HPV

Question 0

What are a) alpha PV and b) beta PV also known as?

Answer 0

a) mucosal

b) cutaneous

Question 1

What is the family PV is classified to?

Answer 1

Papillomaviridae

Question 2

Give a brief structure of the PV genome?

Answer 2

8 kb, circular, double stranded, 7904 bp, URR (upstream regulatory region), many proteins: early (e1, 2, 4, 5, 6, 7) and late (L1 and L2).

Question 3

Which early protein is commonly not expressed?

Answer 3

e5

Question 4

How is HPV transmitted?

Answer 4

Sexual contact, micro-tears and abrasions in the epithelial layer

Question 5

Name some cofactors that work with PV to cause benign or malignant cancers?

Answer 5

Bracken, sunlight, hormones, smoking and host genotype

Question 6

How is HPV usually detected?

Answer 6

Smear tests

Question 7

We’re are the ORFs of the HPV genome located?

Answer 7

On one strand of the DNA

Question 8

What does the coding strand contain?

Answer 8

Approximately 10 designated ORFs, classified as early or late

Question 9

What are early expressed ORFs?

Answer 9

E proteins: 1, 2, 4, 5, 6, 7 and 8.
They are expressed early, in non-productively infected cells.
Includes regulatory proteins and those required for genome replication.

Question 10

What are late expressed ORFs?

Answer 10

L1 and L2.
Capsid proteins.
Expressed late only in productively infected cells.

Question 11

What is LCR?

Answer 11

The long coding region, approximately 1 kbp, one in each genome, contains the Ori of DNA replication as well as important transcription factor control elements.

Question 12

Which protein is for transcriptional regulation and DNA replication?

Answer 12

E2

Question 13

Which proteins are for oncoprotein and cell cycle modulation/genome amplification? (Specify)

Answer 13

E5, genome amplification, and E6 and E7,cell cycle modulation.

Question 14

Which protein has enzymatic activity?

Answer 14

Only E1, helicase activity for the initiation of DNA replication. All other enzymatic activation has to come from host.

Question 15

What is E4 for?

Answer 15

Late expression to help the virus assemble and exit the cell

Question 16

What are L1 and L2 functions?

Answer 16

L1: major capsid protein
L2: minor capsid protein

Question 17

How do epithelial cells grow?

Answer 17

From basal stem layer and reserve to suprabasal then granular and then stratum corneum with cornification and sloughing.

Question 18

How does HPV replicate with epithelial differentiation?

Answer 18

At the basal layer the genome is simply maintained and at suprabasal level the cell begins to proliferate causing genome amplification. At the granular level virus is assembled and released with cornification.

Question 19

How can differentiating keratinocytes be looked at?

Answer 19

Using calcium in a mono layer however HPV cannot be produced using this method only certain stages of viral replication can be studied.

Question 20

How can HPV be grown?

Answer 20

Organothpic raft culture: add HPV to epithelial cells, plate with collagen matrix, fibroblasts and culture media, grow to confluence and transfer submerged mono layer matrix to support grid to create a differentiating epithelium. In two weeks a striated epithelium will have formed.

Question 21

What does confluence mean?

Answer 21

The proportion of the surface of the culture disk covered in cells. 100% is covered, 50% is half covered etc.

Question 22

How does HPV enter?

Answer 22

At a micro-tear or abrasion in the epithelium, enters via a variety of methods, clathrin and non-clathrin coated vesicles, calveolae, via the endosomal pathway.

Question 23

What does HPV do when it has entered?

Answer 23

Disassembly of the capsid occurs in the endosome and exposes the genome. L2 utilises the retro ER complex to traffic the genome into the cytoplasm, enter the nucleus and co localise with ND10 bodies, which may be the site of virus replication. L2 may enhance nuclear trafficking of the genome to transcriptionally active regions of the host genome, as it is inefficient.

Question 24

Describe transpiration of PV?

Answer 24

Complex. Mulitple promoters and alternate and mulitple splicing patterns and differentiation of mRNA production in different cells which produces a potentially huge variety of alternatively spliced proteins.

Question 25

What is a major promoter in undifferentiated cells?

Answer 25

P97 which directs expression of E6 and E7.

Question 26

What is a late promoter?

Answer 26

In immortalised differentiated cells, P670 promoter drives expression of E4 and L1/2 (and E1).

Question 27

What are the Orf of PV?

Answer 27

Polycistronic: meaning the encode two or more proteins.

Question 28

How is transcription of PV regulated?

Answer 28

LCR: contains enhancer elements, responsible for viral and virus-encoded transcriptional regulatory factors, also plays an important role in the initial expression of the viral gens and virus latency and includes binding sites for E2.
E2 are regulatory proteins.

Question 29

Explain how E2 is regulatory?

Answer 29

They are a DNA binding protein that contain sequence-specific DNA binding and dimerisation domains and a trans-activating domain in their amino terminus which are joint by a hinge region. Truncated E2 can act as a repressor of transcription, can repress the P97 promoter to regulate E6 and E7 expression.

Question 30

What does DNA replication require?

Answer 30

E1 and E2 in trans and DNA ori in cis. E1 is a nuclear phospho protein which is essential for virus-encoded replication and functions as an ATP-dependent helicase and interacts with a number of cellular proteins. E2 acts to stabilise E1-Ori interaction. Requires the association of E1 with the S-phase specific cyclin E complex.

Question 31

What is the structure of PV?

Answer 31

Icosahedral capsid (2 major capsid protein, L1 and L2), non-enveloped protein shell