Lecture 4 - Structure and Composition of Viruses Flashcards
What is metastable?
How virions must be both stable and unstable.
Stable in that they need to be able to protect genome.
Unstable to be infectious.
How can potential energy drive viral invasion?
Surmounting an unfavourable energy barrier allows virus to unfold, potential energy converting to kinetic energy to allow invasion, uncoating, etc.
How can viruses be purified?
1) Plaque purified
2) Limiting dilution
3) Generation from a molecular clone (plasmid)
Plaque purification
Select virus from a single plaque, grow in cell culture
Limiting dilution
Biological cloning in chicken eggs (EG: for influenza)
Usual steps for studying viral structure
1)
2)
3)
1) Cell disruption
2) Centrifugation
3) Density gradient centrifugation
Methods of cell disruption
1)
2)
3)
1) Safest is repeated freeze/thaw cycles (two or three cycles)
2) Non-ionic detergents such as Triton X-100 or Nonidet P40 lyse plasma membrane, but not nuclear membrane
3) Homogenisers chop up cells with little blades
Effects of homogenisation of cell culture
Cell membranes form vesicles.
Microsomes - vesicle-bound sacs containing ribosomes, from homogenisation of rough ER
How do you centrifuge out nuclei and large cell fragments from a sample?
Low speed centrifugation (15 minutes at 1000g)
Sedimentation coefficient
Function of size and density
Virus window
When plotting sedimentation coefficient vs density, viral particles occupy a certain place on the graph
Ultracentrifugation
Spin samples at over 100,000g.
Need a vacuum (reduce friction), temperature controlled
Pellets virus particles from fluid.
Contents of ultracentifuge supernatant
Ribosomes, other cellular components
How are samples prepared for density gradient centrifugation?
Purify viral sample.
Ultracentrifugaiton. Discard supernatant.
Resuspend viral pellet in small volume of buffer
Two types of density gradient centrifugation
Rate zonal
Equilibrium
How does rate zonal centrifugation separate particles?
By density coefficient (density and size)
How does equilibrium centrifugation separate particles?
By density only
Rate zonal centrifugation process
1)
2)
3)
1) Preformed gradient in test tube (EG: of sucrose)
2) Centrifuge for a short amount of time (EG: 2 hours)
3) Sedimentation gradient of particles determines how far they migrate during limited spin time (THINK GEL ELECTROPHORESIS)
Equilibrium density gradient centrifugation process
1)
2)
3)
1) Liquid of a uniform density in test tube (EG: caesium chloride)
2) During long spin time (EG: 72 hours), caesium ions form a density gradient (densest at bottom)
3) Viral particles migrate, cluster at a similarly dense part of the test tube
Relative density of sample versus medium in rate zonal centrifugation
Sample is denser than medium
How can tubes that have been density centrifuged be used?
Can form distinct bands of viral particles. Can assay different density bands, see which are infectious.
How can viral proteins be analysed?
Using SDS (a detergent that breaks down viral proteins) PAGE
In SDS page, what does band intensity correlate with?
Protein density (more protein –> more intense band)
What does triangulation (T) describe?
How many capsomeres form a triangular subunit of an icosahedron
Outcome of triangulation
Very different sizes of capsids can be formed using different configurations of the same capsomeres
Features of poliovirus capsid proteins
Four proteins.
Present in equimolar amounts
T number of adenovirus
T=25
Features of adenovirus capsid proteins
Larger number of proteins than poliovirus.
Non-equimolar
Papillomavirus capsid protein features
72 capsomeres
All capsomeres are pentons, but arrange in two different ways. Some capsomeres have five neighbours, others have six neighbours
Virus with symmetrical arrangement of membrane spikes
Sindbis, of togaviridae.
Membrane spikes symmetrical according to icosahedral rules
Virus that undergoes a proteolytic step that forms a complex rod shaped capsid
HIV
Layout of HIV capsid
Fullerene cone
Pentamers cluster at base of cone (12 pentons)
Hexamers form continuous bands
How can helical capsids be described? 1) 2) 3) 4)
1) Left or right handed turn
2) Number of subunits per turn
3) Axial rise per subunit
4) Pitch (nM) per turn
Family that measles belongs to
Paramyxoviridae
Measles virus structure
Nucleocapsid. Not a strict capsid.
Envelope.
Ribonucleoprotein complex.
Measlesvirus nucleoprotein characteristics
60kDa, 525aa
Nuclear, cytosolic localisation
Most abundant protein in infected cells
Self assembles with viral RNA (forms a helical structure, does this without RNA)
Difference between structural and non-structural proteins
Structural - Proteins making up virion. Identified by SDS-PAG of isolated viral particles.
Non-structural - Proteins coded by virus, found in infected cells, but not pure viral culture
Is a viral genome structural or non-structural?
Structural
How can structural and non-structural proteins be differentiated?
Run a gel with infected cell, uninfected cell, pure viral culture. Compare
