11. Papillomavirus 3

Question 1

where does papillomavirus DNA replication occur?

Answer 1

in the nucleus of infected keratinocytes

Question 2

what are 3 things required for papillomavirus DNA replication?

Answer 2

1. E1
2. E2
3. host DNA replication machinery

Question 3

where is papillomavirus DNA replication initiated?

Answer 3

at viral origin (Ori)

Question 4

does papillomavirus DNA replication occur in one direction or bidirectional?

Answer 4

bidirectional

Question 5

what type of protein is E1? what does this mean?

Answer 5

E1 is an initiator protein –> initiates replication

Question 6

what are the 3 main functions of E1?

Answer 6

1. E1 binds ori
2. E1 has helicase activity to melt ori and unwind DNA ahead of replication fork
3. interact with cellular replication factors

Question 7

how many E1 is involved in replication?

Answer 7

there are 2 hexamers, 1 for each strand

Question 8

describe the 3 components of ori

Answer 8

1. E1 binding site
2. E2 binding site
3. AT-rich region

Question 9

what are the 3 general things that are assembled throughout replication?

Answer 9

1. E1-E2-ori complex
2. E1 double hexamer
3. replication-competent complex

Question 10

how is the E1-E2-ori complex assembled?
what is the role of E2?

Answer 10

E2 binds with high affinity and specificity to origin AND interacts with E1 to recruit it to ori

E2 acts as helicase loader

Question 11

what would happen to E1 if E2 wasn’t there to load E1 at ori?

Answer 11

E1 wouldn’t know where to dock at the DNA

Question 12

how is the E1 double-hexamer assembled?

Answer 12

ATP binds and is hydrolyzed by E1, allowing E1 to form a hexamer for each strand and then each hexamer encircles a DNA strand

Question 13

what does the replication-competent complex do?

Answer 13

E1 can unwind DNA and interact with host DNA replication factors

Question 14

what 3 host DNA replication factors does E1 interact with?

Answer 14

1. RPA –> covers ssDNA
2. Polymerase alpha primase –> initiates replication
3. Topoisomerase I –> relieves stress of unwinding DNA

Question 15

Does E2 bind ori and E1 at the same domain?

Answer 15

no –> separate domains

Question 16

how does E2 bind ori?

Answer 16

DNA binding domain of E2 binds E2 binding site of ori

Question 17

how does E2 bind E1?

Answer 17

E1 binding domain of E2 binds helicase domain of E1

Question 18

what are the 2 domains of E2?

Answer 18

1. E1 binding domain / transactivation domain
2. DNA binding domain

Question 19

describe the E1 binding domain / transactivation domain of E2

Answer 19

1 face of the domain interacts with E1, 1 face of the domain is a transcription REPRESSOR for E6/E7

Question 20

What are the 3 functional domains of E1 helicase?

Answer 20

1. N-terminal region
2. DNA-binding domain
3. helicase domain

Question 21

what is the role of E1’s N-terminal domain?

Answer 21

contains motifs for E1 nuclear import and export

Question 22

what is the other name for E1’s DNA-binding domain?

Answer 22

origin-binding domain

Question 23

what is the role of E1’s DNA-binding domain?

Answer 23

4 DNA binding domains bind to 4 E1 binding sites in ori to allow assembly of double-hexamer

Question 24

what are the 3 roles of E1’s helicase domain?

Answer 24

1. enzymatic part –> ATPase and helicase activity
2. allows assembly into hexamers
3. interacts with E2 and host DNA replication factors

Question 25

describe the ATP binding sites at the helicase domain

Answer 25

there are 6 ATP-binding sites made from residues from 2 adjacent monomers

Question 26

describe the mechanism of DNA unwinding (4 general steps)

Answer 26

1. E1 assembles as hexamer around ssDNA 2. E1 translocates along ssDNA 3. 6 ssDNA-binding hairpins of E1 that interact with ssDNA undergo conformational changes upon ATP-binding and hydrolysis 4. unwinding!

Question 27

why does the virus need a special mechanism during mitosis?

Answer 27

the viral genome held in episomes can be lost during mitosis

Question 28

why is it important to properly segregate the viral genome during mitosis

Answer 28

allows equal partitioning of viral episomes to daughter cells during mitosis

Question 29

how does HPV prevent loss of episomes/viral genomes during mitosis?

Answer 29

E2 transactivation domain interacts with C-terminal 20 amino acids of Brd4 to tether the viral genome to mitotic chromosomes

Question 30

what is Brd4?

Answer 30

Brd4 = Bromodomain-containing protein 4 chromatin reader that binds acetylated chromosomes, and coats them during mitosis

Question 31

what are the 2 promoters used in HPV gene transcription?

Answer 31

1. promoter for Early genes (pE) 2. promoter for Late genes (pL)

Question 32

where is pL located? what does its activity depend on?

Answer 32

located on E6 gene dependent on keratinocyte differentiation

Question 33

why is pL dependent on keratinocyte differentiation?

Answer 33

pL is only active when keratinocytes are differentiated, i.e. when higher up in epithelium bc thats when E6 is active

Question 34

what is the location and role of the keratinocyte-specific enhancer?

Answer 34

enhancer is located in LCR and regulates Early and Late promoters

Question 35

what regulates viral gene transcription? (2)

Answer 35

1. UBIQUITOUS CELLULAR transcription factors that bind to LCR 2. E2 acting as a repressor

Question 36

which transcription factors are not cellular?

Answer 36

only E2!

Question 37

why can the HPV viral genome only be active in keratinocytes?

Answer 37

there is a specific combination of cellular transcription factors which lets the virus only be active in keratinocytes

Question 38

describe the mRNAs produced

Answer 38

many early and late mRNAs are made, most are alternatively spliced and poly-cistronic