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Lecture 13. Retroid virusesSee chapter 7, and appendix 1 pp. 835
– 837.
The retroviral life cycleSalient features:• Viral RNA genome is reverse
transcribed into cDNA• The cDNA is integrated into the host cell
genome• Integrated proviral genome transcribed
by host RNA pol. II
REVERSE TRANSCRIPTION
Discovery• 1970: Simultaneous reports in Nature from the Temin
and Baltimore labs• Howard Temin: addressed the question of how
certain RNA viruses (“Slow viruses”) could permanently alter the heredity of cells. Proposed that RNA genomes must become integrated into the DNA genome. Corollary to this is that somehow a DNA copy of the RNA genome must be made. The technology at the time however, prevented being able to detect the viral sequence in the cellular genome.
• David Baltimore: Interested in virus-associated polymerases. Directly observed RNA dependent DNA polymerase activity from viruses of this type.
• Putting 2+2 together, they shared the Nobel Prize in 1975.
REVERSE TRANSCRIPTION
Impact– Changed the “Central Dogma” of Molecular
Biology. • From Watson and Crick, central dogma was
DNARNA protein.• Now, it is DNA RNA Protein• “Retrograde” flow of information “Retrovirus”
– Study of reverse transcription and integration has vastly increased our understanding of cancer
– Allowed us to understand how these viruses can persist in a patient
– RT has become an indispensable tool in molecular biology.
REVERSE TRANSCRIPTION
General: • RNA dependent DNA polymerase
activity copies genetic information from an RNA template to a DNA copy. The copy is called cDNA (complementary DNA).
• RT’s cannot initiate polymerization de novo, but require a specific primer
Essential Components for reverse transcription
Genomic RNA (Fig. 7.1)• Retrovirus particles contain two copies of
genomic (+) strand RNA• Sediments as 70S complex composed of a
dimer of 35S genomes: These viruses are diploid– Annealed head to head
• Includes two molecules of a specific tRNA primer
Essential Components for reverse transcription
Primer tRNA (Figs. 7.1, 7.2)• Virions also contain specific tRNAs: these act as primers
for initiation of reverse transcription.• Primer tRNAs are partially unwound• Base-paired near the 5’ end of each RNA genome at the
Primer Binding Site (PBS)
Essential Components for reverse transcription
Reverse transcriptase• RTs are complex molecular machines
with moving parts and multiple activities• 4 distinct catalytic activities combined
in 1 protein:– RNA- directed DNA polymerase – DNA-directed DNA polymerase– Helicase (unwinding)– Hydrolysis of RNA in RNA-DNA
heteroduplexes (RNaseH)
Recombination during reverse transcription
–Diploid nature of retroviral geneomes allows for high levels of recombination during replication.
–A mechanism for mutation and rapid evolution
Recombination during reverse transcription
2 mechanisms:1. Copy choice: during (-)
strand synthesis
2. Strand assimilation: during (+) strand synthesis
(Fig. 7.5)
CATALYTIC PROPERTIES: Reverse transcriptase
• Slow: – in vitro rate of DNA polymerization = 1 to 1.5 nt/sec. 1/10 rate of cellular DNA polymerase.
• Low Fidelity: – Lack editing (3.g. 5’ 3’ exonuclease activity) – Especially prone to misincorporation, dissociation and
slippage. See Fig 7.7.
•Degrades RNA portion of RNA-DNA duplex
•Produces 5’ PO4 and 3’ OH ends. The latter can be used as primers for extension by RT
CATALYTIC PROPERTIES: RNaseH
STRUCTURAL PROPERTIES
• RT domain– Looks like a polynucleotide polymerase– Thumb, palm and fingers domains.– Active site in the palm domain
• RNaseH domain– Located “behind” RT domain– Follows newly synthesized DNA strand
to access RNA-DNA duplex
INTEGRATION
General comments• Retroviruses integrate their cDNA genomes
into the host genome• The underlying reason for the persistence of
these viruses• Process mediated by viral protein called
Integrase• Integration changes the host cell genome• Can cause cancer• Germline mutation
Characteristic features of retroviral integration
• LTRs of viral cDNA targeted to specific sequences/structures in host genome
• Viral cDNA is integrated into host DNA to produce proviral DNA• Integration mediated by integrase.
– Requires ssDNA cleavage of host DNA.• Sequence changes:
– Host target sequence duplicated– Proviral DNA loses 2 bp from each end.
• Proviral genome serves as template for transcription of new viral RNA genomes.
• Transcription mediated by host cell RNA polymerase II
Fig. 7.14
RETROELEMENTS
• Mobile genetic elements, selfish DNAs, jumping genes.
• Dispersed throughout the genomes in very high copy numbers (up to 10% of mammalian genomes)
• Can jump from one locus to another, causing mutations.
• Major driving forces in evolution.