Virus structure, attachment and entry

Question 1

Viruses come in different sizes and shapes, but all virions are designed for ___________ and effective _________ of viral genome from one host cell to another.

Answer 1

Protection; transmission (delivery)

Question 2

What are the requirements of virions designed for protection?

Answer 2

1) Recognition and packaging of the nuclei acid genome
2) Stable protective protein shell
3) Interaction with host cell membrane for the incorporation of the envelope (if it’s an envelope virus)

Question 3

What are the requirements of virions designed for delivery?

Answer 3

1) Recognition of susceptible cells and performing entry
2) Uncoating of the genome and delivery to the site of replication

Question 4

Virus particles are __________ structures. Define what that means.

Answer 4

Metastable

They are structures that are stable but can reach a lower free energy conformation which can be attained when an unfavourable energy barrier has been surmouted (it’s stable initially (protection state) but when u provide it enough energy, it is going to go to an even more stable conformational state (release state)). This is an IRREVERSIBLE PROCESS.

Question 5

Why are virus particles (protein shells) metastable structures?

Answer 5

They must be stable for the protection of the genome.

However, they must NOT be TOO STABLE to allow entry and genome uncoating in the host cell.

Question 6

How can metastability be achieved for the protein coats?

Answer 6

To make it stable:
Provide maximum contact (non-covalent bonds) between viral proteins that make the protein coat.

Each subunit has identical/quasiquivalent bonding contacts with its neighbours, created by SYMMETRICAL arrangement of many similar/identical proteins.

To make it unstable:
The structure is not permanently bonded together with non-covalent interactions. They can be taken apart or loosened during infection to release/expose the genome.

Question 7

What basic principle of viral protein shells does the HPV vaccine made in yeast take advantage of?

Answer 7

It takes advantage of the fact that many capsid proteins can self assemble into virus like particles (VLPs).

It makes VLPs that resembles the virus with no genome; so very safe!

Question 8

Repeated interactions of chemically complementary surfaces (of the capsid proteins) at the subunit interfaces leads to a ___________ ____________.

What are the two types of structures seen in viruses in nature?

Answer 8

SYMMETRICAL ARRANGEMENT

1. Helical structures
2. Icosahedral structures

Question 9

Helical structures are ____-like or _________ structures. It has a _______ ______ of symmetry.

They are also ______ structures, meaning any volume can be enclosed by varying the length of the helix.

Give an example of a virus with this structure.

Answer 9

rod; filamentous; screw axis

open

An example is: tobacco mosaic virus

Question 10

(T/F) In helical structures, capsid proteins associate with other capsid proteins and often with the genome. Hence, disrupting these interactions and exposing the genome can be used as a pharmacological effect.

Answer 10

True!

Question 11

In the formula P = p x μ; what do each variable mean?

Answer 11

P = the pitch (height) of the helix

p = the axial rise per unit

μ = number of structural units per turn of the helix

Question 12

Vesicular stomatitis virus, part of rabies family, has a ____________ with a helical symmetry. Therefore, it has a repetitive protein-_______ and protein-_____ interactions.

Answer 12

Nucleocapsid

Protein-protein; protein-RNA

Question 13

While capsid is synonymous with _____, nucleocapsid is synonymous with _____.

Answer 13

coat (it’s outside); core (there is multiple layers on top of it)

Question 14

What kind of viral genomes contain a nucleocapsid with helical symmetry?

Give four examples.

Answer 14

Negative ssRNA

1) Paramyxoviridae (measles)
2) Rhabdoviridae (rabies)
3) Filoviridae (ebola)
4) Orthomyxoviridae (influenza)

Question 15

The average capsid protein size is ____-____ (for viruses of all sizes).

All round capsids have _______ number of proteins and multiple of ____ are the most common. What’s the exception??

Answer 15

20-60kDa

precise; 60

Exception: 120 is skipped!!! Therefore, it is 60, 180, 240, 960, etc.

Question 16

What are the characteristics of platonic solids?

Answer 16

1) All faces are regular polygons
2) All faces are congruents (shapes that are exactly the same)
3) All corners are congruents and the same number of faces meet at every vertex

Question 17

Round capsids are built using which one of the 5 platonic solids?

Answer 17

Icosahedron

Icosahedral symmetry

Question 18

Icosahedron is a solid with ___ triangular faces. It has ____ vertices related by _____-, ____-, and _____ axes of symmetry.

It has a _______ axes of symmetry.

Answer 18

20; 12; twofold; threefold; fivefold

Rotation

twofold means ?

Question 19

Icosahedral capsid are ______ structures.

To be able to make icosahedral structure, capsid subunits tend to arrange as _________ and ________.

Answer 19

CLOSED (limits how much DNA/RNA that you can put into it)
while helical is open

Pentamers; hexamers

Question 20

What is the minimal number of protein subunits needed to build a capsid with icosahedral subunits?

Answer 20

3 proteins per face x 20 faces = 60 protein subunits!

Question 21

(T/F) Simple icosahedral capsid is made of 60 identical proteins (3 per face), and the interactions of all proteins with their neighbours are all different.

Answer 21

False!

Though Simple icosahedral capsid is made of 60 identical proteins (3 per face), and the interactions of all proteins with their neighbours are all IDENTICAL (tail-tail, head-head).

Question 22

How can we make bigger particles for icosahedral symmetry?

Answer 22

By ADDING MORE PROTEINS! Not bigger proteins; so you are not increasing the size of your capsid!

So you still have 20 faces but instead of having only 3 proteins per face, you will have more.

Question 23

(T/F) In bigger icosahedral particles (pentmaes with 5-fold axis or hexamers with 6-fold axe of symmetry), the bonding environment is also identical like with pentamers. Therefore, just as stable.

Answer 23

False! They are NOT identical but they are similar: QUASIEQUIVALENT (they are still head-head, tail-tail).

Quasiequivalent are not as stable as identical interactions.

Question 24

Each icosahedral has ____ five-fold axis/vertices and ____ faces!!

Answer 24

12; 20

Question 25

What is triangulation number (T) in simple terms?

What does it represent?

Answer 25

Triangulation is the description of the triangular face of a large icosahedral structure in terms of its subdivision into facets made of 3 (T1) viral subunits.

The triangulation number represents the number of unique environment(s) that subunits occupy.

T = 4 means 4x3 proteins in one face + 4 different environments in one face!.

Question 26

Match the following scenarios to the total umber of subunits:

1) T = 1
2) T = 3
3) T = 4
4) T = 13

A) (60x4) - 240
B) 60
C) (60x13) - 780
D) (60x3) - 180

Answer 26

1) T = 1: 60

2) T = 3: (60x3) - 180

3) T = 4: (60x4) - 240

4) T = 13: (60x13) - 780

Question 27

Match the two round viruses whose protein interactions follow the icosahedral symmetry to their definition:

1) Adeno-associated type 2 virus
2) SV40

A) 50 nm in size, T = 6; 360 copies of VP1 that assemble as pentamers
B) 25 nm in size, T = 1; 60 copies of a single protein

Answer 27

Adeno-associated type 2 virus: 25 nm in size, T = 1; 60 copies of a single protein

SV40: 50 nm in size, T = 6; 360 copies of VP1 that assemble as pentamers

Question 28

Match the different types of viruses with structurally complex capsids to their definitions:

1) Adenovirus
2) Renovirus and Rotavirus
3) Herpesvirus
4) HIV-1

A) T =16 and has one copy of a portal in the capsid for encapsulation.

B) well defined icosahedral capsid with T=25. It has long fibers with a distal knob attached to each of the 12 vertices (so 12 fibers). It also has multiple other proteins involved interacting with the genome or keeping the shell together.

C) Built from a single capsid protein, which can form both pentamers and hexamers. Combines principles of both icosahedral and helical symmetry.

D) contain multiple protein shells. Outer layer has T=13 coat while inner has T=1 nucleocapsid. These viruses are segmented and each segment is close to one 5-fold axis (there is a total of 12 of these). Each RdRp associates with one segment and makes + releases mRNA at each 5-fold axis. Therefore, number of segments can’t be higher than 12.

Answer 28

Adenovirus: well defined icosahedral capsid with T=25. It has long fibers with a distal knob attached to each of the 12 vertices (so 12 fibers). It also has multiple other proteins involved interacting with the genome or keeping the shell together.

Renovirus and rotavirus: contain multiple protein shells. Outer layer has T=13 coat while inner has T=1 nucleocapsid. These viruses are segmented and each segment is close to one 5-fold axis (there is a total of 12 of these). Each RdRp associates with one segment and makes + releases mRNA at each 5-fold axis. Therefore, number of segments can’t be higher than 12.

Herpesvirus: T =16 and has one copy of a portal in the capsid for encapsulation.

HIV-1: Built from a single capsid protein, which can form both pentamers and hexamers. Combines principles of both icosahedral and helical symmetry.

Question 29

(T/F) An adenovirus has 12 fibers which interact with receptors for viral entry. As an antivirus structure, you can calculate the number of molecules of a soluble receptor needed to saturate the one particle.

Answer 29

True!

Question 30

What are poxvirus and pithovirus?

Answer 30

Large viruses with complex structures with no symmetry that we can tell of.

Question 31

How is the viral membrane (envelope) acquired?

Answer 31

Acquired from host cell during budding

Question 32

Viral glycoproteins that protude from the viral membrane are essential for entry.

Briefly explain how the external and internal regions of a glycoprotein assist in entry.

Answer 32

External region:
Contains binding sites for virus receptors and regions critical to mediate membrane fusion.

Internal region:
Makes contact with other component of the virion and often important for assembly.

*The spike protein is the glycoprotein of covid

Question 33

When the glycoproteins directly contact the ________, these are also ordered following the icosahedral symmetry.

However, enveloped viruses with ________ ______ ______ do not follow symmetrical arrangements and neither do the envelope proteins

Answer 33

Capsid

Additional protein layers (like membranes)

Question 34

What are the other components of viral particles? What can they be important for?

Answer 34

Other components:
1) Viral genome
2) Specialized viral proteins associated with genome
3) Enzymes and nongenomic viral nucleic acid
4) Cellular proteins and macromolecules

They can be important for: entry, replication, or have roles to play in the immune response.

Question 35

Viral entry is not a _______ process, but rather an _______ process that involves viral and cellular proteins.

Viral entry encompasses a series of events; from viral ________ to the ________ of the viral genome to the site of replication.

Answer 35

passive; active

attachment; delivery

*viruses are not inert, they change conformations! their entry is an active process triggered by different interactions in which the virus is conversating with the cell!

Question 36

What is a viral receptor?

What can it determine?

Answer 36

A viral receptor is a molecule that binds to the virus particles and participate in entry.

It can determine the HOST RANGE and TISSUE TROPISM of a virus.

Question 37

What is the difference between ATTACHMENT receptors and ENTRY receptors? Include their roles.

Answer 37

ATTACHMENT RECEPTORS:
- Promote attachment to the cell surface

ENTRY RECEPTORS:
- Induce intracellular signalling cascades that cause uptake (endocytosis)
- Induce conformational changes in surface viral proteins that lead to viral membrane fusion (in enveloped viruses) or penetration (in non-enveloped viruses)

Attachment receptors promote binding to surface but are not essential for infection but can enhance it. However, without entry receptors, entry/infection is not possible!

Question 38

(T/F) Entry receptors may be sufficient on its own for entry or may require expression of a co-receptor (ex: HIV-1).

Answer 38

True!

Question 39

While the viral protein is the ______, the entry receptor is the ______.

Answer 39

Key; lock

Question 40

(T/F) A molecule interfering with viral entry can block infection and thus can be a potential antiviral.

Answer 40

True!

Question 41

While viral capsid proteins/surface features such as CANYON and PLATEAU are _______ ______ capsids, PROTRUDING FIBERS are _______ capsids found in NON-enveloped viruses.

Answer 41

simple; icosahedral

complex

Question 42

Fill in the blanks regarding capsid proteins of non-enveloped viruses:

1) The capsids of polioviruses have deep _______ formed by a prominent _______ peaks of ______ subunits. The poliovirus receptor _______ binds to the central position of the _______.

2) The rhinovirus type 2 exhibits a star-shaped ______ at the ______ axis as well as ________. The binding site of the receptor, the low density _______ receptor, is located on the _______.

3) For protruding fibers, initial interactions with the host cell receptors occur at the _____ of the fiber proteins.

Answer 42

1) canyons; five-fold; VP1, CD155; canyon

2) plateau; five-fold; canyons, lipoprotein; plateau (not the canyon)

3) knob

Question 43

Out of the three domains (I, II, III) of the poliovirus receptor, CD155, which one binds the viral capsid?

Answer 43

Domain I

Question 44

What are the viral envelopes made of?

Answer 44

A lipid membrane derived from the host cell (virus producer cells).

Membrane viral glycoproteins are also inserted in the viral membrane with ATTACHMENT SITES (receptor binding domains) to allow for the lock and key interactions with receptors.

Question 45

Briefly answer the questions regarding influenza.

1) What proteins are found in the surface of influenza? Which one is the most important for entry of the virus?

2) Which cell receptor does the influenza protein (HA) bind?

3) What does the sequence in the sialic acid binding site in HA determine?

Answer 45

1) Neuraminidase (NA), hemagglutinin (HA), and ion channel (M2). HA is the most important for entry (it is the viral fusion protein).

2) Sialic acid. HA has two subunits, the subunit that contains the receptor binding domain binds to it.

3) The sequence in the sialic acid binding site in HA determines SIALIC ACID PREFERENCES and strongly INFLUENCES the VIRAL HOST RANGE!

Question 46

_______ ______ virus is a large DNA virus that makes use of ______ viral glycoproteins and _________ entry receptors. Because of this, it has a _______ tropism.

Answer 46

Herpes Simplex; multiple; multiple broad

Question 47

Which one of the statements regarding penetration at the plasma membrane is true?

1) Both enveloped and non-enveloped viruses can undergo penetration.

2) It occurs in acidic conditions.

3) It is mediated via interactions with receptors/co-receptors.

Answer 47

3!

Only ENVELOPED VIRUSES undergo penetration! Non-eveloped viruses do not use penetration because their genome can lead to cell death right away; there will be no time for viral replication.

It occurs at neutral pH!

Question 48

Despite the fact that penetration at the plasma membrane is a very fast method into entering the host cell, why do most enveloped viruses not use this mechanism but rather use endocytosis?

Answer 48

1) Fusion at the cell surface leaves viral glycoprotein at the cell surface. These are detected by the immune system!

2) The viral nucleocapsid delivered at the cell surface must navigate through CORTICAL ACTIN meshwork barrier. This is a very problematic barrier for viruses and not many viruses can over come it.

Question 49

(T/F)

There are many types of endocytosis of viral particles. For most of them, it has to be triggered via a signalling cascade!

Once the viral particles have entered the host cells via endocytosis, entry is over!

Answer 49

False!

Though there are many types of endocytosis of viral particles for which most have to be triggered via a signalling cascade, for full viral entry THE VIRUS MUST ESCAPE INTRACELLULAR ORGANELLES FOLLOWING ENDOCYTOSIS!

This allows them to release their genome to begin replication.

Question 50

Answer the following questions regarding the fusion of lipid bilayers:

1) Does it require energy? If so, why?

2) Briefly describe the steps of the fusion of lipid bilayers. Where does it become irreversible?

3) What drives membrane fusion?

Answer 50

1) Fusion of lipid bilayers REQUIRES ENERGY! Getting two membranes nearby requires lots of energy as the process has to remove the water molecules, the ions, etc that is in between.

2) Separated membranes <–> Membrane contact <–> fusion stalk <–> hemifusion <–> initial fusion pore <–> small fusion pore –> large fusion pore!

3) Conformational changes in the viral fusion proteins drive membrane fusion!

Viral fusion proteins PRE-fusion are at a high (but stable) energy confirmation. Viral fusion proteins POST-fusion are at a lower energy conformation (even more stable).

Receptor interaction gives them the push to go from the high energy conformation to low energy conformation.

Question 51

Process of metastability is _________.

Answer 51

Irreversible.

Metastable: chemically unstable in the absence of certain conditions that would induce stability, but not liable to spontaneous transformation.

Question 52

Out of the three classes of viral fusion proteins, which one is the major one?

Answer 52

Class I

Question 53

Briefly describe the overall structure of the class I viral fusion proteins.

Answer 53

It starts off as a precursor bound to a signal peptide. At this stage, it is not metastable.

Then, it gets cleaved by FURIN-LIKE ENZYME and becomes metastable. The receptor binding domain is linked to the rest of the structure with several domains with a DISULFIDE BOND.

The several domains include; Fusion peptide (F), C/N-Heptad Repeats (HR) and Transmembrane domain (TM).

All viruses with class I viral proteins have SP, F, HR and TM as a result of convergent evolution. These are very important for fusion activity!

Question 54

Briefly describe the conformational changes that occur in class I viral fusion proteins using influenza as an example.

Answer 54

Influenza’s key binding protein is HA, which contains two domains: HA1 with the receptor binding domain and HA2 with the fusion subunit.

A. Prefusion - HA1 binds to sialic acid; internalization occurs!

B. Low pH, dissociation of HA1 - In the endosomes, the pH is low. This causes some of the histidine resides of HA1 to get protonated which changes its conformation, causing it to get displaced, releasing the fusion subunit, HA2.

C. Formation of the rod-like structure, Insertion of the fusion peptide - HA2 extends towards the target membrane (the cell). Fusion peptide is composed of hydrophobic amino acids. Since it loves lipids, it inserts itself to the cell membrane.

D. Formation of the six-helix bundle, Pore formation - Changes of confirmation in HA2 results in a new helix. This helix has an affinity for the other helix, causing them to interact with each other, bringing the cell membrane towards the viral membrane. This forms a hairpin (trimer); 6 helix bundle - 3 hairpins made of 2 helices.

E. Interaction between the fusion peptide and the transmembrane region.

Question 55

Class I viral fusion proteins are reminiscent of cellular _______.

Answer 55

SNARES!

These allow fusion in our cells but in 4 HELIX BUNDLE.

Question 56

(T/F) Viral fusion proteins are not one time proteins; triggering of fusion can occur whenever!

Answer 56

False!

They are ONE TIME PROTEINS; triggering of fusion has to be really timed and in the right place. Therefore, viruses use receptors.

Question 57

(T/F) While the trigger of fusion for influenza is the low pH in the endosomes, receptor/co-receptor interactions can be a trigger of fusion for other viruses.

Answer 57

True!

Receptor binding can lead to the receptor binding subunit to get displaced, exposing the fusion subunit.

Question 58

There can be combination of triggers for fusion. Match the viruses to their triggers:

1) Avian leukosis and sarcoma virus
2) Ebola virus

A) (Cathepsin) protease cleavage + receptor binding
B) Receptor binding + low pH

Answer 58

Avian leukosis and sarcoma virus: Receptor binding + low pH

Ebola virus: (Cathepsin) protease cleavage + receptor binding

Question 59

(T/F) Same family of viruses can have different triggers! Triggers are absolutely required for infection. Therefore, inhibition of receptor binding or protease cleavage (types of triggers) is a potential antiviral!

Answer 59

True!

Question 60

The entire endosome membrane can be disrupted or a pore can be formed to release the viurs/genetic material of the virus.

Briefly describe both.

Answer 60

Endosome disruption (ADENOVIRUS):

Binds to viral receptors –> endocytosis —> multiple sequencial uncoating steps as structural proteins are removed –> low pH destabilizes the capsid —> viral protein VI is released causing the disruption of the endosomal membrane

Pore formation (POLIOVIRUS):

Interaction with the virus entry receptor, CD155, leads to a conformation change in the virus particle —-> sphingosine molecules are released (sphingosine switch) –> inner capsid protein VP4 and an inner capsid portion of VP1 move to the outside —> formation of a pore allowing delivery of the viral RNA in the cytoplasm.

Question 61

Some viruses need to enter the nucleus for replication. Briefly describe the two ways to do so (i.e, how the viruses move in the cells).

Answer 61

1) INDIRECTLY: within a membrane vesicle such as endosomes

2) DIRECTLY: by binding directly to the transport machinery (ex. adenovirus, binding of a capsid protein with dynein and transport along microtubules).

In both cases, microtubules are being used.

Question 62

Why do most simple retroviruses restrict infection to dividing cells?

Answer 62

This is because some viruses (most simple retroviruses) enter the nucleus after the nuclear envelope breaks down during cell division.

However, other viruses need to exploit the cellular pathway for protein import into the nucleus.

Question 63

What are the four strategies to enter the nucleus?

Answer 63

1) Uncoating in the cytoplasm and the genome goes through the nuclear pore complex (influenza).

2) Virus capsid docks onto the nuclear pore complex and the genome is injected in the nucleus.

3) Subviral particle docks onto the nuclear pore complex, is disassembled, and the viral genome is transported into the nucleus.

4) Particle binds the nuclear pore complex and disrupt the nuclear envelope.

Question 64

Why is it important to know the receptor of viruses?

Answer 64

1) It explains tropism (target organ/cells) - this helps understand the pathogenesis and host range (determines if it’s a threat to humans)

2) Development of animal models

3) Development of therapeutic strategies (for HIV-1, MARAVIROC is a CCR5 antagonist and there are receptor mimetics that bind to HIV and inactive its glycoprotein)

Question 65

(T/F) Change of conformation of viral entry proteins is IRREVERSIBLE! Thus, if you trigger this change prematurely, you inactivate the virus. This would be a potential antirviral!

Answer 65

True! This is how receptor mimetics work.