Cowpea Chlorotic Mottle Virus: Difference between revisions
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===Self-Assembling Cowpea Chlorotic Mottle Virus Capsid: Nanoreactor and Scaffold for Molecular Synthesis=== | ===Self-Assembling Cowpea Chlorotic Mottle Virus Capsid: Nanoreactor and Scaffold for Molecular Synthesis=== | ||
<scene name='Cowpea_Chlorotic_Mottle_Virus/Full_capsid/2'>Assembled Capsid</scene> | <scene name='Cowpea_Chlorotic_Mottle_Virus/Full_capsid/2'>Assembled Capsid</scene> | ||
'''Introduction''' | '''Introduction''' | ||
The cowpea chlorotic mottle virus is a plant virus that infects the cowpea plant. It is a naked (non-enveloped), icosahedral virus | The cowpea chlorotic mottle virus is a plant virus that infects the cowpea plant. It is a naked (non-enveloped), icosahedral virus. Its capsomeres are stabilized by cationic metal binding (a motif similar to calmodulin in eukaryotic cells)<ref name=Speir>PMID:7743132</ref> and by the interactions of the negatively charged nucleic acid with the basic amino- terminus arms. CCMV contains a positive-sense RNA genome. Therefore, its encapsidated nucleic acids can be directly translated by host machinery to make the protein components for progeny virions. Its replication strategy requires the formation of a negative-sense template that can be used to synthesize new positive-sense RNA. | ||
An intriguing feature of virus is its ability to undergo a major structural change without destroying the tertiary structure of its subunits under certain environmental conditions. Specifically, when conditions are acidic (pH 5) the capsid is in a stable, assembled form. However, when the pH is raised ~6.5-7 or placed into solution of low ionic strength, the capsid undergoes a concerted structural change called swelling. <ref name=Speir>PMID:7743132</ref> | An intriguing feature of virus is its ability to undergo a major structural change without destroying the tertiary structure of its subunits under certain environmental conditions. Specifically, when conditions are acidic (pH 5) the capsid is in a stable, assembled form. However, when the pH is raised ~6.5-7 or placed into solution of low ionic strength, the capsid undergoes a concerted structural change called swelling. <ref name=Speir>PMID:7743132</ref> | ||
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In the field of virology, CCMV is relevant to mammalian research due to the similarity | In the field of virology, CCMV is relevant to mammalian research due to the similarity in structural transitions that human polio virus undergoes (picornaviridae family)<ref name=Speir>PMID:7743132</ref> | ||
(another naked icosahedral virus). | (another naked icosahedral virus). | ||
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First, virus can be directly extracted from plant leaves in high yields (1 kg of infected plant tissue can yield 1–2 g of virus<ref name=Liepold>PMID:Liepold16280622</ref>). By raising the pH to 7.5 and dissociating the capsid into dimers, the protein can be isolated from the RNA. The purified proteins can then self-assemble into functional virus-like particles when restored to more acidic conditions. | First, virus can be directly extracted from plant leaves in high yields (1 kg of infected plant tissue can yield 1–2 g of virus<ref name=Liepold>PMID:Liepold16280622</ref>). By raising the pH to 7.5 and dissociating the capsid into dimers, the protein can be isolated from the RNA. The purified proteins can then self-assemble into functional virus-like particles when restored to more acidic conditions. | ||
A more modern strategy involves the use of recombinant DNA technology. In this approach, the capsid-encoding genes of the virus can be transfected into a yeast expression vector.<ref name=Liepold>PMID:Liepold16280622</ref>) As the yeast replicate, they amplify the number of protein producing units, and the secreted protein products can be readily collected from the media. The major benefit to this method, aside from high yield production, is that the genome can be modified prior to insertion into the vector. This can be used to | A more modern strategy involves the use of recombinant DNA technology. In this approach, the capsid-encoding genes of the virus can be transfected into a yeast expression vector.<ref name=Liepold>PMID:Liepold16280622</ref>) As the yeast replicate, they amplify the number of protein producing units, and the secreted protein products can be readily collected from the media. The major benefit to this method, aside from high yield production, is that the genome can be modified prior to insertion into the vector. This can be used to generate capsid subunits with differing chemical properties. Such a procedure was performed by Dr. J. J. L. M. Cornelissen in the Netherlands. He attempted to solve the problem that many reactants of interest, particularly enzymes, are sensitive to the and the acidic requirements for capsid stability by looking for other stabilization strategies. He found that nickel atoms could stabilize the capsid at pH 7.5. This was a promising alternative to the large polyelectrolytes that were being used to stabilize capsids because they do not fill the capsid volume. | ||
Histidine residues were able to readily coordinate with nickel, and this property was used to purify the modified proteins from the media of their expression vectors. This purification was performed with a column of nickel atoms immobilized to a resin enriched with a metal chelating agent (NTA). Once the proteins were isolated, a experimental, pH-sensitive, protein (EGFP) was coupled with the capsomeres.<ref name=Liepold>PMID:Liepold16280622</ref>) | Histidine residues were able to readily coordinate with nickel, and this property was used to purify the modified proteins from the media of their expression vectors. This purification was performed with a column of nickel atoms immobilized to a resin enriched with a metal chelating agent (NTA). Once the proteins were isolated, a experimental, pH-sensitive, protein (EGFP) was coupled with the capsomeres.<ref name=Liepold>PMID:Liepold16280622</ref>) | ||
