1. 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

3. 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.

4. 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.

5. 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

6. 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

7. 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)

8. 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)

9. Critical reactions in reverse transcription RNA priming

10. Critical reactions in reverse transcription Template strand exchange

11. Critical reactions in reverse transcription Strand displacement synthesis

12. Critical reactions in reverse transcription LTR formation

13. Recombination during reverse transcription –Diploid nature of retroviral geneomes allows for high levels of recombination during replication. –A mechanism for mutation and rapid evolution

14. Recombination during reverse transcription 2 mechanisms: 1.Copy choice: during (-) strand synthesis 2.Strand assimilation: during (+) strand synthesis (Fig. 7.5)

15. 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.

16. Degrades RNA portion of RNA- DNA duplex Produces 5’ PO 4 and 3’ OH ends. The latter can be used as primers for extension by RT CATALYTIC PROPERTIES: RNaseH

17. 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

18. Figs. 7.10 & 7.12

19. 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

20. 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

21. 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.

22. RETROELEMENTS