Lecture 4 - Structure and Composition of Viruses

Question 1

What is metastable?

Answer 1

How virions must be both stable and unstable.
Stable in that they need to be able to protect genome.
Unstable to be infectious.

Question 2

How can potential energy drive viral invasion?

Answer 2

Surmounting an unfavourable energy barrier allows virus to unfold, potential energy converting to kinetic energy to allow invasion, uncoating, etc.

Question 3

How can viruses be purified?

Answer 3

1) Plaque purified
2) Limiting dilution
3) Generation from a molecular clone (plasmid)

Question 4

Plaque purification

Answer 4

Select virus from a single plaque, grow in cell culture

Question 5

Limiting dilution

Answer 5

Biological cloning in chicken eggs (EG: for influenza)

Question 6

Usual steps for studying viral structure
1)
2)
3)

Answer 6

1) Cell disruption
2) Centrifugation
3) Density gradient centrifugation

Question 7

Methods of cell disruption
1)
2)
3)

Answer 7

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

Question 8

Effects of homogenisation of cell culture

Answer 8

Cell membranes form vesicles.

Microsomes - vesicle-bound sacs containing ribosomes, from homogenisation of rough ER

Question 9

How do you centrifuge out nuclei and large cell fragments from a sample?

Answer 9

Low speed centrifugation (15 minutes at 1000g)

Question 10

Sedimentation coefficient

Answer 10

Function of size and density

Question 11

Virus window

Answer 11

When plotting sedimentation coefficient vs density, viral particles occupy a certain place on the graph

Question 12

Ultracentrifugation

Answer 12

Spin samples at over 100,000g.
Need a vacuum (reduce friction), temperature controlled

Pellets virus particles from fluid.

Question 13

Contents of ultracentifuge supernatant

Answer 13

Ribosomes, other cellular components

Question 14

How are samples prepared for density gradient centrifugation?

Answer 14

Purify viral sample.
Ultracentrifugaiton. Discard supernatant.
Resuspend viral pellet in small volume of buffer

Question 15

Two types of density gradient centrifugation

Answer 15

Rate zonal

Equilibrium

Question 16

How does rate zonal centrifugation separate particles?

Answer 16

By density coefficient (density and size)

Question 17

How does equilibrium centrifugation separate particles?

Answer 17

By density only

Question 18

Rate zonal centrifugation process
1)
2)
3)

Answer 18

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)

Question 19

Equilibrium density gradient centrifugation process
1)
2)
3)

Answer 19

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

Question 20

Relative density of sample versus medium in rate zonal centrifugation

Answer 20

Sample is denser than medium

Question 21

How can tubes that have been density centrifuged be used?

Answer 21

Can form distinct bands of viral particles. Can assay different density bands, see which are infectious.

Question 22

How can viral proteins be analysed?

Answer 22

Using SDS (a detergent that breaks down viral proteins) PAGE

Question 23

In SDS page, what does band intensity correlate with?

Answer 23

Protein density (more protein –> more intense band)

Question 24

What does triangulation (T) describe?

Answer 24

How many capsomeres form a triangular subunit of an icosahedron

Question 25

Outcome of triangulation

Answer 25

Very different sizes of capsids can be formed using different configurations of the same capsomeres

Question 26

Features of poliovirus capsid proteins

Answer 26

Four proteins.

Present in equimolar amounts

Question 27

T number of adenovirus

Answer 27

T=25

Question 28

Features of adenovirus capsid proteins

Answer 28

Larger number of proteins than poliovirus.

Non-equimolar

Question 29

Papillomavirus capsid protein features

Answer 29

72 capsomeres
All capsomeres are pentons, but arrange in two different ways. Some capsomeres have five neighbours, others have six neighbours

Question 30

Virus with symmetrical arrangement of membrane spikes

Answer 30

Sindbis, of togaviridae.

Membrane spikes symmetrical according to icosahedral rules

Question 31

Virus that undergoes a proteolytic step that forms a complex rod shaped capsid

Answer 31

HIV

Question 32

Layout of HIV capsid

Answer 32

Fullerene cone
Pentamers cluster at base of cone (12 pentons)
Hexamers form continuous bands

Question 33

How can helical capsids be described?
1)
2)
3)
4)

Answer 33

1) Left or right handed turn
2) Number of subunits per turn
3) Axial rise per subunit
4) Pitch (nM) per turn

Question 34

Family that measles belongs to

Answer 34

Paramyxoviridae

Question 35

Measles virus structure

Answer 35

Nucleocapsid. Not a strict capsid.
Envelope.
Ribonucleoprotein complex.

Question 36

Measlesvirus nucleoprotein characteristics

Answer 36

60kDa, 525aa
Nuclear, cytosolic localisation
Most abundant protein in infected cells
Self assembles with viral RNA (forms a helical structure, does this without RNA)

Question 37

Difference between structural and non-structural proteins

Answer 37

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

Question 38

Is a viral genome structural or non-structural?

Answer 38

Structural

Question 39

How can structural and non-structural proteins be differentiated?

Answer 39

Run a gel with infected cell, uninfected cell, pure viral culture. Compare