Egress

Question 1

Describe the steps of HSV-1 assembly

Answer 1

1. VP22a (scaffold) + VP5 (major capsid protein) and VP22a-VP24 fusion (protease) + VP5 are transported into the nucleus
2. procapsid assembly where VP24 is in the centre
3. procapsid goes under maturation: VP24 activity to create an empty capsid (loss of scaffold fragements)
4. the genome is inserted into the capsid via UL6 portal vertex protein

Question 2

What are some characterisitics of chaperone assisted assembly (what provides these proteins, what do they do, give three examples of chaperone proteins)

Answer 2

• chaperones can be provided by the HC or encoded by the virus
• assist in protein folding and assembly of multi-protein complexes
• e.g. influenza’s HSP90 for nuclear imporat of its genome segments and rdRNAP
• e.g. groEL in E. colu is a proteolytic chaperone required for protein folding
• e.g. adenovirus L4 100kDa protein

Question 3

Describe the adenovirus capsid (symmetry, hexamers, pentamers, triangulation number)

Answer 3

symmetry = complex icosahedral
hexons = 240 hexons (each a trimer of the hexon protein L3 pll)
pentons = bases come out
T = 25 (sort of)

Question 4

Describe why it is important for adenovirus’ hexon trimer’s curvature to be perfect

Answer 4

Curvature needs to be perfect so that the curvature of the capsid is perfect, if not, there will be holes/gaps

Question 5

What is adenvirus’ hexon protein’s name and what is the chaperone required to make the folding process efficient?

Answer 5

hexon protein = L3 pll
chaperone = L4 100kDa

Question 6

What naked viruses have capsids proteins that “self assemble”? Describe the make up of the capsid, how the self assembly process occurs, and how we take advantage of self assembly

Answer 6

virus = papillomaviruses
capsid = L1 + L2 proteins

Process:
1. capsid undergoes repeated rounds of assembly and disassembly in the cell nucleus sampling both cellular DNA and the viral genome if they are < 8kb
2. host nucleic acid is too large however, so L1 and L2 nucleate around the viral genome
3. disassemble and assemble until the ideal capsid conformation (lowest energy) is achieved

Advantage:
- make papillomaviruses a convenient gene therapy vector

Question 7

Where do enveloped viruses assemble and name some examples?

Answer 7

assemble at cellular membranes prior to budding (e.g. p.m, nuclear memb, GA, vesicles, ER)

Question 8

What is the viral envelope made of (generally) and what is the origin of the components?

Answer 8

cellular lipids and viral proteins

Question 9

Which env virus buds from the nuclear membrane?

Answer 9

herpesviruses

Question 10

Which env virus buds from the ER?

Answer 10

coronaviruses and flaviviruses

Question 11

Which env virus buds from the GA?

Answer 11

poxviruses (infectious with any number of envelopes)

Question 12

Which env virus buds from vesicles?

Answer 12

herpesviruses

Question 13

Which env virus buds from the p.m?

Answer 13

retroviruses (proteins aggregate at the surface and virion bulges out)

Question 14

Describe the egress process of herpes virus from the nucleus

Answer 14

1. formation of empty capsids
2. DNA packaging
3. 1o envelopment by budding through inner leaflet of the nuclear membrane
4. fusion with the outer leaflet of the nuclear membrane -> loss of primary env
5. capsid associates with tegment proteins and buds into vesicles from trans-GA -> 2o envelopment (this is where it gets it’s envelope ultimately)
6. fusion of vesicel containing the virus with the p.m, resulting in release of the virus

Question 15

Why do herpesvirus and other viruses have to bud through the nuclear membrane and not go through the nuclear pore complex?

Answer 15

too large

Question 16

When looking at TEM of herpesvirus in the nucleus, how can we tell the difference between an empty capsid and a capsid with the genome in it?

Answer 16

genome in capsid has greater electron density to it will appear very dark; whereas an empty capsid will appear hollow

Question 17

What are the domains/proteins in the Gag polyprotein, and what does each one do? How are the polyprotein made into seperate proteins? What viruses have Gag proteins?

Answer 17

Domains:
- matrix (MA) -> targets gag to the p.m, anchoris it via N-term myrisitic acid
- capsid (CA) -> forms the core that contains the RNA genome
- nucleocapsid (NC) -> binds genomic RNA
- P6 -> virus budding (recruits escrt)
- P1 and P2 -> spacer regions

Polyportein is cleaved by proteases

All retroviruses express a Gag protein

Question 18

What does ESCRT stand for, and what processes is it required for (name three)?

Answer 18

ESCRT = endosomal sorting complex required for transport

Required for memb. scisson during:
1. vesicle budding (e.g. into endosomes)
2. separation of daughter cells at mitosis (abscisson)
3. viral budding

Question 19

How does ESCRT work during viral budding?

Answer 19

shrinks down the neck of the bud

Question 20

How is ESCRT recruited in retroviruses? In other enveloped viruses?

Answer 20

retroviruses:
- recruited by the p6 domain of Gag, using specific sequence motifs termed “late” domains

other env viruses:
- use “late” domains

Question 21

What proteins make up ESCRT machinery?

Answer 21

ESCRT1, ALIX, CMPs and VSP4

Question 22

How does maturation (post-release) in retroviruses occur?

Answer 22

cleavage of Gag protein via viral proteases

Question 23

How do naked viruses exit the cell?

Answer 23

lysis