Lecture 7 - Reverse Transcription Virus Replication Flashcards
Fork replication picture
Displacement replication picture
Function of topoisomerase
Relaxes supercoils in dsDNA for replication
Enzyme that relaxes supercoiling in dsDNA for replication
Topoisomerase
How can topoisomerase work? 1) 2)
1) When a covalently-closed circular dsDNA genome is replicated, topoisomerase I or II cleaves one strand of DNA, to relax supercoils 2) When two daughter dsDNA are catenated, topoisomerase II cleaves two strands of DNA (uses ATP) and reseals them to separate the daughter strands.
Viruses that form proteins from spliced RNA transcripts from DNA
Papovaviridae Herpesviridae Adenoviridae
Viruses that form proteins from unspliced RNA, which came from dsDNA
Poxviridae
How do parvoviridae form proteins?
ssDNA made into dsDNA. RNA transcribed, spliced.
Do hepadnaviridae splice RNA?
No
How does hepadnaviridae genome replication differ from Baltimore I viruses?
Hepadnaviridae make RNA transcripts from dsDNA, but some of these transcripts (pre-genomic RNA) are reverse-transcribed to form (-)ssDNA
Baltimore class of hepadnaviridae
VII
Hepatitis B viral structure
Core containing genome. Hepatitis B surface antigen lines exterior of capsid.
Hepatitis B genome 1) 2) 3) 4) 5)
1) 3kb, relaxed circle 2) most of genome is dsDNA, but a section is ssDNA 3) Capped with a viral polymerase 4) 2 short direct repeats (~12bp) 5) RNA primer (~18 nucleotides) on 5’ end
What is the RNA primer on hepatitis B genome?
A remnant of pregenomic RNA
Hepatitis B genome gene architecture 1) 2) 3) 4)
1) Can make 4 different sized mRNAs using 4 different dsDNA promotor/enhancer transcription start sites 2) All mRNAs end at the same poly-A sequence 3) Genes can have overlapping reading frames, alternative splice sites 4) Pre-genomic RNA is longer than DNA genome (transcription involves more than one revolution of genome)
Roles of pre-genomic mRNA 1) 2)
1) Transcript for translation of core and reverse transcriptase proteins 2) Synthesis of viral DNA genome
Hepatitis B genome replication 1) 2) 3) 4) 5) 6) 7) 8)
1) Relaxed circle genome enters host cell nucleus
2) Genome is repaired into two continuous strands by host cell enzymes. This forms covalently closed circular DNA (cccDNA)
3) Pre-genomic RNA is formed from cccDNA transcript.
4) Pre-genomic RNA enters cytoplasm. Self-annealing parts of RNA form loops that act as binding sites for viral RT polymerase.
5) When short DNA reaches 5’DR1, a template switch to the 3’ DR1 occurs.
6) (-) DNA elongation occurs. RNase H activity of viral polymerase cleaves RNA.
7) Remaining 15-18nt from before template switch forms an RNA primer for (+) DNA elongation. About 10% of the time the RNA primer stays in the same place, and linear dsDNA is formed that will become cccDNA
8) 90% of the time primer translocates, so that DR1 binds to DR2 (second template exchange). This (+) strand of DNA is extended.
Hepatitis B reverse transcription 1) 2) 3) 4) 5)
1) Pre-genomic RNA enters cytoplasm. Self-annealing parts of RNA form loops that act as binding sites for viral RT polymerase. 2) When short DNA reaches 5’DR1, a template switch to the 3’ DR1 occurs. 3) (-) DNA elongation occurs. RNase H activity of viral polymerase cleaves RNA. 4) Remaining 15-18nt from before template switch forms an RNA primer for (+) DNA elongation. About 10% of the time the RNA primer stays in the same place, and linear dsDNA is formed that will become cccDNA 5) 90% of the time primer translocates, so that DR1 binds to DR2 (second template exchange). This (+) strand of DNA is extended.
How does the structure of the hepatitis B genome reflect it’s genomic replication?
The section of ssDNA is from the second template exchange. where DS1 and DS2 bind. The section of RNA left on the 5’ end of (+) DNA is from reverse transcription, and acted as a primer for DNA elongation.
Hepatitis B viral replication 1) 2) 3) 4) 5) 6) 7) 8)
1) Attachment, uptake by RME 2) + DNA repair by cellular polymerases into cccDNA (covalently closed cirucular DNA) 3) RNA transcription from (-)DNA 4) RNA enters cytoplasm. Some transcripts are translated into hepatitis B surface antigen (HBsAg) in the ER. 5) Pregenomic RNA is translated into capsid, reverse trascriptase 6) Pregnome is packaged into capsid 7) Reverse transcription of genome 8) EITHER cccDNA is amplified, goes to nucleus, OR capsid matures, enveloped, released from cell.
- Attachment 2. Uptake 3. + DNA gap repair by P 4. Covalently closed circular (CCC) DNA 5. RNA transcription from - DNA 6. RNA transport 7. HBsAg translation 8. Pregenome translation - polymerase (P) 9. - capsid 10. Pregenome packaging 11. Reverse transcription by P 12. CCC DNA amplification 13. Nucleocapsid matures 14. Envelope addition 15. Particle release.
Hepatitis B first template exchange