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
Hepatitis B second template exchange
Possible options for hepatitis B genome during reverse transcription
Features of retroviridae 1) 2) 3) 4) 5)
1) 80-130nm 2) Icosahedral 3) Enveloped 4) Diploid linear (+)ssRNA 5) Genome replicated in nucleus, virion assembly in cytoplasm
Diseases caused by retroviruses 1) 2) 3)
1) AIDS 2) Neurologic arthritis 3) T cell lymphoma
Structure of a simple retrovirus
Structure of a simple retrovirus genome
Shape of a retrovirus genome 1) 2) 3) 4)
1) Two copies of (+)ssRNA genome. 2) RNA is capped and poly-adenylated 3) Both RNAs are joined loosely at the 5’ end 4) Both RNAs have an attached tRNA at the 5’ end
Which retroviral genes form a polyprotein?
gag and pol. Autocleave to form separate proteins
When are env proteins expressed?
When mRNA transcript is spliced.
How are pol proteins differentially expressed from env?
Frameshift readthrough of genome-length mRNA
Mechanism by which pol genes are translated 1) 2) 3)
1) The reading frame of pol is shifted back -1 from that of gag. 2) A highly-stable RNA structure can stall RNA polymerase at the ‘U-rich slippery sequence’. 3) Frameshift occurs in between 1/5 - 1/20 translations
Host cell structural component of a retrovirus
tRNA (acts as a primer for reverse transcription)
Only way that new (+)ssRNA retroviral genome can be made
From integrated dsDNA provirus in host cell genome, using host RNA polymerase II.
Retroviral replication 1) 2) 3) 4) 5) 6) 7)
1) Cell receptor bound, transmembrane protein drives viral fusion.
2) Reverse transcription occurs in the cytoplasm
3) (-)ssDNA becomes dsDNA, then is transported to the nucleus
4) Integrase enzyme catalyses integration of proviral DNA into genome of host
5) Unspliced mRNA transcripts from provirus can become either gag, gag/pol precursor proteins, or move to plasma membrane for packaging into a virion.
6) Spliced mRNA is translocated to ER, where it becomes env glycoprotein. env glycoproteins are modified in the Golgi.
7) New virus buds, undergoes proteolytic maturation.
How are env made?
Spliced mRNA enters the ER, becomes env glycoproteins. Modified in the Golgi
Reverse transcription of viral RNA to proviral DNA 1) 2) 3) 4) 5) 6) 7) 8) 9)
1) tRNA primer binds to primer-binding site on 5’ end of RNA
2) tRNA primer is extended to 5’ end of RNA. RNAse H removes hybridised RNA
3) R sequence on DNA hybridises with R sequence on RNA. First ‘jump’
4) DNA is extended.
5) Most of hybridised RNA removed, except for a primer beside U3
6) 3’ end of second DNA strand synthesised
7) RNA and tRNA primer removed
8) Second jump. PBS (primer binding site) hybridise
9) Extension of DNA (both strands)
Retroviral reverse transcription up to first jump
Retroviral reverse transcription from the first jump to the second jump
Formation of retroviral long terminal repeats
Restriction on which cells simple retroviruses can integrate into
Only dividing cells. HIV isn’t a simple retrovirus, and so can integrate into terminally differentiated or resting cells
Long terminal repeat structure
3’ U3 | R | U5 | PBS ……… U3 | R | U5 5’ (of -DNA)
Effect of integration on retroviral provirus
4bp loss from proviral sequence. 2bp from each terminus.
How does integrase work? 1) 2) 3)
1) Monomer binds each terminus of provirus. Removes one nucleotide from each 3’ end. 2) Cuts host genome, ligates host genome to 3’ ends of provirus 3) Host enzymes repair nicked ends. Leads to characteristic flanking repeats in host cell DNA.
Features of long terminal repeat when integrated into host genome as a provirus 1) 2) 3) 4) 5)
1) U3 of 3’ end has enhancer sequences
2) U3 of 3’ end has TAATA transcription start sequence
3) Cap site sits between 3’ U3 and R
4) Polyadenylation sequence in 5’ R (AATAAA) 5) Poly-(A) addition site between 5’ R and U5
Pictorial representation of integrated proviral LTR features
Endogenous retroviruses
Retroviral genomes that have integrated into germline DNA, and are passed down through generations.
Proportion of genome that is endogenous retroviral
More endogenous retroviral genes than human genes
Characteristics of endogenous retroviruses
RT, LTR, env
Characteristics of retrotransposons
RT, LTR
Characteristics of LINES
RT, internal pol III promotor, A-rich sequence at the end
Characteristics of SINES
Internal pol III promotor, A-rich sequence at the end
Characteristics of processed pseudogenes
A-rich sequence
Retrovirus affecting koalas
Koala retrovirus (KoRV)
KoRV characteristics
1) Originally thought to be an endogenous retrovirus, but can replicate and transmit 2) Less common in southern Australia
