12. RNA virus replication structures Flashcards
What viruses tend to make replication structures?
+ sense RNA viruses
Why don’t some viruses make replication structures?
- DNA viruses replicate in the nucleus so don’t need to change cellular architecture.
- We don’t really know why - sense RNA viruses don’t.
Does cell morphology change when they are infected with a virus?
- Yes infected cells look very different to uninfected cells.
- Often there are massive rearrangements around membranes like the mitochondria.
- These rearrangements are to aid the virus replication.
Why is studying virus replication structures tricky?
- Because viruses replicate in close association with membranes.
- It is hard to isolate viral proteins from membranes without disrupting the structure of the protein.
What are virus replication factories?
- Specialised intracellular compartments that viruses use to replicate their genome and assemble new infectious particles.
- They are large scaffolds to which virus and host factors/proteins are recruited.
- They involve cell membranes and cytoskeleton.
- Inter-organelle contacts occur that are not present in uninfected cells.
- Host metabolic and signalling pathways are manipulated to form these.
- This best characterised replication complexes are those in the cytoplasm.
What is the general process of RNA virus replication?
- The virus enters the cell.
- The virus uncoats somewhere in the cell. This process is transient and poorly understood.
- Once the viral genome is translated to produce the RNA-dependent RNA polymerase.
- The virus associates with a membrane for replication.
- This causes curves in the membrane to form a replication vesicle.
- This vesicle contains viral RNA and host and viral proteins.
- The RNA is then released from the vesicle and then packaged into the virus particle.
What are the advantages of viral replication factories?
- Enhanced replication efficiency
- Protection from host defences
- Spatial co-ordination of virus replication, translation and assembly
Advantages of viral replication factories: Enhanced replication efficiency
- This concentrates replication substrates in one place.
- They contain everything that is needed for replication in a compartment.
- It ensure the correct topology.
Advantages of viral replication factories: Protection from host defences
- It prevents the activation of host cell defence mechanisms that can be triggered by dsRNA or intermediates of RNA-virus replication.
- They hide dsRNA to prevent triggering of type 1 interferon response.
Advantages of viral replication factories: spatial co-ordination of virus replication, translation and assembly
- Ribosomes and RNA binding proteins are excluded from the replication complex.
- There needs to be this separation between RNA replication and packaging.
- The replication organelle regulates the amount of RNA made and released.
How are virus factories visualised?
- Using transmission electron microscopy
- Using Cryo EM/ET
What are the main viral replication structures?
- Spherules - a membrane enclosed vesicle, most common.
- Cubic membranes
- Lattice structures which hold the viral replication proteins in high concentration
What is cryo-EM and what is it used for?
- It is used to look at protein structures in combination with alpha fold.
- It is used to look at symmetrical viruses/structures.
- The virus is rapidly frozen then imaged in different planes
- A larger image is built up from all the images.
What is cryo-ET and what is it used for?
- It is used to look at non-symmetrical viruses/structures.
- You freeze and rotate the particle to get images of each slice of the virus.
- You can then build up the structure.
What is focus iron beam milling?
- You take a whole cell and use the iron beams to shave away a tiny layer
- As you do this you image the layer.
- This is used to image through the whole cell at atomic level.
- You then build up a 3D image of the cell.
- You can use this to see all stages of the viral lifecycle.
What can you see about viral replication factories using focus iron beam milling?
- You can reconstruct the replication factories in 3D.
- You can see double membrane vesicle in the ER which associates with the viral particle.
- Both coronaviruses and flaviviruses use these.
What is flock house virus?
- A model RNA virus that only encodes 4 proteins.
- It infects insects.
- Protein A is multifunctional and is responsible for replication.
- Capsid protein.
- B1 protein
- B2 protein which is important in viral defence against host immunity.
What did cyro-ET show about flock house virus replication factories?
- Cyro-ET was used to image the replication factory.
- Flock house virus forms replication structures in the mitochondrial membrane.
How did cyro-ET show how RNA leave the flock-house virus replication factory?
- It showed the formation of protein A into a crown structure.
- This acted as a channel that the RNA can enter and exit from once replicated.
- Protein A is also the polyermase
How does protein A form the replication vesicle for flock house virus?
- Protein A has a target signal that interact with the mitochondrial membrane and localise it there.
- Protein A then also interacts with the viral RNA.
- This triggers a change in conformation of the protein to start to shape the membrane.
- Protein A then pulls the RNA to the membrane and distorts the membrane to form the replication vesicle.
- Host factors are recruited.
- Protein A changes conformation depending on if its bound to RNA or itself.
- Once the RNA is replicated ssRNA can be released through the crown of protein A and then packaged.
What replication structures to coronaviruses use?
- They replicate in double membrane vesicles at the ER.
- The RNA also exist through a pore at the crown of the vesicle.
What makes up the coronavirus replication structure pore?
- It is made of a number of coronavirus proteins.
- NS3, NS4 and others proteins make up the pore.
- It is a multimeric structure.
- Many other RNA viruses also have this structure.
How is siRNA screening used to identify host factors associated with viral replication factories?
- Libraries of siRNAs against each protein in a cell and down regulate these proteins.
- Then infect the cells with the virus.
- Use an assay were each cells has a different protein knocked down.
- A knock down of a single protein can effect viral replication and you can observe this.
- You then use this information to work out which host proteins are important for viral replication.
What types of proteins are involved in virus replication complexes?
- RNA binding proteins for RNA recruitment and +/- strand RNA synthesis.
- Cellular chaperones and trafficking proteins for complex assembly, polyprotein folding and polymerase conformation.
- Protein targeting for replication protein targeting and anchoring proteins to membranes.
- Membrane remodelling and lipid synthesis for fatty acid synthesis and PI4P enrichment in membranes used for replication (altering membranes)
- Membrane-shaping proteins
What are membrane shaping proteins?
- They induce positive or negative curves in the membrane.
- Protein scaffolds
- Amphipathic helix insertion
- Transmembrane protein insertion and oligomerisation.
- Proteins that alter lipid head group for acyl chain composition
What 3 ways can curves be induced in membranes?
- Changing the composition of the membrane.
- Inserting proteins directly into the membrane.
- Proteins that interact with the lipid bilayer
Do all viruses replicate associated with the same membrane?
No
What membrane does Rubella associate with during replication?
the late endosome
What membrane does flock house virus associate with during replication?
mitochondria
What membrane do coronaviruses associate with during replication?
The ER membrane
What membrane does poliovirus associate with during replication?
the ER membrane
What membrane does dengue associate with during replication?
the ER membrane
How quickly does poliovirus take over host cells?
It causes massive changes and kills the host cell in about 6 hours.
What is the normal cell secretory pathway?
- Secreted or glycosylated proteins get inserted into the ER lumen for modification and then secretion.
- Proteins are then transported to the Golgi through the ER-Golgi intermediate compartment.
- COP1 and COP2 vesicle are important to move these proteins and fuse membranes to deliver proteins.
- COP2 transports proteins from ER to Golgi and COP1 recycles cellular material and proteins by taking them back to the Golgi
- ARF1 interacts with COP1 to recruit it back to the ER.
How does poliovirus subvert the normal cell secretory system?
- Initially the virus starts to replicate in association with the Golgi network.
- Lots of different viral proteins are made including 3A.
- 3A interacts with ARF1.
- This interaction stops the interaction of ARF1 with COP1 and stops the cell recycling process of cellular material.
- ARF1 also recruits other host cell molecules including PI4K3ß.
- PI4K3ß changes the composition of the lipid membrane of the ER.
- This then leads to the formation of viral replication vesicles which is a brand new organelle for viral replication.
What mechanisms does poliovirus used to interact with ARF1 and COP1?
- Protein 3A inserts itself into the ER-Golgi intermediate compartment membrane.
- It then interacts with ARF1 via GBF.
- This displaces the COP1 complex.
- This disrupts the recycling in ER-Golgi transport.
- This then recruits PI4K3ß.
- PI4K3ß changes the lipid composition of the membrane to allow viral proteins to bind.
- This includes the viral polymerase which then assembles the replication complex and the replication factory.
What host pathway do coronaviruses subvert for replication?
the endoplasmic reticulum associated proteins degradation pathway (ERAD)
How does the ERAD pathway normally work?
- Normally proteins made in the ER lumen and if they are damaged for too many the cell can get rid of them by unfolding or tuning.
- EDEM1 associates with misfolded proteins
- HRD1 is an E3 ubiquitin ligase that binds to the membrane and degrades proteins.
- Adaptor proteins like OS9, SEL1L and HERP are also recruited.
- This complex then degrades misfolded proteins.
- When all proteins are okay the complex dissociates.
- Some parts get degraded by the proteasome.
- EDEM1, OS9 and SEL1L are put into a vesicle by LC3-1 for lysosomal degradation.
How do coronaviruses subvert the ERAD pathway?
- Somehow coronaviruses replicate in the vesicles containing EDEM1, OS9 and SEL1L.
- This was found by discovering large amounts of viral proteins in these vesicles.
- Major coronavirus proteins that control this are NSP3/4/6.
- Instead of degradation the vesicles are used for viral replication.
What subcellular structure does hepatitis C virus use for replication?
Membranous webs
How does hepatitis C form membranous webs for replication?
- NS5a interacts with host membrane lipids.
- This mediates interactions with PI4Ka.
- This modifies the host membrane lipids.
- This recruits other host factors, lipid droplets and viral NS5b polymerase to the membrane.
- This induces curves in the membrane to form the replication membrane web.
- This is a very large distortion of the membrane.
