7. Viral uncoating Flashcards
Why does viral uncoating need to be carefully controlled?
- Viral particles are carefully assembled during replication.
- They are very stable particles in the environment that need to rapidly fall apart once they enter the cell.
- This is programmed.
- Uncoating is triggered and mediated by host cell proteins as you know that you are in a cell.
- Some other cues like pH drop can be used.
What is the purpose of viral uncoating?
- Viruses need to get to the site of replication for their genome.
- Cells can be different shapes so the nucleus can be far away from the edge of the cell.
- This creates a problem for viruses trying to find the nucleus for replication.
- Uncoating releases proteins that aid the virus to move towards the nucleus.
What is the ubiquitin ligase system?
- A system for degrading and recycling proteins in cells.
- Ubiquitin can covalently bind to proteins to flag them for the proteasome. They usually bind to a lysine residue.
- These proteins are then degraded and recycled.
- This process constantly occurs in cells.
- Ubiquitin can also be ubiquitinated to make ubiquitin chains which drives proteins into the proteasome.
What is ubiquitin?
A short 76 amino acid long peptide that exist free in cells.
What is the mechanism of the ubiquitin ligase system?
- E1 is ubiquitinated which requires ATP.
- E1 passes the ubiquitination onto E2.
- E3 picks up a target protein and brings it to E2.
- E2 then ubiquitinates the target protein.
- The E3 ubiquitin ligases target individual proteins for E2 to degrade.
What is the ubiquitin system essential for in host cells?
- Cell cycle control
- controlling proliferation.
How can adenoviruses hijack the ubiquitin ligase system?
- They direct ubiquitin ligases to degrade proteins it wants to get rid of quickly.
- These are usually proteins that will interfere with viral replication.
- This is much quicker then targeting the production of these proteins.
- Over ubiquitination rapidly get rid of host cell proteins.
- This is useful for viral entry
What are microtubules?
- The main way for moving things around the cell.
- They spread out from the microtubule organising centre (MTOC).
- MTOC is associated with the nucleus and the microtubules radiate out from this point.
- The + end is dynamic and undergoes polymerisation and depolymerisation.
- The - end is anchored at the MTOC and is less dynamic
- Microtubules help form the shape of the cell.
Why do viruses associate with the microtubules?
- If a virus associates with the microtubules and makes its way towards the - end, it will end up near the nucleus of the cell.
- Most helpful to associate with dynactin to move towards the nucleus.
How is cargo moved along the microtubules?
- Via motor proteins called dynactin and kinesin.
- Kinesin moves cargo towards the positive end and away from MTOC.
- Dynactin moves towards the negative end and towards MTOC.
- They attach substrate by various adaptor proteins and move cargo around the cell.
What is the nuclear pore complex?
- It is a very complex protein made up of many different proteins.
- It is embedded in the nuclear membrane and allows movement in and out of the nucleus.
- There are a few thousand nuclear pores per cell.
Do all proteins need to use the nuclear pore to enter the nucleus?
- No
- Proteins less than 40KDa can diffuse across the pore.
- This is passive.
- 40KDa is a loose boundary and diffusion can also depend on shape can charge of the protein.
How do proteins cross through the nuclear pore complex?
- They are actively transported through the poor.
- Proteins interact with the cytoplasmic filaments of the nuclear pore and trigger nuclear import.
- Cellular and viral proteins need to bind an import/export factor to enter/leave the nucleus.
- This requires energy.
What are nuclear localisation sequences?
- These are short arginine or lysine-rich sequences in proteins.
- These bind import factors.
- This allows proteins to enter the nucleus.
(This is a slight simplification.)
What are adenoviruses?
- Non-enveloped, linear dsDNA genome virus.
- Viral replication and assembly of particles occurs in the nucleus of infected cells.
- It is most famous for its potential as a gene therapy vector.
What is the structure of adenoviruses?
- Hexons form the bulk of the virus particle.
- The hexons make the icosahedral shape.
- Core proteins like protein 5 and 7 wrap and condense the genome.
- Cement proteins stabilise the viral particle by associating with the hexons.
What are the most common adenoviruses?
- Type 2 and type 5 adenoviruses.
- These are very common.
- These cause very mild colds.
What is the adenovirus protein 6?
- It is a cement protein.
- It is associated with the inside of the hexons.
- It plays an important role in viral uncoating.
What is the adenovirus penton base protein?
- It makes the points of the icosahedral shape.
- It also has the fibre that allows the virus to attach to host cells.
How are most adenovirus proteins made?
- As immature precursors.
- These are used to assemble the virus.
- This includes protein 6, protein 7, protein 8 and terminal protein.
How are the adenovirus precursor proteins activated?
- A viral protease called AVP is also packaged into the adenovirus particle.
- AVP is activated as the host cell dies and the virus is released.
- AVP cleaves specific sites in the precursor proteins inside the immature virus.
- All the proteins need to be cleaved and are needed to make the mature virus particle.
- Once the proteins are cleaved, the structure of the virion changes to make the mature virus.
What happens if the adenovirus precursor proteins aren’t cleaved?
- The viral proteins can’t mature.
- So the virus particle can’t mature.
- This virus can still enter the endosome but it can’t escape the endosome so the virus dies.
- If the virus has no mature proteins it is not infectious.
How does adenovirus enter the host cell?
- It attaches to the host cell via the penton fibre.
- The virus particle is taken up by clathrin-mediated endocytosis.
- The endosome is trafficked along the microtubules.
- The pH starts to drop.
How do adenoviruses escape the endosome?
- As the endosome moves along the microtubules the pH drops to pH 5.
- This triggers the virus particle structure to change and start to come apart.
- This allows protein 6 to escape the virus and lyse the endosome.
- The virus particle can then escape the endosome.
What do adenoviruses do once they have escaped the endosome?
- The virus associates with the microtubules and moves to the MTOC.
- Then it associates with the nuclear pore complex and the virus is further degraded and the genome is delivered to the nucleus.
How is protein 6 released from adenoviruses?
- Protein 6 starts to escape from the virus particle by itself.
- A massive drop in pH helps the release of protein 6 by loosening the virus structure.
How does protein 6 trigger the endosomal escape of adenoviruses?
- Protein 6 is released from the virus particle.
- It inserts in the endosome membrane and induces a strong negative curvature in the membrane.
- This causes disruption of the endosome membrane and lysis.
- This allows viral escape.
What can make adenovirus protein 6 hard to study?
- It is very effective at lysing membranes.
- It can break open any membrane, including ones in bacterial expression systems used to study viruses.
What modification is essential for the function of protein 6?
- Ubiquitination.
- This is essential to allow the virus to associate with the microtubules.
- We don’t know why this is needed.
Can adenovirus protein 6 embed in other membrane?
Yes, protein 6 can embed in the plasma membrane
What does adenovirus protein 6 do in the plasma membrane?
- Small amounts of protein 6 escapes the virus particle and embed in the plasma membrane as the virus enters the cell.
- This causes uptake of Ca2+ uptake.
- We don’t really know what function this has.
- It could be causing small breaks in the plasma membrane to allow Ca2+ in to trigger cellular processes.
- It triggers processes that later aid endosomal escape.
- This occurs before the pH drop.
What is the main function of protein 6?
To break the endosomal membrane to escape the endosome and attach the virus to the microtubules.
What is assisted diffusion?
- The random movement of viral particles along the cell’s microtubules.
- The virus associates with either kinesin or dynactin to move along the microtubules.
- As the MTOC is located by the nucleus, at some point the virus will make contact with a nuclear pore complex and trigger nuclear up take.
How does adenovirus get from the microtubules to the nuclear pore complex?
- We don’t know
- We can’t find the directed mechanisms of how this works.
- It could be that microtubules lie across the nucleus surface, causing active or passive association with the nuclear pore.
How long does viral uncoating take?
30 minutes from attachment to DNA in the nucleus
What is a theory of how the viral genome can exit the viral particle?
- Active movement from the motor proteins physically pulls apart the hexon.
- This is due to kinesin and dynactin binding to different parts of the virus particle.
- This loosens up the viral particle and allows the genome to escape.
What happens once the adenovirus viral genome associates with the nuclear pore complex?
- Nup214 and Nup358 are the external part of the pore which the viral particle 1st associates with.
- Ubiquitin ligases and the proteasome are recruited to degrade host or viral proteins.
- This then allows the dynactins and kinesins to further pull the virus apart.
- The viral DNA and associated proteins can then interact with the nuclear pore complex.
- Interactions particularly occur through protein 5 and 7.
What happens once the adenovirus genome and associated proteins is associated with the nuclear pore complex?
- Protein 5 is ubiquitinated to flag it for degradation.
- This pulls the genome out of the virus particle.
- The viral DNA is imported through protein 7.
- Protein 7 associates with the DNA import factor, transportin 1.
- This pulls the DNA through the nuclear pore complex by pulling protein 7 through.
What is adenovirus protein 7?
- A core protein that binds to the viral DNA a bit like histones.
- It contains a nuclear localisation sequence.
What is MIB1?
- Mind bomb 1
- It is a ubiquitin ligase that degrades protein 5.
- This reveals protein seven and the DNA and ensures it can be pulled into the nucleus.
What is essential for adenoviral nuclear import?
- Protein 5
- If there is no protein 5 the genome falls back into the cytoplasm.
- We don’t know why.
What is adenovirus protein 5?
- It is a core protein
- It is associated with the viral capsid and protein 7.
- It is the bridge between the genome and the viral particle.
How do protein 5 and protein 7 work together to release the adenovirus genome?
- Protein 5 is associated with protein 7 and the capsid.
- Protein 7 tightly wraps the DNA.
- As the capsid is degraded protein 5 is pulled out with it and this pulls protein 7 and the DNA with it.
- This unwinds the genome and aids nuclear import.
What are most steps in viral uncoating for?
They are for increasing efficiency of processes as well as triggering them.
Do we fully understand how viral uncoating works?
- No
- We have experiments that tell us which proteins and processes are essential.
- There are still significant gaps in our knowledge, and we don’t understand what happens.
What is influenza virus?
- An enveloped, segmented RNA virus.
- It causes serious disease
Where does influenza deliver its genome?
- Into the nucleus.
- This is unusual for an RNA virus as most don’t bother entering the nucleus.
What is the structure of influenza?
- Its genomes is in 8 segments that cluster together as the polymerase complex associates with the M1 protein
- M1 associates to the inside of the membrane and brings everything together as the virus leaves the cell.
- HA mediates attachment to host cells.
- NA cleaves sialic acid residues.
- The M2 ion channel allows the acidification of the inside of the viral particle.
How is the influenza virus different from adenovirus?
- Enveloped influenza vs non-enveloped adenovirus.
- Influenza also has lots of cellular proteins within its particle.
- This includes ubiquitin, actin, tubulin and annexin.
Do adenoviruses use cellular proteins?
no
How does influenza enter cells and escape the endosome?
- It attaches to the cell through the HA protein.
- It is taken up into an early endosome.
- Acidification of the endosome and inside the virus particle changes the structure of HA.
- This exposes the fusion motif on HA and causes membrane fusion of the virus and endosome.
- The pH in the virus particle also drops.
- This triggers the genome to dissociates from M1.
How does a drop in pH cause influenza M1 to dissociate from the genome?
- M1 contains histidine-rich sequences.
- The change in pH causes these histidines to push away from each other and dissociate from the genome.
What does influenza release once it has fused with the endosome membrane?
Free ubiquitin
Why does influenza release ubiquitin?
To recruit HDAC6
What is HDAC6 and what does it do?
- HDAC6 binds to free ubiquitin.
- HDAC6 also associates with motor proteins like dynein or myosin and microtubules and actin.
- This creates a physical shearing effect. Pushing and pulling apart the virus particle.
- HDAC6 triggers the recruitment of the aggresome components.
- HDAC6 can also associate with M1.
What is the aggresome?
- A structure that forms at the MTOC to degrade misfolded ubiquitinated proteins.
- Usually forms when the normal protein degradation is overwhelmed.
How does M1 recruit HDAC6?
- M1 is ubiquitinated by the E3 ligase Itch.
- This recruits HDAC6 to M1.
What does HDAC6 recruited to M1 do?
- It helps to pull apart the viral particle.
- It also pulls the virus towards the nucleus.
- It recruits ESP8, which also pulls M1 to the nuclear pore complex.
What happens to the influenza genome once its associates with the nuclear pore complex?
- Importin alpha and importin beta bind to the RNA genome.
- This pulls the genome into the nucleus.
- Transportin 1 is also important as it helps pull M1 proteins away from the viral genome.
What is the summary of influenza uncoating?
- The virus escapes the endosome through membrane fusion.
- Influenza releases lots of ubiquitin that recruits HDAC6.
- Itch ubiquitinates M1 so HDAC6 binds.
- This associates with the microtubules and actin motor proteins and pull them towards the nucleus.
- This also helps pull apart the virus particle.
- ESP8 and transportin 1 aids in the disassembly of the viral particle.
- The RNA is released from the viral particle, and importin alpha and beta bring the genome into the nucleus.
What is essential for the disassembly of influenza and association with the nuclear pore complex?
Ubiquitin ligases.
What does viral uncoating involve?
Complex interplay between viral and cellular proteins.
