Lecture 13. Retroid viruses
See chapter 7, and appendix 1 pp. 835 –
837.
The retroviral life cycle
Salient 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 DNARNA
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)
Critical reactions in reverse
transcription
•
RNA priming
Critical reactions in reverse
transcription
•Template strand exchange
Critical reactions in reverse
transcription
• Strand displacement synthesis
Critical reactions in reverse
transcription
• LTR formation
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.
CATALYTIC PROPERTIES:
RNaseH
• Degrades RNA portion of RNADNA duplex
• Produces 5’ PO4 and 3’ OH ends.
The latter can be used as
primers for extension by RT
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
Figs. 7.10 & 7.12
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.
•
Sequence changes:
•
Proviral genome serves as template for transcription of new viral RNA
genomes.
Transcription mediated by host cell RNA polymerase II
•
– Requires ssDNA cleavage of host DNA.
– Host target sequence duplicated
– Proviral DNA loses 2 bp from each end.
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.
RETROELEMENTS