11. Papillomavirus 3 Flashcards
where does papillomavirus DNA replication occur?
in the nucleus of infected keratinocytes
what are 3 things required for papillomavirus DNA replication?
- E1
- E2
- host DNA replication machinery
where is papillomavirus DNA replication initiated?
at viral origin (Ori)
does papillomavirus DNA replication occur in one direction or bidirectional?
bidirectional
what type of protein is E1? what does this mean?
E1 is an initiator protein –> initiates replication
what are the 3 main functions of E1?
- E1 binds ori
- E1 has helicase activity to melt ori and unwind DNA ahead of replication fork
- interact with cellular replication factors
how many E1 is involved in replication?
there are 2 hexamers, 1 for each strand
describe the 3 components of ori
- E1 binding site
- E2 binding site
- AT-rich region
what are the 3 general things that are assembled throughout replication?
- E1-E2-ori complex
- E1 double hexamer
- replication-competent complex
how is the E1-E2-ori complex assembled?
what is the role of E2?
E2 binds with high affinity and specificity to origin AND interacts with E1 to recruit it to ori
E2 acts as helicase loader
what would happen to E1 if E2 wasn’t there to load E1 at ori?
E1 wouldn’t know where to dock at the DNA
how is the E1 double-hexamer assembled?
ATP binds and is hydrolyzed by E1, allowing E1 to form a hexamer for each strand and then each hexamer encircles a DNA strand
what does the replication-competent complex do?
E1 can unwind DNA and interact with host DNA replication factors
what 3 host DNA replication factors does E1 interact with?
- RPA –> covers ssDNA
- Polymerase alpha primase –> initiates replication
- Topoisomerase I –> relieves stress of unwinding DNA
Does E2 bind ori and E1 at the same domain?
no –> separate domains
how does E2 bind ori?
DNA binding domain of E2 binds E2 binding site of ori
how does E2 bind E1?
E1 binding domain of E2 binds helicase domain of E1
what are the 2 domains of E2?
- E1 binding domain / transactivation domain
- DNA binding domain
describe the E1 binding domain / transactivation domain of E2
1 face of the domain interacts with E1, 1 face of the domain is a transcription REPRESSOR for E6/E7
What are the 3 functional domains of E1 helicase?
- N-terminal region
- DNA-binding domain
- helicase domain
what is the role of E1’s N-terminal domain?
contains motifs for E1 nuclear import and export
what is the other name for E1’s DNA-binding domain?
origin-binding domain
what is the role of E1’s DNA-binding domain?
4 DNA binding domains bind to 4 E1 binding sites in ori to allow assembly of double-hexamer
what are the 3 roles of E1’s helicase domain?
- enzymatic part –> ATPase and helicase activity
- allows assembly into hexamers
- interacts with E2 and host DNA replication factors
describe the ATP binding sites at the helicase domain
there are 6 ATP-binding sites made from residues from 2 adjacent monomers
describe the mechanism of DNA unwinding (4 general steps)
- E1 assembles as hexamer around ssDNA
- E1 translocates along ssDNA
- 6 ssDNA-binding hairpins of E1 that interact with ssDNA undergo conformational changes upon ATP-binding and hydrolysis
- unwinding!
why does the virus need a special mechanism during mitosis?
the viral genome held in episomes can be lost during mitosis
why is it important to properly segregate the viral genome during mitosis
allows equal partitioning of viral episomes to daughter cells during mitosis
how does HPV prevent loss of episomes/viral genomes during mitosis?
E2 transactivation domain interacts with C-terminal 20 amino acids of Brd4 to tether the viral genome to mitotic chromosomes
what is Brd4?
Brd4 = Bromodomain-containing protein 4
chromatin reader that binds acetylated chromosomes, and coats them during mitosis
what are the 2 promoters used in HPV gene transcription?
- promoter for Early genes (pE)
- promoter for Late genes (pL)
where is pL located? what does its activity depend on?
located on E6 gene
dependent on keratinocyte differentiation
why is pL dependent on keratinocyte differentiation?
pL is only active when keratinocytes are differentiated, i.e. when higher up in epithelium bc thats when E6 is active
what is the location and role of the keratinocyte-specific enhancer?
enhancer is located in LCR and regulates Early and Late promoters
what regulates viral gene transcription? (2)
- UBIQUITOUS CELLULAR transcription factors that bind to LCR
- E2 acting as a repressor
which transcription factors are not cellular?
only E2!
why can the HPV viral genome only be active in keratinocytes?
there is a specific combination of cellular transcription factors which lets the virus only be active in keratinocytes
describe the mRNAs produced
many early and late mRNAs are made, most are alternatively spliced and poly-cistronic
