Lecture 6 - DNA Virus Replication Flashcards
Cellular polymerase that makes mRNA from DNA
RNA polymerase II
Where does RNA polymerase II terminate elongation?
At AATAAA (poly-A signal)
Parts of the core promoter
1) TATA sequence
2) Initiator sequence
Role of the initiator sequence
Lies just before what is to be transcribed into mRNA.
Occupies +1 position of gene
Role of TATA sequence
Recruits RNA polymerase II
Components of the promoter
1) Local regulatory sequences
2) Core promoter
Local regulatory sequences in the promotor
Factors that define whether a gene is transcribed or not.
Often binding sites for transcription factor proteins.
Components of the transcriptional control region upstream of a gene
1) Distant regulatory sequences
2) Promoter
Distant regulatory sequences
Enhancers, silencers
Ways in which enhancer-binding proteins can act
1) Additively, where their action adds together
2) Synergistically, where they complement each others enhancement
ds DNA replication 1) 2) 3) 4) 5)
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
Name for short, newly synthesised, discontinuous portions of DNA on lagging strand
Okazaki fragments
Baltimore I
dsDNA –> (+) mRNA
Examples of Baltimore I viruses
Papovaviridae
Adenoviridae
Herpesviridae
Poxviridae
Baltimore II
ssDNA –> dsDNA –> (+) mRNA
Which Baltimore class does papovaviridae belong to?
Baltimore I
Example of a Baltimore II virus
Parvoviridae
Where do DNA viruses replicate?
In the nucleus
EXCEPT POXVIRUSES
Example of a papovavirus
Simian virus 40 (SV40)
SV40
Naked, dsDNA papovavirus
SV40 genome
dsDNA, circular, complexed with histones to form a mini-chromosome
SV40 genome gene architecture
Circular.
Counterclockwise are early genes (LT, ST)
Clockwise are structural proteins (VP2, VP3, VP1).
There are overlapping reading frames of genes (VP2 and VP3).
LT role
Large T antigen.
Non-structural SV40 protein. Promotes genome replication.
SV40 replication 1) 2) 3) 4) 5)
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
How does LT promote genome replication?
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.
Motif in SV40 that promotes RNA pol II transcription of early genes
72bp enhancer repeat. Increases RNA synthesis by 100x
How does LT switch the activity of the promoter?
1)
2)
3)
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.
How does SV40 keep the leading strand and lagging strands together in DNA synthesis?
A loop of template strand is formed at the replication complex
Two types of viral DNA synthesis
1) Fork
2) Displacement
Fork DNA synthesis
1)
2)
3)
1) Can initiate at an origin sequence
2) Always uses an RNA primer
3) Always has a leading and lagging strand
Displacement DNA synthesis
1)
2)
3)
1) Doesn’t use an RNA primer
2) No lagging strand
3) Commonly initiates at free DNA ends, uses a viral polymerase
Viruses that use fork DNA synthesis
1) Papovaviruses
2) Herpesviruses
Viruses that use displacement DNA synthesis
1) Adenoviruses (protein)
2) Parvoviruses (DNA hairpin)
Herpesvirus genome
dsDNA, linear.
126kb.
Complex. Many open reading frames, internal inverted repeats.
Herpesvirus replication
1)
2)
3)
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
Type of viral replication used by herpesviruses
Fork replication
Direction that DNA is assembled in
5’–>3’
Adenovirus genome structure
dsDNA, linear.
Origins at extremes of genome, use terminal proteins rather than RNA primer to initiate transcription
Adenovirus replication 1) 2) 3) 4) 5) 6)
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
Where does adenovirus virion assembly occur?
Nucleus
How is DNA replicated in adenoviruses?
No lagging strand.
Continuous DNA synthesis from each end of the genome.
Displacement.
Parvovirus DNA replication
1)
2)
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
Inverted terminal repeat
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
Example of a parvovirus
Adeno-associated virus
Poxvirus replication oddity
DNA virus, but replicates in the cytoplasm.
Therefore RNAs don’t splice alternatively
How can poxviruses replicate in the cytoplasm?
Encode their own polymerases
