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MIMM 324 > 11. Papillomavirus 3 > Flashcards

11. Papillomavirus 3 Flashcards

1
Q

where does papillomavirus DNA replication occur?

A

in the nucleus of infected keratinocytes

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2
Q

what are 3 things required for papillomavirus DNA replication?

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

where is papillomavirus DNA replication initiated?

A

at viral origin (Ori)

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4
Q

does papillomavirus DNA replication occur in one direction or bidirectional?

A

bidirectional

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5
Q

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

A

E1 is an initiator protein –> initiates replication

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6
Q

what are the 3 main functions of E1?

A
  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
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7
Q

how many E1 is involved in replication?

A

there are 2 hexamers, 1 for each strand

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8
Q

describe the 3 components of ori

A
  1. E1 binding site
  2. E2 binding site
  3. AT-rich region
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9
Q

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

A
  1. E1-E2-ori complex
  2. E1 double hexamer
  3. replication-competent complex
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10
Q

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

A

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

E2 acts as helicase loader

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11
Q

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

A

E1 wouldn’t know where to dock at the DNA

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12
Q

how is the E1 double-hexamer assembled?

A

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

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13
Q

what does the replication-competent complex do?

A

E1 can unwind DNA and interact with host DNA replication factors

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14
Q

what 3 host DNA replication factors does E1 interact with?

A
  1. RPA –> covers ssDNA
  2. Polymerase alpha primase –> initiates replication
  3. Topoisomerase I –> relieves stress of unwinding DNA
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15
Q

Does E2 bind ori and E1 at the same domain?

A

no –> separate domains

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16
Q

how does E2 bind ori?

A

DNA binding domain of E2 binds E2 binding site of ori

17
Q

how does E2 bind E1?

A

E1 binding domain of E2 binds helicase domain of E1

18
Q

what are the 2 domains of E2?

A
  1. E1 binding domain / transactivation domain
  2. DNA binding domain
19
Q

describe the E1 binding domain / transactivation domain of E2

A

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

20
Q

What are the 3 functional domains of E1 helicase?

A
  1. N-terminal region
  2. DNA-binding domain
  3. helicase domain
21
Q

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

A

contains motifs for E1 nuclear import and export

22
Q

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

A

origin-binding domain

23
Q

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

A

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

24
Q

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

A
  1. enzymatic part –> ATPase and helicase activity
  2. allows assembly into hexamers
  3. interacts with E2 and host DNA replication factors
25
Q

describe the ATP binding sites at the helicase domain

A

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

26
Q

describe the mechanism of DNA unwinding (4 general steps)

A
  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!
27
Q

why does the virus need a special mechanism during mitosis?

A

the viral genome held in episomes can be lost during mitosis

28
Q

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

A

allows equal partitioning of viral episomes to daughter cells during mitosis

29
Q

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

A

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

30
Q

what is Brd4?

A

Brd4 = Bromodomain-containing protein 4

chromatin reader that binds acetylated chromosomes, and coats them during mitosis

31
Q

what are the 2 promoters used in HPV gene transcription?

A
  1. promoter for Early genes (pE)
  2. promoter for Late genes (pL)
32
Q

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

A

located on E6 gene

dependent on keratinocyte differentiation

33
Q

why is pL dependent on keratinocyte differentiation?

A

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

34
Q

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

A

enhancer is located in LCR and regulates Early and Late promoters

35
Q

what regulates viral gene transcription? (2)

A
  1. UBIQUITOUS CELLULAR transcription factors that bind to LCR
  2. E2 acting as a repressor
36
Q

which transcription factors are not cellular?

A

only E2!

37
Q

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

A

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

38
Q

describe the mRNAs produced

A

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

MIMM 324 (37 decks)

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