Lecture 3 Flashcards
What is major difference in packaging DNA vs RNA?
DNA is pumped into an already formed capsid due to stiffness of DNA, whereas, RNA is packaaged as the capsid assembles (Coassembly)
How can virus control what RNA is packaged?
Use of packaging signals on the RNA (e.g. stemloops) will ensure that only the viral RNA is packaged
Why is it important that capsid assembly is reversible?
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
How has the reaction been shown to occur quickly?
Intermediates rarely seen; either individual coat proteins for fully formed capsid
What have molecular dynamics simulations shown?
Building blocks can form triangles and then icosahedrals quickly without any additional interaction by other cell components
What was shown by Fraenkel-Conrat and Willaims?
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
Describe the genome and capsid structure of TMV
6400 bases of ssRNA
Helical symmetry
2130 molecules of coat protein, which is 158 amino acids long.
Virion is 300nm long and 18nm wide
Outline the process of TMV particle formation
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.
What are required for larger icosahedral viruses to be assembled?
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.
What occurs during procapsid maturation?
Process which makes the virion infectious
Small proteolytic cleavages lead to increased stability
Cross linking
Give an example of targeting maturation for treating a viral disease
Inhibition of HIV protease, an enzyme involved in maturation, leads to virion being ‘dead-on-arrival’
What about the nucleic acid backbone makes packagaing difficult?
How can this be overcome?
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
How is vNA specificty achieved?
- 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
What are the baltimore classes?
- Class I: dsDNA
- Class II: SSDNA
- Class III: dsRNA
- ClassIV: (+) ssRNA
- ClassV: (-) ssRNA
- ClassVI: (+) ssRNA-RT
- ClassVII: dsDNA-RT
How is the bacteriphage T4 assembled?
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.
Outline the encaupsulation of M13
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.
Why can M13 be used in recombinant DNA techniques?
A little extra nucleic acid can be encapsulated and transferred with the virion
Describe the genome of rotavirus
dsRNA virus- which has 12 genome segments, with each encoding a single gene
What is interesting about the rotavirus capsid?
Why is it essential for disease progression?
Forms 3 concentric icosahedral shells
Protects virus from pH of stomach and digestive enzymes in the gut. So can be delivered to target cells
What are the advantages and disadvantages of dsRNA?
How are the challenges overcome by rotavirus?
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.
How are new rotavirus particles encapsulated for release?
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.
What are the steps in mass spec?
Ionisation of the particle
Seperation of particle by size and charge
Outline the life cycle of hepatitis B
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.
What role does the TR sequence play in MS2 capsid formation?
The TR sequence forms a stem loop which interacts with the capsid proteins; changing the conformation from symmetrical C/C to asymmetric A/B.
