Flashcards in Papillomaviruses (HPV)- lifecycle and replication strategy Deck (23):
The HPV genome is a small dsDNA genome with around 10 ORF (varies between types). What do the three regions encode?
Early region- non-structural proteins- change the wiring of the host cell and also encode the viral replication machinery
Late region- only expressed in a productively infected cells and encode capsid proteins (L1 and L2)
Untranslated or long control region- Does not make proteins, contains all sequence elements needed for viral transcription and genome replication
The genome only encodes one enzyme, which one?
E1- helicase activity to unwind the DNA
What do each of the genes do in the HPV genome?
E1-Initiation of DNA replication (helicase)
E2-Transcriptional regulation/DNA replication
E3- There is no E3
E4-Late early protein implicated in virus assembly/release
E5- Oncoprotein and genome amplification
E6-Oncoprotein and cell cycle modulation
E7-Oncoprotein and cell cycle modulation
E8- unclear function
L1- Major capsid protein
L2- Minor capsid protein
Why does HPV infect basal layers of epithelial cells?
It exploits the differentiation process of the cells. Epithelial cells start as basal stem cells, division results in one cell staying in basal layer and one leaves and moves up(cannot divide once it has left the basal layer). Stops receiving growth signals from epidermis and down regulates growth factor receptors whilst increasing differentiation markers. Keratin is important for the end of the life cycle. The virus therefore only infects the basal layers as they are the only cells that continue to divide. The late proteins of the virus are only expressed in the top layers of the surfaces whereas early ones are lower layers or throughout
What's the difference between the epithelial layer of the skin (cutaneous) and mucosal type?
Cutaneous one has a much larger cornified layer (squamous/dead cells) than the mucosal one
What cells are used in labs for studing HPV lifecycle?
Primary Keratinocytes from foreskin.
How can the virus life cycle be studied in labs?
Infect the cells with HPV, then there are two ways:
Monolayers grown on cultures/ media then tricked into differentiating by increasing calcium levels in the media. This gives a pretty close to normal viral life cycle but does not give virus out of those cells.
The other system used is the organotypic skin raft culture. This is a 3D cell culture. There is a layer of fibroblasts which feed the primary cells on top with appropriate hormones. The cells are then raised to an interface with the media and the air which causes the cells to differentiate. After 2 weeks time, the result is a completely differentiated skin that will allow the complete virus life cycle!
What are monolayers useful for then?
To see when certain proteins are expressed but if you want to look in a temporal fashion then raft cultures are used
What is immediately evident morphologically when looking at raft cultures of normal and HPV-infected epithelium?
The HPV-infected epithelium is much thicker than the non-infected one
How does the virus get into the cell? (remember this is just a theory)
It is a slow process (can take a few days). Needs several receptors to get in (one thought to be EGFR) and induces a conformational change in the capsid structure. This reveals parts of the capsid that is not usually exposed and therefore induces endocytosis.
What are the three different views on how the HPV particle is wrapped?
Clatherin-coated, non- clatherin+non caveolae and caveolae
Could all be true since there are many types
At some point in this, you get disassembly of the virus, the L2 protein uses the retromer complex to get into the cytoplasm and enter the nucleus for replication. L2 also helps direct the genome to get to the nucleus
How can the number of proteins made from such few elements that the HPV genome has be increased?
Through multiple splicing patterns can produce many (around 30) proteins from just the 10 elements
What are the 2 major promoters in HPV transcription?
Early promoter P97- drives transcription of E6 and E7
Late promoter P970- drives expression of E4/L1/L2 and E1
What is the LCR important for?
Initiation of expression of the viral genes after infection
What do E2 proteins regulate?
Viral replication, transcription and long term plasmid maintenance. The E2 protein can bind to DNA so grabs hold of the viral genome and the host cell so when the cell divides, the virus goes with it so you get persistent infection. E2 can upregulate and downregulate expression of genes (and is important in down regulating E6 and E7)
How many copies of the viral genome are there in one infected cell?
50-100 in the lower layers of the epithelium
How do E1 and E2 work together in DNA replication?
E2 binds to DNA and brings E1 to form a double hexamer on the DNA. It then moves along the DNA unwinding it. E1 brings host cell machinery like DNA polymerase, histone H1, cyclin E, Ubc9 for the replication
Why do HPVs delay differentiation of basal cells?
To hold them in a state of cell cycling so they can have the machinery to replicate so cells near the top are found to still be dividing
Viruses need the cell to be in G2 stage of their cycle. What marker is used to see if this is what is happening?
Cyclin B protein expression. Usually, only seen in basal cells but in infected cells it is seen throughout. E5 and 7 drive the production of cyclin B
What is vegetative viral DNA replication?
Where most of the viral DNA replication occurs (substantial amplification- 1000s of copies per cell). It usually happens in the top layers, terminally differentiated cells
What does BrdU do?
Marks newly synthesised DNA so if HPV infected, all cells show up with this. Knocking out viral proteins severely reduced the ability of the virus to replicate
Where are late genes such as E4 and L1 expressed?
In top layers/ differentiated keratinocytes. One of the jobs of E4 is to break open the keratin surrounded tough cells for release of the virus