Biomedical Engineering Reference
In-Depth Information
stable.at.60°C.for.up.to.1.h.and.are.stable.indeinitely.in.a.pH.range.from.3.5.to.9.0.at.room.
temperature.(Lin.and.Johnson.2003).
The. CPMV. genome. is. bipartite,. containing. two. individual. single-stranded,. positive-
sense. RNA. molecules,. packaged. into. separate. capsids.. The. larger. RNA. molecule,.
RNA-1,. is. 5889. nucleotides. in. length,. and. the. smaller. RNA-2. is. 3481. nucleotides. in.
length.(Lomonossoff.and.Johnson.1991)..Both.RNA.molecules.are.required.for.infection,.
exist.in.a.polyadenylated.state,.and.are.covalently.linked.to.a.small.protein,.(VPg,.virus.
genome-linked.protein).at.the.5′.end.(Sainsbury.et.al..2010)..Puriication.of.CPMV.virions.
from. infected. plant. tissue. by. centrifugation. on. a. density. gradient. yields. three. distinct.
populations:.the.top,.and.the.least.dense.population,.is.composed.of.empty.capsids.with.
no.packaged.RNA;.the.middle.population.is.made.up.of.virions.containing.RNA-2;.and.
the.bottom.population.contains.virions.encapsidating.RNA-1..Both.the.middle.and.bottom.
components.are.required.for.infection.of.whole.plants.(Lomonossoff.and.Johnson.1991)..
The. RNA-1. molecule. encodes. proteins. required. for. viral. RNA. replication. and. protein.
processing,.while.RNA-2.encodes.proteins.for.systemic.infection,.namely.the.movement.
protein,. which. is. required. for. transmission. from. cell. to. cell;. and. the. capsid. protein.
subunits.(Sainsbury.et.al..2010)..It.has.been.demonstrated.that.the.RNA.genome.can.be.
removed.yielding.intact,.empty.capsids.by.subjecting.the.virions.to.alkaline.conditions.
at.pH.9.4.(Ochoa.et.al..2006).
To. date,. little. is. known. about. the. assembly. mechanism. of. CPMV.. Some. evidence.
indicates.that.the.carboxyl-terminus.of.the.S.subunit.plays.a.role.in.the.encapsidation.of.
RNA.(Taylor.et.al..1999)..The.comoviruses.as.a.group.share.several.structural.and.genetic.
similarities.to.the.picornavirus.group,.(poliovirus,.rhinovirus,.and.coxsackie.virus,.etc.);.
and. while. some. information. regarding. self-assembly. of. the. picornaviruses. has. been.
elucidated,. it. is. premature. to. assume. that. CPMV. use. the. same. mechanisms. (Lin. et. al..
2003,.Manchester.and.Singh.2006)..Furthermore,.it.is.still.unknown.how.CPMV.manages.
to. recognize. and. encapsidate. only. the. two. viral. RNAs. and. whether. the. empty. capsids.
formed. are. the. result. of. an. error. or. a. byproduct. of. assembly.. Other. questions. yet. to. be.
answered.are.whether.a.speciic.length.of.RNA.is.needed.for.assembly.or.whether.there.
exists.a.speciic.RNA.packaging.signal,.either.in.the.primary.sequence.of.nucleotides.or.in.
the.secondary.sequence.structure.(Lomonossoff.and.Johnson.1991).
7.5.2 Material Templating
Initial.work.on.CPMV.explored.chemical.and.genetic.modiication.strategies.to.tailor.the.
surface.of.the.capsid..As.the.crystallographic.structure.of.CPMV.had.been.solved.to.2.8.Å,.
it.was.thought.that.there.were.no.available.cysteine.residues.available.for.modiication.on.
the.capsid.exterior..This.was.made.clear.as.1.4.nm.gold.nanoclusters.with.maleimide.groups.
failed.to.bind.to.the.capsid..However,.it.was.shown.that.cysteines.located.on.the.capsid.
interior.could.be.modiied.with.ethyl.mercury.phosphate,.a.compound.with.subnanometer.
dimensions.. It. is. possible. to. introduce. cysteines. on. the. capsid. exterior. through. peptide.
insertion. in. the. C. domain. (Cys295).. Through. this. approach,. Wang. demonstrated. that. it.
was.possible.to.conjugate.approximately.60.larger.gold.nanoclusters.to.the.exterior.(using.
maleimide.chemistry),.while.due.to.size.restraints,.cysteine.residues.on.the.interior.did.not.
react.(Wang.et.al..2002b)..Furthermore,.it.is.possible.to.control.the.position.and.number.of.
nanoparticles.bound.to.the.capsid.surface.by.changing.the.location.and.number.of.cysteine.
modiications.on.the.CPMV.capsid.(Figure.7.10).(Blum.et.al..2004)..The.cysteine-modiied.
CPMV.virions.have.also.been.used.to.generate.virus.arrays.on.gold.substrates.by.using.
