5. Virus entry Flashcards
What is virus entry essential for?
- Replication
- Establishing infection
What does virus attachment on the cell surface do?
- It enriches the virus on the cell surface.
- It can be very specific or non-specific.
- It can determine what cells the virus infects.
How is the virus stabilised on the cell surface?
Multiple receptor-virus interactions
What does initial attachment of the virus determine?
The tropism of the virus (what cells it infects)
What does the virus binding to the receptor cause?
- It triggers a series of signalling events that change the structure of the virus.
- This allows the virus to enter the cell.
What methods can a virus used to enter a cell?
- Fusion and uncoating at the plasma membrane
- Being taken up by receptor-mediated endocytosis or macropinocytosis.
How do viruses escape from endosomal compartments?
By fusing membranes with the endosome/pinosome OR breaking the endosome/pinosome
Overview of the steps in virus entry
- Initial binding and attachment via specific receptor interactions.
- The virus particle is taken up by direct membrane fusion or endosomal pathways.
- The virus needs to escape the endosome or it will just be degraded.
- Some viruses traffic to the nucleus
- Then, the viral genome needs to get into the right place in the cell for replication. eg a DNA genome needs to get into the nucleus.
Why is viral entry still not very well understood?
It is a rapid transient process which makes it hard to study some steps in the process.
What kinds of molecules are viral receptor/attachment factors?
- Proteins
- Glycoproteins
- Glycolipids
Where are viral receptors and attachment factors present?
- They are cell surface molecules.
- Some are present on lots of cells so viruses that bind these can infect lots of cells.
- Some are very cell specific so these viruses are very cell specific.
Do viruses only use 1 receptor?
- No
- Viruses may use one or more receptors or attachment factor
- Some viruses use different receptors and attachment factors on different cells.
- Some viruses also use primary receptor and co-receptors.
How do viruses bind their receptors/attachment factors?
- Electrostatic interactions
- Includes ionic strength, pH and specific ions
- These attachments are often reversible
What viral structures bind to their receptor/attachment factors?
- Non-enveloped viruses bind using surface structures.
- Enveloped viruses bind using envelope glycoproteins
How does HIV attach to host cells?
- It uses CD4 as the primary receptor
- It uses CCR5 or CXCR4
- This shows a virus can use multiple co receptors
What is sialic acid?
- An attachment factor
- It is not a receptor
- Viruses need other interactions to enter the cell.
Is the receptor for a specific virus always known?
We don’t always understand what receptors mediate some viruses uptake
How do viruses interact with virus receptors/co-receptors?
- They interact directly with the virus through multivalency binding.
- Binding is typically highly specific but low affinity.
- Once the virus is stably attached on the cell surface, it induces signals.
- This promotes endocytosis/pinocytosis or membrane fusion.
How do viruses interact with virus attachment factors?
- Attachment factors are not always essential for viral entry.
- They concentrate the virus particles on the cell surface so they can interact with the true receptor.
- Binding is non-specific and low affinity.
- These can interact with lots of different viruses
What are some examples of virus attachment factors?
- Heparan sulfate
- carbohydrates
- Sialic acids
What are the different ways viruses can attach to host cells?
- Binding to a single primary receptor.
- Binding to a primary receptor and a co-receptor
- Binding to different attachment factors than the primary receptor.
What can make the viral receptor hard to identify?
High concentrations of attachment factors can make it hard to identify the true receptor.
How does influenza virus enter cells?
- Haemagglutinin binds multivalently to sialic acids that contain the receptor.
- This causes receptor clustering on the cell surface as the receptors migrate through the membrane.
- often, multiple interactions with receptors are required before the virus enters the cell.
- The virus is taken up into the cell.
- The virus needs to traffic to the right intracellular compartment.
What do viruses take advantage of when entering host cells?
They take advantage of host cell mechanisms that naturally uptake things.
What mechanisms do viruses use to enter the host cell?
- Plasma membrane fusion
- Endocytosis
- Macropinocytosis
- Lipid rafts.
What can the different uptake mechanisms determine?
Different results in the cell
What do viruses need to breach to infect cells?
- The actin cortex forms a barrier to prevent viral entry.
- This needs to be overcome for the virus to enter the cell.
- This can be breached by endocytosis signalling or cortex disassembly for membrane fusion.
What are cell cues?
- These are host cell signals that cause changes in the virus particle
- This causes the virus to change its surface structure so it can interact with the host cell
- Triggers things like uncoating
What are examples of cell cues?
- Receptors/co-receptors
- Ca2+
- actin
- Microtubules
- dynein/myosin
- Low pH
- Ions like K+
- Enzymes or proteases
- Endosome maturation receptor switch
- Chaperone proteins
What do viruses do once they escape the endosome?
- They interact with other host proteins.
- The nucleocapsid needs to be rearranged so the genome can be accessed for replication.
What is macropinocytosis?
- A typical process of taking up particles from the environment or environmental sampling.
- The cells change their actin cytoskeleton to engulf particles
What viruses are typically taken up by macropinocytosis?
- Larger viruses due to the size limit of endocytosis.
- This includes smallpox and Ebola.
How are viruses taken up by macropinocytosis?
- The virus binds to a receptor.
- This triggers receptor tyrosine kinase signalling.
- This signalling causes actin dependent formation of membrane protrusions or ruffles.
- There is then non-specific uptake of the virus as the particle is engulfed.
- A pinosome containing the virus, surface receptors and plasma membrane is formed.
- This virus needs to escape the pinosome.
When does membrane fusion occur?
- At the cell surface
- When the virus needs to escape the endosome, they fuse with the endosomal membrane to release the inner viral particle.
What is the viral mechanism of membrane crossing?
- Glycoproteins on the viral cell surface interact with the receptor.
- This causes a change in the virus structural protein so the virus can interact with the cell membrane.
- Dynamic rearrangement of the virus proteins causes the membrane to come together.
- This forms a hemifusion intermediate.
- Then it forms a full fusion pore.
- The nucleocapsid can be released into the cytoplasm.
How do non-membrane viruses interact with the plasma membrane?
- There are some interactions with the membrane and changes in the virus surface.
- This can cause pore formation, membrane disruption or modification.
- The viral genome is then often delivered straight into the cytoplasm.
- These mechanisms are less understood then enveloped viruses.
When can viral membrane fusion occur?
- At the plasma membrane
- Within an endosome
What mediates viral membrane fusion?
- A fusion peptide
- These are often within viral glycoproteins.
How do viruses take advantage of normal cell uptake processes?
They evolved specific proteins that manipulate and use host cell mechanisms.
How are the viral membrane and host cell membrane bought together?
- By viral glycoprotein-receptor interactions
- The viral glycoprotein or separate fusion proteins catalyses fusion of the membranes.
Why does viral fusion need to be regulated?
- Different structures or mechanisms are needed to prevent premature fusion.
- Premature fusion would destroy the virus.
How is viral fusion regulated?
- Fusion proteins may be masked until receptor binding.
- Low pH can be used to expose the fusion domain.
- Activity of fusion proteins may be regulated by cleavage of a precursor.
How does fusion occur at the plasma membrane?
- Many enveloped viruses particles fuse directly at the plasma membrane at a neutral pH.
- This fusion is mediated by a dedicated fusion protein.
- This fusion protein is made as a precursor F0 and cleaved to make F1 and F2 during maturation.
- The fusion peptide is believed to be hidden through its conformation until it is needed.
- This is triggered by receptor binding.
- The fusion peptide is often hydrophobic and only in active conformation when it is needed.
What is an example of a virus that fuses at the plasma membrane?
Measles or mumps
How does a virus with a single primary receptor fuse with the host cell membrane using mumps as an example?
- Haemagglutinin-neuraminidase protein binds to the primary cell receptor.
- The fusion protein is cleaved on maturation by a cellular enzyme. It is still linked by a disulphide bridge.
- HN binding to the receptor causes a conformational change in the fusion peptide.
- The fusion peptide then inserts into the host cell membrane.
- This causes membrane fusion
How does a virus with a single primary receptor and co-receptor fuse with the host cell membrane?
- The viral glycoprotein binds to the primary receptor.
- This binding causes the fusion protein to bind to the co-receptor.
- This causes a conformational change in the fusion peptide.
- The fusion peptide then inserts into the cell membrane.
What is plasma membrane fusion simpler than?
Going through the endosome
How does membrane fusion in the endosome occur?
- The virus binds to a receptor on the cell surface.
- The virus is internalised by clathrin-dependent receptor-mediated endocytosis.
- A drop in pH in the late endosome results in a conformational change in the envelope glycoprotein which exposes the fusion peptide.
What pathways can SARS-CoV-2 use to enter cells?
- Plasma membrane fusion. This more common in later SARS-CoV-2 strains.
- Receptor mediated endocytosis. This is more common in early SARS-CoV-2 strains.
Why do you need to be aware if a virus can enter by multiple pathways?
If you use drugs that block 1 pathway the virus can still enter cells via other methods.
Why do viruses need to escape the endosome?
- The innate immune system has molecules that interact with viruses in the endosome to contain them.
- Viruses need to escape this.
- They do this by fusing with the plasma membrane or escaping the endosome.
How does SARS-CoV-2 enter cells via plasma membrane fusion?
- SARS-CoV-2 binds to ACE2.
- It needs to interact with other proteins as well.
- This includes TMPRSS2.
- TMPRSS2 cleaves the fusion protein
- SARS-CoV-2 can then rearrange its structure and enter at the plasma membrane.
What is TMPRSS2?
- A host cell protease
- It does proteolytic cleavage of proteins including fusion proteins.
How is SARS-CoV-2 taken up by receptor mediated endocytosis?
- This can happen in the presence or absence of TMPRSS2.
- The virus binds to ACE2 and is taken into the endosome.
- The cleavage of the fusion protein can then occur in the endosome by other host cell proteases.
What must happen for membrane fusion in SARS-CoV-2?
The fusion peptide must be cleaved
What can determine the tropism of SARS-CoV-2?
The host cell proteases present at the cell surface
What do some coronaviruses like SARS-CoV-2 require?
- An initial cleavage event before TMPRSS2 cleavage.
- This can occur in the extracellular space before TMPRSS2 cleavage or as the mature virus leaves the cell.
Why does SARS-CoV-2 undergo an extra cleavage event?
- It has an extra furin cleavage site
- Furin is a host cell protease
- Once it is cleaved, it is better primed for TMPRSS2 cleavage.
- This can occur at the cell surface, in the endosome, as it leaves the cell, or as it enters the cell.
- This extra cleavage makes SARS-CoV-2 much more transmissible.
How does influenzae fuse with the endosome membrane?
- The H protein initially interacts with the cell receptor via the head region and then it gets displaced.
- The fusion peptide gets projected up and inserted into the endosomal membrane.
- Further structural rearrangement brings the 2 membranes together to allow fusion.
How was the function of different fusion proteins discovered?
By looking at their structure under different pH conditions
What is the mechanism of class 1 fusion proteins?
- They often act as trimers.
- The initial interaction is between the head region and the receptor.
- Low pH in the endosome triggers conformational change and the fusion peptide is projected upwards into the endosomal membrane.
- A 2nd structural rearrangement bends the protein back on itself to bring the 2 membranes into close contact.
- This allows membrane fusion and the viral genome to be passed into the cell.
What is the structure of class 1 fusion proteins?
- 6 alpha helices
- This is common for a lot of fusion protein.
- These all evolved to be similar as it’s the most efficient means of entry.
How can membrane fusion be blocked?
- Small peptides can bind to the 6 alpha helices bundles to block fusion and virus entry.
- They mostly block the 2nd rearrangement.
- This includes T20, a small molecule inhibitor of HIV fusion.
What are class 1 fusion proteins?
- Used by orthomyxoviruses, retroviruses, paramyxovirus and filoviruses.
- These fusion proteins mature by proteolytic cleavage of a precursor.
- This results in a membrane anchored subunit with an amino-terminal fusion peptide.
- Application of the fusion trigger (low pH) results in the formation of a characteristic trimeric structure.
How do Flavivirus fusion proteins work?
- The fusion peptide is part of the envelope protein that lies flat at a neutral pH.
- In acidic environment the fusion peptide is projected up in trimeric formation.
- This exposes the fusion peptide
What is the mechanisms of class 2 fusion proteins?
- The 1st rearrangement is an upward projection of the envelope protein and fusion peptide.
- The 2nd rearrangement is the envelope protein bends back on itself to cause fusion.
What are class 2 fusion proteins?
- Used by alphaviruses and flaviviruses.
- They are not proteolytically cleaved and have an internal fusion domain to protect the fusion peptide.
- They are synthesised as a complex with a 2nd protein. Cleavage of the accessory protein results in activation of the fusion protein.
- Formed mostly of ß sheets
What are class 3 fusion proteins?
- They are used by herpesviruses and rhadoviruses (VSV).
- They are more complex and less understood. They fall between class 1 and 2.
- They are trimeric proteins that sit perpendicular to the membrane.
- Formed of both alpha helices and ß sheets containing one or more fusion loops.
- The protein containing the fusion proteins doesn’t require proteolytic maturation and isn’t co-synthesised with another protein.
- They undergo reversible conformational changes at low pH.
What are fusion inhibitors?
- We understand the fusion step in virus entry so we can create drugs that target it.
- These prevent membrane fusion.
- As many viruses use similar fusion mechanisms this could create broad spectrum anti-virals
How do non-enveloped viruses like picornavirus enter host cells?
- They don’t have a membrane so there is no fusion event.
- They interact with a host cell receptor and a conformational rearrangement causes binding.
- A viral protein inserts into the host cell membrane. This protein needs to be hydrophobic.
- They can create a pore or enter the endosome.
- We don’t know when the genome is released. In the endosome or at the membrane.
