrough proteins are thought to have a function in the transmission by aphids, which are the vectors that spread the virus. We expect that these so-called spikes can be changed without having a large effect on VLP formation. It is shown that Polero VLPs can be formed either with or without the readthrough protein [4]. We wanted to know to what extent we could modify this spike for application of the Plug and Apply (PnA) System. Therefore, we isolated the PLRV coat protein including the readthrough part. PCR with primers specific to the start of the coat protein and to the end of the readthrough protein showed DNA segments of various sizes (Figure 6). This might indicate that there is large natural variation in the size of the readthrough. Unfortunately, all attempts to clone and sequence the PLRV readthrough failed. For the close relative TuYV though, we did manage to brick part of the readthrough.

Figure 6: Isolation of PLRV coat protein (CP) and coat protein with readthrough from (CP-RT)infected potato plants

Isolation of the TuYV constructs

The viral genes encoding the TuYV constructs were 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. Using procedures similar to those for PLRV, we successfully made four TuYV BioBricks (TuYV Coat Protein (CP), TuYV CP with N-terminal His-Tag (HT), TuYV CP with partial readthrough sequence (RT) and TuYV CP with both HT and RT) from the genome plasmid 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.

5. Mao, P.L., et al., Predicting the efficiency of UAG translational stop signal through studies of physicochemical properties of its composite mono- and dinucleotides Computational Biology and Chemistry, 2004. 28: p. 245–256.

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