Assembly, exit and maturation

Question 1

What are viral factories?

What is their function?

Answer 1

Viral factories are also known as viroplasms or viral inclusion bodies. It is an intense are of replication and assembly.

Their functions:
1) Location for efficient viral RNA synthesis (has all machinery)
2) Protect nucleic acids from nucleases
3) Prevent viral RNAs from activating host intrinsic immune defences
4) Possibility to coordinate different steps in the viral life cycle (protein synthesis, genome replication and exit)

Question 2

(T/F) Each sub factory of viral factories have a speciality. Ratio of them changes as viral life cycle progresses!

Answer 2

True!

Some are needed more than the others depending on the stage of the viral life cycle.

Question 3

(T/F) Viral factories are not present in normal cells! It is part of the cytopathic effects.

Answer 3

True!

Question 4

Briefly describe the assembly of protein shells/capsids.

Answer 4

The first steps are the formation of the various components of virus particles from their parts.

These intermediates must then associate in ordered fashion.

These reactions need to proceed with reasonable efficiency, and the overall pathway need to be IRREVERSIBLE.

*it is kind of like putting furniture together
*viral factories help co-ordinate these steps

Question 5

Why does the overall pathway for assembly of protein shells need to be irreversible?

Answer 5

To drive it to the final product.

Or it needs to be highly concentrated.

Question 6

What are the three ways of formation of structural units? Briefly describe each.

Answer 6

1) Assembly from individual protein molecules: they assemble individually and have complementary surfaces where they bind to. the structures are generally stable but can disassemble (intermolecular).

2) Assembly from a poly-protein precursor: all structural units are encoded in one transcript that is covalently linked with no stop codons in between. when it gets translated, it is one big polypeptide. after folding, a viral protease cleaved linker region and now the process is IRREVERSIBLE! (intramolecular)

3) Chaperone-assisted assembly: proteins have a chaperone with them that catalyze their formation, making no mistakes.

Question 7

What are the advantages and disadvantages of assembly from individual protein molecules?

Answer 7

Advantages: no energy required, subunits simply interlock

Disadvantages: proteins need to be produced in excess and concentrated (it must be saturated because two molecules have to come together and interact with each other).

Question 8

What are the advantages and disadvantages of assembly from poly-protein precursor?

Answer 8

Advantages: the intramolecular interaction avoids the need for high concentration of protein. the process is irreversible after protease cleavage.

Disadvantage: need to encode protease

Question 9

What are the advantages and disadvantages of chaperone-assisted assembly?

Answer 9

Advantages: highly specific

Disadvantages: need to encode chaperone

Question 10

(T/F) Vaccines use assembly from individual protein molecules to make protein shells.

Answer 10

True!

First, capsid proteins are in excess and then they self-assemble and are purified and are used in vaccines!

Question 11

Assisted assembly reactions (for assembly of protein shells) need:

Answer 11

1) participation of viral genome (helical nucleocapsid symmetry require protein-protein interaction and protein-genome (mostly RNA) interactions)

2) cellular or viral chaperone assisted assembly

Question 12

What are viral scaffolding proteins?

Answer 12

These participate in the reactions by which the capsid/nucleocapsid is constructed (assembly of protein shells) but are then removed (usually degraded by proteases)

They are especially prevalent in complicated and larger protein coats.

Question 13

For the herpes virus:

Pre-VP22a, a viral _________ protein, self-associates and stimulates binding to VP5, which regulates the ______ ______ of nucleocapsid proteins for self-assembly.

Other proteins such as _________ ______ is incorporated. Via interactions with pre-vp22a, a _______ is created. Then Pre-VP22a is ______ by the protease to accommodate the viral ________.

Answer 13

Scaffolding; intrinsic capacity

Protease Vp24; portal; degraded; genome

*all occurs in the nucleus

Question 14

What are the two mechanisms of selective packaging of the viral genome in virus particles?

Answer 14

1) Concerted: the structural units of the protective protein shell assemble productively ONLY IN ASSOCIATION WITH GENOMIC NUCLEIC ACID.

2) Sequential: The genome is inserted into a preformed protein shell.

Question 15

Which kinds of viruses use the concerted packaging?

Answer 15

All (-) ssRNA viruses, retroviruses, and other RNA viruses.

(viruses with a helical nucleocapsid requires protein-protein and protein-RNA interactions with the genome)

(formation of protein coat and genome interaction occur at the same time)

Question 16

Which kinds of viruses use the sequential packaging?

Answer 16

Most DNA viruses and dsRNA viruses use the sequential packaging mechanism.

For example, ADENOVIRUS (a DNA virus) capsid assembly is followed by genome packaging. The capsid proteins need to go into the nucleus via the nuclear pore complex!

Question 17

What are packaging signals?

What kinds are there?

Answer 17

Packaging signals are specific recognition of sequences or structures UNIQUE to the viral genome.

The nucleic acid packaging signals: DNA signals and RNA signals.

*unique = not found in cellular RNA or DNA!

Question 18

Match the terms to their definitions:

1) DNA packaging signals
2) RNA packaging signals
3) Packaging of segmented genomes

A) Secondary STRUCTURES

B) To be infectious, each segment (whether DNA or RNA) of a virus has to be encapsulated. RNA SEQUENCES and INTERMOLECULAR INTERACTIONS among RNA segments allow this to happen.

C) Repeats of short SEQUENCES positioned close to Origin of Replication. They are recognized directly or indirectly by viral proteins (allowing the packaging of the genome; mostly sequential).

Answer 18

DNA packaging signals: Repeats of short SEQUENCES positioned close to Origin of Replication. They are recognized directly or indirectly by viral proteins (allowing the packaging of the genome; mostly sequential).

RNA packaging signals: Secondary STRUCTURES

Packaging of segmented RNA genomes: To be infectious, each segment (whether DNA or RNA) of a virus has to be encapsulated. RNA SEQUENCES and INTERMOLECULAR INTERACTIONS among RNA segments allow this to happen.

Question 19

How do the different segments of the a segmented virus (i.e, influenza, an enveloped -RNA virus that undergoes concerted packaging) come together in the cytoplasm?

Answer 19

Each segment of a segmented virus has to be ACTIVELY transported from the nucleus to the plasma membrane through the membranes of vesicles (endosomes mostly) on the microtubules (not directly).

As they move close to the cytoplasm, they interact with each other, through FUSION EVENTS of the endosomes, until they make a stable structure. The most stable structure occurs when one copy of each segment is bound!

Question 20

In many cases, the production of infectious particles requires essential viral enzymes.

1) What do (-)ssRNA and dsRNA need?

2) What do retroviruses need?

3) What does influenza need?

Answer 20

1) RdRp

2) RT, Integrase, protease, cellular tRNA primer

3) Because it is a (-) RNA segmented virus, it need RdRp.

Question 21

Enveloped viruses can bud from the plasma membrane and release the viral particle from the host cell. However, they can also bud from the __________ membrane which requires _________ to release from the host cell.

Budding in general is ___________ and allows the infected cells to ____________ and continuously _________ viral particles.

Answer 21

Intracellular; exocytosis

Nondestructive; survive; release

*sometimes apoptosis can occur.
*nondestructive also means no pores being formed

Question 22

Non-enveloped viruses undergo host ____ ____ that releases the viral particles from the host cell causing the cell to die.

Answer 22

cell lysis

Question 23

(T/F) All non-enveloped viruses undergo host cell lysis.

Answer 23

False! A few non-enveloped viruses use other mechanisms.

Question 24

(T/F) Large quantities of assembled viruses may accumulate within infected cells for hours and days before viral release through host cell lysis. It is timed!

Answer 24

True!

Lysis occurs at the peak of production to maximize the amount of viral particles released as after lysis the cell dies.

Question 25

Briefly answer the following questions about acquisition of the viral envelope:

1) What does it require?

2) How is the site of budding generally determined?

3) What are the two mechanisms?

Answer 25

1) Acquisition of the viral envelope requires budding and pinching off

2) The site of budding is generally determined by the LOCALIZATION of viral GLYCOPROTEINS.

3) Sequential and Simultaneous

Question 26

Describe the sequential assembly of internal components and budding from a cellular membrane.

Give an example. Which viruses do this?

Answer 26

The assembly of internal structures and budding to cellular membranes are SPATIALLY and TEMPORALLY separated.

For influenza, the formation of ribonucleocaspid (viral RNA segments, NP, polymerase) occurs in the nucleus. The sorting of RNA segments occurs in transit to the cell surfaces where the HA, NA and M2 proteins are localized and where budding takes place.

Most ssRNA viruses do this!

Question 27

Describe the simultaneous assembly of internal structures with acquisition of the envelope. Give an example.

Answer 27

Assembly of internal structures and budding from a cellular membrane are largely COINCIDENT IN SPACE AND TIME.

Retroviruses; everything occurs at the same time.

Question 28

What are the advantages and disadvantages of assembly and budding at internal membranes?

Answer 28

Advantage: The glycoproteins are not exposed at the cell surface (hide from immune cells)

Disadvantage: It requires exocytosis for virus release.

Question 29

Which viruses undergo budding at internal membranes. Describe the more complicated one.

Answer 29

SARS‑CoV‑2 and Herpes

The assembly of the nucleocapsid occurs at the nucleus for herpes. To exit the cell, it first buds from the nucleus. Since the nuclear membrane is associated with the ER, there is a fusion of the nucleocapsid with the ER membrane, allowing it to be in the cytoplasm. Exocytosis then occurs at the golgi, where the nucleocapsid acquires the glycoproteins important for infectivity.

Question 30

What are the three steps of budding?

Budding can be _________ or ________ of host proteins.

Answer 30

1) Induction of membrane curvature by viral components (bud formation)

2) Bud growth

3) Scission of the membrane (bud off)

Dependent; independent

Question 31

(T/F) There is an unusual acquisition of the viral envelope and release of large viruses and there are four distinct routes for their release.

Answer 31

False!

Though there is an unusual acquisition of the viral envelope and release of large viruses and there are THREE distinct routes for their release.

Question 32

Which 2 different infectious particles need to be assembled for the unusual acquisition of the viral envelope and release of large viruses?

Answer 32

1) Intracellular mature virions

2) Extracellular enveloped virions

Question 33

What are viroporins?

Give an example.

Answer 33

Viroporins are viral proteins forming pores that induce cell lysis in non-enveloped viruses.

They allow timing for cell lysis; because once the cell dies, the virus can’t make more particles, thus it has to be done when there is an accumulation!

An example is SV40.
SV40 VP4 protein associates with the nuclear membrane, perforates it by forming pores which induce the release of nuclear content in the cytoplasm (escape of the viral particles and cell lysis).

Question 34

What are the two non-lytic ways of release of non-enveloped viruses? Describe them.

Answer 34

1) AUTOPHAGOSOME-LIKE VESICLES; viral proteins of POLIOVIRUS induce the formation of double-membrane vesicles that resemble autophagosomes.

2) Enveloped in particles resembling EXOSOMES; Hepatitis A virus may use the exosome biosynthesis pathway to be released from cells and may help virus spread in the liver.

Both cases are not for host-to-host transmission. It is only within the host where these can occur!

Question 35

(T/F) The acquisition of a membrane protects the virions against neutralizing antibodies

Answer 35

True!

Question 36

Many viruses require a MATURATION step which involves __________ _______ necessary to convert the particles into infectious virions.

Maturation can occur late in ________ of particles or following the ________ from the host cell.

Answer 36

PROTEOLYTIC PROCESSING

assembly; release

Question 37

How does maturation of viruses facilitate entry?

Answer 37

By breaking COVALENT linkages (metastability requires non-covalent) between specific proteins leads to weaker interactions

Proteolysis may allow new interactions between viral proteins

Question 38

Following or during release of most RETROVIRUS particles, the _____ polyprotein is _______ by the viral protease leading to substantial conformational changes.

What would be a good antiviral?

Answer 38

Gag; cleaved

Protease inhibitor; so the virus is immature at all times and can not be infectious