Polyomavirus (Dr. Frappier)

Question 1

Features of capsid of polyomaviruses

Answer 1

nonenveloped icosahedral

Question 2

features of genome for genome

Answer 2

circular, dsDNA with histons

Question 3

How is genome organized?

Answer 3

early region and late region separated by regulatory region

Question 4

what is found in regulatory region?

Answer 4

origin of DNA replication, promoter, enhancer

Question 5

What is found in late region?

Answer 5

structural proteins like 3 capsid proteins (VP1, VP2, VP3)

Question 6

What is found in early regions?

Answer 6

T antigens (types depending on specific polyomaviruses)

Question 7

Difference in early region of SV40 and polyoma

Answer 7

i) SV40 don’t have tiny and middle T
ii) Polyoma have all tiny, small, middle and large T antigens

Question 8

What is used for transcription of polyomavirus?

Answer 8

cellular RNA polymerase II

Question 9

What does middle T antigen do?

Answer 9

i) bind PP2A (phosphatase) and SRC kinase to affect their activity
–> SRC kinase activated – phosphorylate T and other cellular protein

Needed for cell transformation

Question 10

similarities between small t and large T

Answer 10

their N-terminal region

Question 11

Special about small t of SV40

Answer 11

C terminal can bind and inhibit PP2A
–> contribute to cell transformation

Question 12

Main functions of large T antigen

Answer 12

i) stimulate resting cells to enter cell cycle
ii) regulates early mRNA production
iii) needed for viral DNA replication

Question 13

How does large T antigen stimulate resting cells to enter cell cycle?

Answer 13

i) bind pRb
ii) bind p53 (only for SV40 T)
iii) interact with p300/CBP
iv) bind hsp70

Question 14

How does binding to pRB stimulate resting cells to enter cell cycle?

Answer 14

pRb released from E2F
–> activate genes involved in cell proliferation by E2F

Question 15

What kind of infection occur in primate of polyoma?

Answer 15

non permissive

Question 16

How does binding to p53 stimulate resting cells to enter cell cycle?

Answer 16

i) inhibit p54 binding to DNA
–> lower expression of cell cycle inhibitors
ii) inhibit p53 mediated apoptosis

Question 17

How does binding to p300 stimulate resting cells to enter cell cycle?

Answer 17

changes the histone acetylation and expression of specific genes (e.g. E1A)

Question 18

How does binding to hsp70 stimulate resting cells to enter cell cycle?

Answer 18

i) stimulate ATPase activity of hsp70
ii) N terminal domain of T binding to hsp70
–> important for cell trnasformation

Question 19

Name for N terminal domain of large T

Answer 19

DnaJ

Question 20

How does large T antigen regulate early mRNA production?

Answer 20

Bind site I in regulatory region
–> interferes with RNA pol and TFs binding to early promoter
–> repress early gene expression at end of early phase

Question 21

How does large T antigen required for viral DNA replication?

Answer 21

i) bind origin to initiate DNA replication
ii) melt origin DNA and serve as DNA helicase
iii) bind cellular DNA replication proteins

Question 22

How many origin of replication is found in SV40?

Answer 22

one

Question 23

What is found within the origin of replication?

Answer 23

core + auxiliary sequences

Question 24

Features of SV40 core sequence

Answer 24

i) T binding site II
ii) palindrome on early side
iii) AT-rich on late side

Question 25

Features of SV40 auxiliary sequences

Answer 25

i) transcriptional elements
ii) T binding sites I

Question 26

What kind of infection occur in primate of SV40?

Answer 26

permissive

Question 27

What kind of infection occur in rodent of SV40?

Answer 27

nonpermissive

Question 28

How does DNA replication start in SV40?

Answer 28

i) T forms 2 hexamers on site II of origin
ii) T unwinds at early palindrome
–> RPA bind ssDNA
iii) T recruits polymerase alpha primase to origin to initiate DNA synthesis
iv) T hexamer at each end act as DNA helicase

Question 29

What mechanism does SV40 replication operate on?

Answer 29

theta structure mechanism

Question 30

Cellular proteins needed for SV40 replication

Answer 30

i) RPA (bind ssDNA)
ii) DNA polymerase alpha primase (initiate replication on both strands)
iii) DNA polymerase delta (make leading strand, extend lagging strand)
iv) PCNA
v) RFC (load PCNA on DNA)
vi) Topoisomerase I, II (relieves strained bits)
vII) RNaseI, FEN-1 (Exonuclease to remove RNA primters)
viii) DNA ligase

Question 31

Where does permissive infection occur for each virus?

Answer 31

SV40 –> primate
Polyoma –> rodent

Question 32

Where does nonpermissive infection occur for each virus

Answer 32

SV40 –> rodent
Polyoma –> primate

Question 33

What kind of infection occur in rodent of polyoma?

Answer 33

permissive

Question 34

Difference between permissive and nonpermissive infection

Answer 34

i) virions formed in permissive infection, not formed in nonpermissive
ii) viral DNA replicates in permissive, not in nonpermissive

Question 35

types of nonpermissive infection

Answer 35

i) transient transformation
ii) Permanent transformation

Question 36

Difference between transient and permanent transfomraiton

Answer 36

i) T antigen expression: lack in transient, continued in permanent

Question 37

Which T antigen is expressed during permanent transformation?

Answer 37

SV40: largeT
polyoma: middle T

Question 38

T antigens expressed by merkel cell polyomavirus

Answer 38

small t
large T
57 kT
ALTO

Question 39

contributing factors to merkel cell carcinoma

Answer 39

i) UV exposure
ii) immunosuppresion

Question 40

lethality of merkel cell carcinoma compared to malignant melanoma

Answer 40

twice as lethal as merkel cell carcinoma

Question 41

What is found in tumor cells of merkel cell carinoma?

Answer 41

integrated copies of merkel cell polyomavirus

Question 42

function of small T in merkel cell polyomavirus

Answer 42

protects large T from degradation by inhibiting ubiquitin ligase

Question 43

Difference between largeT found in normal cells and tumor cells

Answer 43

tumor cells
–> truncated
–> N terminal here but lack DNA helicase domain

Question 44

What is important for transformation in merkel cell polyomavirus?

Answer 44

large T binding to Rb

Question 45

What is found in tumor in raccons?

Answer 45

cirular form of polyomavirus
–> nonintegrated