Lecture 5 - Virus Entry and Exit

Question 1

How do enveloped viruses enter and exit cells?

Answer 1

Entry through mebrane fusion

Exit using budding to acquire envelope

Question 2

How do naked viruses enter and exit cells?

Answer 2

Entry through interaction but not fusion with cell membrane

Exit by membrane lysis or cell death

Question 3

What type of interactions initiate virus attachment to host cells? What type of interactions occur afterwards?

Answer 3

Weak, electrostatic, reversible binding to a host receptor

Strong, multivalent irreversible interactions follow and sometimes occur with a second receptor

Question 4

Receptor binding is the first trigger for ____ changes in viral ___ ___ and facilitates subsequent ____ and ____ steps

Answer 4

Receptor binding triggers conformational changes in viral surface proteins and these are often important for subsequent penetration and uncoating steps

Question 5

What part of the poliovirus binds what receptor on the host cell?

Answer 5

Conserved amino acids in the vertice canyons on the virus capsid bind to the CD155 host receptor

Question 6

What are the 3 penetration and uncoating pathways that viruses use and what are the physiological conditions required at these locations to make it possible?

Answer 6

1) penetration and uncoating at the plasma membrane requires neutral pH
2) penetration and uncoating at the endosomal membrane requires an acidic pH, a secondary signal and/or host proteases
3) penetration at the plasma or endosomal mebrane and stepwise uncoating intracellularly requires

Question 7

How does a large, hydrophilic structure such as a naked virus, get across a hydrophobic structure such as the plasma membrane?

Answer 7

Binding and post-binding steps involved with host receptors induce conformational changes in virus particle or spike proteins to expose hydrophobic residues that are able to interact with the membrane

Question 8

What are the 3 known triggers that can induce conformational changes in structural proteins required for virus entry?

Answer 8

• receptor/co-receptor binding
• acidic pH
• extracellular or endosomal proteases

Question 9

How do enveloped viruses fuse to plasma membranes?

Answer 9

Enveloped viruses bind to a host receptor with the spike protein (surface glycoprotein).
Receptor binding triggers conformational changes in the spike protein, exposing a hydrophobic fusion peptide that facilitates membrane fusion

Question 10

Enveloped viruses can either fuse with endosomal or plasma membranes to enter a cell. What are 2 requirements to induce function of the fusion peptide at an endosomal membrane?

Answer 10

Receptor-mediated endocytosis and acidic pH

Question 11

What is metastability and how do viral fusion proteins achieve it?

Answer 11

Metastability is the ability of viral proteins to undergo triggered conformational changes, allowing them to be stable in certain environments and readily able to uncoat in other environments. Host proteolytic cleavage of viral fusion proteins allows for their activation and metastability.

Question 12

Why must viruses regulate membrane fusion?

Answer 12

So that it occurs in the right location (replication competent genome must be delivered to the correct subcellular compartment in order for the virus to continue its life cycle)

Question 13

How is viral fusion regulated at the plasma membrane?

Answer 13

With a second protein-receptor interaction

Question 14

How is viral fusion regulated at the endosomal membrane?

Answer 14

Proteolytic cleavage of either the fusion protein or a secondary protein is required to activate the fusion protein

Question 15

What part of a naked virus mediates its attachment?

Answer 15

Canyons or loops on the icosahedral virus capsid

Question 16

What needs to occur for a hydrophilic, naked virus to interact with a hydrophobic membrane?

Answer 16

Conformational changes in the stable hydrophobic capsid that expose hydrophobic domains able to interact with membrane

Question 17

What are 3 ways that the hydrophobic domains of non-enveloped viruses facilitate entry into the cell and which one is the most common?

Answer 17

Hydrophobic domains promote membrane reorganization to pores, membrane dissolution or promote receptor-mediated endocytosis into endosomes
Receptor-mediated endocytosis is the most common

Question 18

What region of poliovirus capsid proteins bind to the host receptor?

Answer 18

Canyon regions in capsid

Question 19

What are the 2 poliovirus capsid proteins required for uncoating?

Answer 19

VP1 and VP4

Question 20

What are the functions of the 2 polio virus proteins required for uncoating? How are they activated?

Answer 20

Receptor binding exposes hydrophobic residues in both proteins to activate
VP1: flips outward from virus to form a pore in the membrane through which viral RNA is extruded into the cytoplasm
VP4: released into membrane to promote interaction

Question 21

Outline the 4 steps in poliovirus entry

Answer 21

• canyon proteins in capsid bind to host receptor
• receptor binding induces conformational changes that expose hydrophobic domains in capsid proteins VP1 and VP4
• VP4 hydrophobic domain is released into the membrane to promote interaction
• VP1 hydrophic domain flips outward from the virus into the membrane to form a pore through which the viral RNA is extruded into the cytoplasm

Question 22

Viral components are often visible by light microscopy as factories or inclusions. Why does virus assembly occur in concentrated regions?

Answer 22

Because assembly would be very slow in a dilute solution of viral components

Question 23

Why do viral proteins often have “addresses”? What are 2 examples of these addresses?

Answer 23

Because they are often synthesized in different compartments and need to be brought together to facilitate assembly of the virus

• Membrane targetting or membrane retention signals
• nuclear localization sequences or nuclear export signals

Question 24

What are the 2 requirements that must be met when assembling a non-enveloped virus capsid in terms of functionality? (think metastability)

Answer 24

1) Must assemble a highly stable capsid that can survive the harsh conditions of the gut
2) Capsid must be assembled in such a way that it can easily disassemble upon entry into a host cell

Question 25

Why are virus capsids built from sub-assemblies? What are 3 examples of sub-assemblies that viruses use?

Answer 25

To ensure orderly formation of virus particles and subunits, this involves formation of discrete intermediate structures

• assembly from individual protein molecules
• assembly from a polyprotein precursor
• chaperone-assisted assembly

Question 26

What are the steps in poliovirus capsid assembly and proteins involved at each step

Answer 26

1) Poliovirus capsid polyprotein P1 is translated
2) P1 is myristylated at amino terminus glycine to stabilize folding
3) P1 folds and is cleaved to form 5S structural unit made up of VP1, VP2, VP3 and VP4
4) 5S units associate to form 14S capsid pentamers

Question 27

How are viral genomes distinguished from host DNA or RNA where assembly occurs?

Answer 27

Cis packaging signals on the viral genome

Question 28

Describe the general difference between non-enveloped and enveloped virus exit

Answer 28

• Non-enveloped viruses exit cells by membrane lysis or cell death and may use viroporins to poke holes in the membrane
• Enveloped viruses use budding to acquire an envelope

Question 29

When do enveloped viruses acquire their envelope?

Answer 29

AFTER assembly of internal structures

Question 30

What enveloped virus family assembles at the plasma membrane?

Answer 30

Retroviruses

Question 31

What are the steps for assembly of Retroviruses (6)

Answer 31

1) Envelope proteins are trafficked through ER-golgi to cell surface
2) Virus assembly occurs from a polyprotein with proteins in order from inner to outer compartment of the virus
3) Gag associates with the plasma membrane and other gag proteins and coats cytoplasmic side of membrane
4) Gag associates with cytoplasmic tails of viral spike proteins and viral RNA via scaffolding proteins
5) Immature virus particle buds from membrane
6) A viral protease cleaves the polyprotein forming the mature, infectious particle