Lecture 13. Retroid viruses
See chapter 7, and appendix 1 pp. 835 – 837.
BSCI437
The retroviral life cycle: Figure 22, p. 837.
The salient features are
The 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.
Impact
1. Changed the “Central Dogma” of Molecular Biology.
 From Watson and Crick, central dogma was DNARNA protein.
 Now, it is DNA  RNA  Protein
 “Retrograde” flow of information  “Retrovirus”
2. Study of reverse transcription and integration has vastly increased our
understanding of cancer
3. Allowed us to understand how these viruses can persist in a patient
4. 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 polymeration 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 dimmer of 35S genomes
 Annealed head to head
 Includes two molecules of a specific tRNA primer
Essential Components for reverse transcription
Primer tRNA (Figs. 7.1, 7.2)
 In addition to genomic RNA, retroviral particles contain a lot of other stuff from
the host cell, e.g. 5S rRNA, 7S rRNA, traces of cellular RNA: functions are
unknown.
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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:
1. RNA- directed DNA polymerase
2. DNA-directed DNA polymerase
3. Helicase (unwinding)
4. Hydrolysis of RNA in RNA-DNA heteroduplexes (RNaseH)
Critical reactions in reverse transcription (Fig. 7.3)
1. RNA priming
2. Template strand exchange
3. Strand displacement synthesis
4. Long Terminal Repeat (LTR) formation
Recombination during reverse transcription (Fig. 7.5)
 The diploid nature of retroviral geneomes allows for high levels of recombination
during replication.
 A mechanism for mutation and rapid evolution
 2 mechanisms:
i. Copy choice: during (-) strand synthesis
ii. Strand assimilation: during (+) strand synthesis
CATALYTIC PROPERTIES
Reverse transcriptase
 Slow:
o in vitro rate of DNA polymerization = 1 to 1.5 nt/sec.
o 1/10 rate of cellular DNA polymerase.
 Low Fidelity:
o Lack editing (3.g. 5’  3’ exonuclease activity)
o Especially prone to misincorporation, dissociation and slippage. See Fig
7.7.
RNase H
 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
STRUCTURAL PROPERTIES (Figs. 7.10, 7.12)
 RT domain
o Looks like a polynucleotide polymerase
o Thumb, palm and fingers domains.
o Active site in the palm domain
 RNaseH domain
o Located “behind” RT domain
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o 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 (Fig. 7.14)
 LTRs of viral cDNA are targeted to specific sequences/structures in host genome
 Viral cDNA is integrated into host DNA to produce proviral DNA
 Integration mediated by integrase.
o Requires ssDNA cleavage of host DNA.
 Sequence changes:
o Host target sequence is duplicated
o 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
RETROELEMENTS (see box 7.2, p. 231)
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.
Rtroelement Families
Endogeneous retroviruses: Proviruses that have been integrated into the germline.
Behave as Mendelian elements, passed onto future generations
Retrotransposons: Lack of envelope genes keeps them intracellular
Retroposons: aka Long interspersed nuclear elements (LINEs). Lack LTRs, A-T
rich ends. Very high copy numbers in the genome.
Retrosequences: aka SINES, Alu elements. Lack RT and LTRs. Very high
genomic copy numbers.
Processed pseudogenes: mRNAs that were reverse transcribed and integrated into
the genome.
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