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Reverse Transcriptase: reversing the dogma

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  1. Reverse Transcriptase:reversing the dogma Ariel (אָרּיּאּלּ) Grostern Seminar for the Guelph Symposium of CHEM*4550 Students

  2. Reversing the Dogma Standard dogma of molecular biology: dsDNA  mRNA protein Reverse transcription: mRNA RNA/DNA  dsDNA

  3. Overview of Reverse Transcriptase (RT) (i) The family of Retroviruses Retroviral Infection Process RNA-> DNA: the role of RT Structure/Function of RT

  4. Overview of Reverse Transcriptase (ii) Structure/Function of RT Inhibition of RT: anti-RT drugs Resistance to anti-RT drugs RT: its other role in biology

  5. Overview of Reverse Transcriptase (i) The family of Retroviruses Retroviral Infection Process RNA-> DNA: the role of RT Structure/Function of RT

  6. The Family of Retroviruses • First discovered in 1908 - chicken leukosis • 1960s - Reverse transcription first proposed • Includes: • Rous sarcoma virus • Human immunodeficiency virus (HIV) • Human T-cell leukemia virus (HTLV) HIV (ICTV database)

  7. Overview of Reverse Transcriptase (i) The family of Retroviruses Retroviral infection process RNA-> DNA: the role of RT Structure/Function of RT

  8. Retroviral Infection Process (Flint et al., Principles of Virology, 2000)

  9. Overview of Reverse Transcriptase (i) The family of Retroviruses Retroviral Infection Process RNA -> DNA: the role of RT Structure/Function of RT

  10. RNA -> DNA: the role of RT +ve strand -ve strand (Gotte, Li and Wainberg, 1999)

  11. RNA -> DNA: the role of RT Summary of role of RT: *RNA-dependent DNA polymerase* *DNA-dependent DNA polymerase* *RNase H activity* *Strand displacement*

  12. Overview of Reverse Transcriptase (i) The family of Retroviruses Retroviral Infection Process RNA-> DNA: the role of RT Structure/Function of RT

  13. Structure/Function of RT Efforts to determine structure of RT: 1990 – first crystal reported by Unge et al – too poor for x-ray diffraction 1991 – RNase H domain structure determined at 2.4 A 1991 – Structure of RT with bound Fab and DNA/DNA strand at 3.5 A 1992 – Structure of RT with bound inhibitor at 3.5 A 1994 – First structure of unliganded RT, at 3.2 A

  14. Structure/Function of RT • Heterodimer • Encoded from pol as 66 kDa preprotein • One subunit cleaved between F440 and Y441: 51 kDa (p51) (415 AAs) • One subunit intact: 66 kDa (p66) (556 AAs) • Orientation: asymmetric • Active sites on p66 only

  15. Structure/Function of RT • Domains: • Fingers • Palm • Thumb • Connection • RNAse H* *in p66 only, not p51

  16. Structure/Function of RT Active sites: DNA polymerase - located at the 6- 10- 9 sheet of palm subdomain p66 p55

  17. Structure/Function of RT Active sites: DNA polymerase • YMDD motif (all polymerases) - catalytic residues: D110*, D185*, D186 * Bind Mg2+ (Coffin, Hughes and Varmus, Retroviruses, 1997)

  18. Structure/Function of RT Active sites: DNA polymerase Fancy version Finger (Huang et al., 1998) palm thumb

  19. Structure/Function of RT Active sites: RNase H • Located in RNase H domain p66 p55

  20. Structure/Function of RT Active sites: RNase H • Catalytic residues: D443, E478, D498 • Binding of divalent cation (not yet known if Mg2+ or Mn2+) • Restoration of 3’-OH at cut (Saraianos et al, 2001)

  21. Structure/Function of RT DNA binding:Fingers, palm and thumb of p66 form binding channel (Coffin, Hughes and Varmus, Retroviruses, 1997) Blue: fingers Red: palm Green: thumb Gray/white:DNA

  22. Structure/Function of RT dNTP binding: Fingers subdomain bends to trap incoming dNTP

  23. Structure/Function of RT Observation: • constant number of 18 base pairs between polymerase and RNase H active sites for both RNA/DNA and DNA/DNA

  24. Structure/Function of RT (ii) Model of activity (steps): • 18 nucleotides bind in central channel of RT, with 3’-OH at polymerase active site (p66) • Open fingers allow dNTP entrance, then close to capture dNTP • Nucleophilic attack of -phosphate of dNTP by 3’-OH of “primer,” facilitated by bound Mg2+ • PPi released • Ribonucleoside cleaved from 3’ end of template at RNase H active site, facilitated by bound metal ion • Movement along template (?) and repeat i-vi

  25. Overview of Reverse Transcriptase (ii) Structure/Function of RT Inhibition of RT: anti-RT drugs Resistance to anti-RT drugs RT: its other role in biology

  26. Inhibition of RT: anti-RT drugs Classes: • Nucleoside Analogs • Non-nucleoside RT inhibitors

  27. Inhibition of RT: anti-RT drugs Nucleoside Analogs • Examples: zidovudine (AZT), lamivudine (3TC), dideoxyinosine (ddI), dideoxycytidine (ddC) • Bind at DNA polymerase binding site • Compete for binding in RT with nucleosides • Lack 3’OH,  chain termination

  28. Inhibition of RT: anti-RT drugs Structures of Nucleoside Analogs (Sluis-Cremer, Arion and Parniak, 2000)

  29. 3’ 5’ 5’ Inhibition of RT: anti-RT drugs Mode of Action of Nucleoside Analogs O

  30. Inhibition of RT: anti-RT drugs Non-nucleoside RT inhibitors • Examples: nevirapine, delavirdine, efavirenz, TIBO • Bind to hydrophobic pocket close (10 A) to polymerase active site • allosteric effects –> repositioning of active site -strands •  DNA polymerase inactivated

  31. Inhibition of RT: anti-RT drugs Structures of Non-nucleoside RT Inhibitors (Flint et al., Principles of Virology, 2000)

  32. Inhibition of RT: anti-RT drugs RT with bound nevirapine nevirapine p66 p51

  33. Overview of Reverse Transcriptase (ii) Structure/Function of RT Inhibition of RT: anti-RT drugs Resistance to anti-RT drugs RT: its other role in biology

  34. Resistance to RT Drugs Considerations: • High number of virus particles in host • RT lacks proof-reading function • High mutation rate: 10-5-10-3 per base pair  ~0.1-10 mutations per replication cycle Result: mutants arise quickly (~1/3 of virions in host)

  35. Resistance to RT Drugs Nucleoside analog resistance mechanisms: • Mutations - Discrimination against analogs due to positioning (e.g. 3’-OH binding pocket) • Phosphorlytic removal of incorporated analog

  36. Resistance to RT Drugs Nucleoside analog resistance: mutated residues (Sluis-Cremer, Arion and Parniak, 2000)

  37. Resistance to RT Drugs Nucleoside analog resistance: Pyrophospholytic cleavage (Sluis-Cremer, Arion and Parniak, 2000)

  38. Resistance to RT Drugs Non-nucleoside analog resistance mechanism: • Mutations alter hydrophobic pocket • esp. single exchange at position 181 in p66

  39. Overview of Reverse Transcriptase (ii) Structure/Function of RT Inhibition of RT: anti-RT drugs Resistance to RT Drugs RT: its other role in biology

  40. RT: its other role in biology RT-PCR (Watson, Gilman, Witkowski and Zoller, Recombinant DNA, 1999)

  41. THE END