Lecture 3

Question 1

What is major difference in packaging DNA vs RNA?

Answer 1

DNA is pumped into an already formed capsid due to stiffness of DNA, whereas, RNA is packaaged as the capsid assembles (Coassembly)

Question 2

How can virus control what RNA is packaged?

Answer 2

Use of packaging signals on the RNA (e.g. stemloops) will ensure that only the viral RNA is packaged

Question 3

Why is it important that capsid assembly is reversible?

Answer 3

To uncoat later in new cell

To allow incorrect bonds to be broken and the correct ones made to result in a fully formed capsid

Question 4

How has the reaction been shown to occur quickly?

Answer 4

Intermediates rarely seen; either individual coat proteins for fully formed capsid

Question 5

What have molecular dynamics simulations shown?

Answer 5

Building blocks can form triangles and then icosahedrals quickly without any additional interaction by other cell components

Question 6

What was shown by Fraenkel-Conrat and Willaims?

Answer 6

Capsid formation for tobacoo mosaic virus occurs spontaneously without other cell machinery. Driven by hydrophobic and electrostatic interactions. Covalent bonds rarely involved in holding subuits together

Question 7

Describe the genome and capsid structure of TMV

Answer 7

6400 bases of ssRNA

Helical symmetry

2130 molecules of coat protein, which is 158 amino acids long.

Virion is 300nm long and 18nm wide

Question 8

Outline the process of TMV particle formation

Answer 8

30 protein subunits form a two-layer disk. Origin of assembly sequence on viral RNA (ensures right RNA is packaged) interacts with disk and induces allosteric conformational change to form the locked washer shape. The OAS loop is threaded through the hole and sits on top of the disk. Individual subunits are added in a spiral staircase manner as the OAS loop travels round.

Question 9

What are required for larger icosahedral viruses to be assembled?

Answer 9

Scaffolding proteins which form a mold which is used by the coat proteins to form the procapsid. Scaffold proteind are degraded and removed. The procapsid then undergoes maturation. DNA may then be added, however, sometimes empty capsids are formed.

Question 10

What occurs during procapsid maturation?

Answer 10

Process which makes the virion infectious

Small proteolytic cleavages lead to increased stability

Cross linking

Question 11

Give an example of targeting maturation for treating a viral disease

Answer 11

Inhibition of HIV protease, an enzyme involved in maturation, leads to virion being ‘dead-on-arrival’

Question 12

What about the nucleic acid backbone makes packagaing difficult?

How can this be overcome?

Answer 12

The negatively charged backbone will repel other sections of backbone; making it hard to package the DNA in small spaces

• Use small positively charged ions
• Positively charged Nucleo(capsid) proteins
• Histone-like or host histone proteins

Question 13

How is vNA specificty achieved?

Answer 13

• Stochastic approach - random which virions are infectious
• Nucleocapsid protein - links right nucleic acid with right coat protein
• Packaging signals
  
  • More in RNA where the nucleic acid is required for packaging

Question 14

What are the baltimore classes?

Answer 14

• Class I: dsDNA
• Class II: SSDNA
• Class III: dsRNA
• ClassIV: (+) ssRNA
• ClassV: (-) ssRNA
• ClassVI: (+) ssRNA-RT
• ClassVII: dsDNA-RT

Question 15

How is the bacteriphage T4 assembled?

Answer 15

Capsid is formed and has DNA added.

At the same time the tail is formed from the base plate up.

The capsid head is added onto the top of the tail.

The tail fibres are added onto the bottom of the tail.

Question 16

Outline the encaupsulation of M13

Answer 16

g5p forms proetective sheath around the ssDNA whilst g8p accumulates at the cytoplasmic membrance. g5p is exchanged for g8p upon exit. ~3000 g8p proteins surrounds the ssDNA.

Question 17

Why can M13 be used in recombinant DNA techniques?

Answer 17

A little extra nucleic acid can be encapsulated and transferred with the virion

Question 18

Describe the genome of rotavirus

Answer 18

dsRNA virus- which has 12 genome segments, with each encoding a single gene

Question 19

What is interesting about the rotavirus capsid?

Why is it essential for disease progression?

Answer 19

Forms 3 concentric icosahedral shells

Protects virus from pH of stomach and digestive enzymes in the gut. So can be delivered to target cells

Question 20

What are the advantages and disadvantages of dsRNA?

How are the challenges overcome by rotavirus?

Answer 20

dsRNA is more stable then both ssRNA and dsDNA, however, this make it difficult to denature, to create mRNA for transcription.

The double layer shell of rotavirus allows the dsRNA to say encapsulated after endocytosis maintaining conditions for denaturation. Also allowing evasion from RNAi.

Question 21

How are new rotavirus particles encapsulated for release?

Answer 21

dsRNA is replicated and double layer particles form in the viroplasm (viral factories made by NSP5 and NSP2). The double layer particles migrate to the ER where they otain their third and final layer (formed by VP7 and VP4). The fully formed virion is then releases by lysis.

Question 22

What are the steps in mass spec?

Answer 22

Ionisation of the particle

Seperation of particle by size and charge

Question 23

Outline the life cycle of hepatitis B

Answer 23

Partial uncoating of the virion causes the partial (+)cDNA to be completed by RT and it binds with the (-)cDNA to from dsDNA.

dsDNA forms mini-chromosome in nucleus using histones.

4mRNAs synthesised

C-mRNA is packaged into capsid of HBc. (-)cDNA fully formed.

RNase H degrades template RNA and partial (+)cDNA is synthesised during maturation.

Question 24

What role does the TR sequence play in MS2 capsid formation?

Answer 24

The TR sequence forms a stem loop which interacts with the capsid proteins; changing the conformation from symmetrical C/C to asymmetric A/B.

Question 25

Why is it preicted that other stem loops in the MS2 nucleic acid are involved in capsid formation?

Answer 25

There are 12 A/B pentamers in the capsid, and if a stemloop is required to turn every C/C dimer into A/B dimer then ~60 stemloops must exist.

Question 26

What affect does symmetry averaging have?

How is this important in virus reconstruction?

Answer 26

Washes out all asymmetric features

Virus’ with asymmetrci features, one distinguished vertex, will appear symmetrical

Question 27

How can the issues with symmetry averaging be overcome?

Answer 27

Pick the distinguished vetex and do 5-fold averaging about that vertex

If the absolute orientation is known an asymmetric recstruction can be completed

Question 28

What has a hamiltonian path shown about MS2 capsid assembly?

Answer 28

There are 40,678 possible routes for MS2 RNA to take to visit all 60 vertices and cause the dimer switch it is responsible for

Question 29

How was the number of possible routes been lowere using biochemical information?

Answer 29

The maturation protein connects to the start and end of the RNA. A hamiltonian path can start and end in the same place but the MS2 RNA does not; only 66 possible paths.

Question 30

How has the number of possible paths in MS2 capsid assembly been further lowered using enzyme kinetics?

Answer 30

Paths which cause continuous completion of hexamers or decamers are preffered by enzyme kinetics Only three possible paths

Question 31

Why is it possible to know the absolute orientation of the MS2 virus?

What does this allow?

Answer 31

When attached to the F-pilus the virus is slightly at an angle, so is not 5-fold symmetric. Allows asymmetric construction

Question 32

What is the outcome of the asymmetric reconstruction of MS2?

Answer 32

5 possible hamiltonian paths

One of which matches what was found according the enzyme kinetics and biochemical information

Question 33

Which stem loop-co-protein interaction should be strongest?

Answer 33

The interaction between the TR loop at the beginning to ensure correct path is followed

Question 34

Why is co-protein concentration important in ensuring correct RNA packaging?

Answer 34

If co-protein concentration constantly high then non-viral RNA becomes involved due to lack of competition. If co-protein levels rise steadily (as they do in real setting) then only viral RNA is encorporated into the path

Question 35

What type of proteins are viruses likely to be expressing at different stages of infection?

Answer 35

Early stage: Host defense suppression proteins

Midle Stage: Genome copying and viral structural proteins

Late stage: Capsid assembly and host exit (e.g. lysis) proteins

Question 36

Which MS2 genes are expressein the three stages of viral infection?

Answer 36

Early stage: RNA dependent RNA polymerase

Middle Stage: Coat protein and maturation protein

Late Stage: Lysis Protein

Question 37

Apart from capsid formation what role does the TR sequence play?

Answer 37

The stem loop that forms hides the start codon for the RNA dependent RNA polymerase gene. Thereofore high levels of co-protein inhibit further translation of the polymerase (negative feedback loop)