Lecture 6 - DNA Virus Replication

Question 1

Cellular polymerase that makes mRNA from DNA

Answer 1

RNA polymerase II

Question 2

Where does RNA polymerase II terminate elongation?

Answer 2

At AATAAA (poly-A signal)

Question 3

Parts of the core promoter

Answer 3

1) TATA sequence

2) Initiator sequence

Question 4

Role of the initiator sequence

Answer 4

Lies just before what is to be transcribed into mRNA.

Occupies +1 position of gene

Question 5

Role of TATA sequence

Answer 5

Recruits RNA polymerase II

Question 6

Components of the promoter

Answer 6

1) Local regulatory sequences

2) Core promoter

Question 7

Local regulatory sequences in the promotor

Answer 7

Factors that define whether a gene is transcribed or not.

Often binding sites for transcription factor proteins.

Question 8

Components of the transcriptional control region upstream of a gene

Answer 8

1) Distant regulatory sequences

2) Promoter

Question 9

Distant regulatory sequences

Answer 9

Enhancers, silencers

Question 10

Ways in which enhancer-binding proteins can act

Answer 10

1) Additively, where their action adds together

2) Synergistically, where they complement each others enhancement

Question 11

ds DNA replication
1)
2)
3)
4)
5)

Answer 11

1) Parental DNA is unwound by a helicase
2) Short RNA primers complementary to the template are synthesised by a primase
3) Other DNA-binding proteins keep the replication bubble open
4) DNA polymerase grows primed template in5’ to 3’ direction for both strands (lagging and leading strand)
5) Free ends are ligated

Question 12

Name for short, newly synthesised, discontinuous portions of DNA on lagging strand

Answer 12

Okazaki fragments

Question 13

Baltimore I

Answer 13

dsDNA –> (+) mRNA

Question 14

Examples of Baltimore I viruses

Answer 14

Papovaviridae
Adenoviridae
Herpesviridae
Poxviridae

Question 15

Baltimore II

Answer 15

ssDNA –> dsDNA –> (+) mRNA

Question 16

Which Baltimore class does papovaviridae belong to?

Answer 16

Baltimore I

Question 17

Example of a Baltimore II virus

Answer 17

Parvoviridae

Question 18

Where do DNA viruses replicate?

Answer 18

In the nucleus

EXCEPT POXVIRUSES

Question 19

Example of a papovavirus

Answer 19

Simian virus 40 (SV40)

Question 20

SV40

Answer 20

Naked, dsDNA papovavirus

Question 21

SV40 genome

Answer 21

dsDNA, circular, complexed with histones to form a mini-chromosome

Question 22

SV40 genome gene architecture

Answer 22

Circular.
Counterclockwise are early genes (LT, ST)
Clockwise are structural proteins (VP2, VP3, VP1).
There are overlapping reading frames of genes (VP2 and VP3).

Question 23

LT role

Answer 23

Large T antigen.

Non-structural SV40 protein. Promotes genome replication.

Question 24

SV40 replication
1)
2)
3)
4)
5)

Answer 24

1) Genome released into nucleus, early RNA transcripts made with cell RNA pol II. Transcription factors (EG: NF-kB) bind promotor of early genes (‘early promoter’).
2) Alternative RNA splicing, RNA moves to cytoplasm, translation of early proteins.
3) Large T protein enters nucleus, binds origin on viral genome, promotes genome replication
4) Late promoter is antirepressed as viral genome reaches a certain concentration.
5) Structural genes transcribed, translated

Question 25

How does LT promote genome replication?

Answer 25

LT binds sequences flanking the core promoter, and assists in unwinding DNA genome, accelerating initiation of replication. Binds as a hexamer. Allows binding of single-stranded DNA binding protein, replication protein A, DNA polymerase alpha-primase.

Question 26

Motif in SV40 that promotes RNA pol II transcription of early genes

Answer 26

72bp enhancer repeat. Increases RNA synthesis by 100x

Question 27

How does LT switch the activity of the promoter? 1) 2) 3)

Answer 27

1) LT initiates viral DNA synthesis. 2) Cellular inhibitory binding proteins (IBP) inhibit late promoter. 3) LT causes genome to increase relative to IBP, diluting IBP, until the late promoter is no longer inhibited.

Question 28

How does SV40 keep the leading strand and lagging strands together in DNA synthesis?

Answer 28

A loop of template strand is formed at the replication complex

Question 29

Two types of viral DNA synthesis

Answer 29

1) Fork | 2) Displacement

Question 30

Fork DNA synthesis 1) 2) 3)

Answer 30

1) Can initiate at an origin sequence 2) Always uses an RNA primer 3) Always has a leading and lagging strand

Question 31

Displacement DNA synthesis 1) 2) 3)

Answer 31

1) Doesn't use an RNA primer 2) No lagging strand 3) Commonly initiates at free DNA ends, uses a viral polymerase

Question 32

Viruses that use fork DNA synthesis

Answer 32

1) Papovaviruses | 2) Herpesviruses

Question 33

Viruses that use displacement DNA synthesis

Answer 33

1) Adenoviruses (protein) | 2) Parvoviruses (DNA hairpin)

Question 34

Herpesvirus genome

Answer 34

dsDNA, linear. 126kb. Complex. Many open reading frames, internal inverted repeats.

Question 35

Herpesvirus replication 1) 2) 3)

Answer 35

1) VP-16 (tegument layer) initiates transcription of immediate early promoter 2) ICP0 and ICP4 change RNA transcription to early genes 3) Early genes activate transcription of late genes

Question 36

Type of viral replication used by herpesviruses

Answer 36

Fork replication

Question 37

Direction that DNA is assembled in

Answer 37

5'-->3'

Question 38

Adenovirus genome structure

Answer 38

dsDNA, linear. | Origins at extremes of genome, use terminal proteins rather than RNA primer to initiate transcription

Question 39

``` Adenovirus replication 1) 2) 3) 4) 5) 6) ```

Answer 39

1) Genome to nucleus. 2) Cellular RNA pol II transcribes immediate early genes, these are alternatively spliced 3) E1A is transcribed and translated, protein brought into nucleus 4) Increased E1A concentrations lead to transcription of early genes, including genes for viral DNA replication 5) Early proteins increase viral DNA replication, lead to transcription of late genes, which encode viral structural proteins 6) Virion assembly occurs in the nucleus

Question 40

Where does adenovirus virion assembly occur?

Answer 40

Nucleus

Question 41

How is DNA replicated in adenoviruses?

Answer 41

No lagging strand. Continuous DNA synthesis from each end of the genome. Displacement.

Question 42

Parvovirus DNA replication 1) 2)

Answer 42

Displacement 1) Terminus of parvovirus ssDNA genome has an inverted terminal repeat 2) ITR folds into a hairpin structure, which serves as both a primer and template for dsDNA synthesis

Question 43

Inverted terminal repeat

Answer 43

Composed of a central and flanking palindromic repeat sequence. In parvoviruses, folds to form a hairpin structure, which acts as a primer and template for ssDNA genome replication

Question 44

Example of a parvovirus

Answer 44

Adeno-associated virus

Question 45

Poxvirus replication oddity

Answer 45

DNA virus, but replicates in the cytoplasm. | Therefore RNAs don't splice alternatively

Question 46

How can poxviruses replicate in the cytoplasm?

Answer 46

Encode their own polymerases