Reverse Transcriptase: reversing the dogma
Ariel (ָאּרּיּאּל) GrosternSeminar for the Guelph Symposium of CHEM*4550
Students
Reversing the Dogma
Standard dogma of molecular biology:
dsDNA mRNA protein
Reverse transcription:
mRNA RNA/DNA dsDNA
Overview of Reverse Transcriptase (RT) (i)
The family of Retroviruses
Retroviral Infection Process
RNA-> DNA: the role of RT
Structure/Function of RT
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
Overview of Reverse Transcriptase (i)The family of Retroviruses
Retroviral Infection Process
RNA-> DNA: the role of RT
Structure/Function of RT
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)
Overview of Reverse Transcriptase (i)
RNA-> DNA: the role of RT
Structure/Function of RT
Retroviral infection process
The family of Retroviruses
Retroviral Infection Process
(Flint et al., Principles of Virology, 2000)
Overview of Reverse Transcriptase (i)
Retroviral Infection Process
Structure/Function of RT
RNA -> DNA: the role of RT
The family of Retroviruses
RNA -> DNA: the role of RT+ve strand
-ve strand(G
otte, Li and Wainberg, 1999)
Summary of role of RT:
*RNA-dependent DNA polymerase*
*DNA-dependent DNA polymerase*
*RNase H activity*
*Strand displacement*
RNA -> DNA: the role of RT
Overview of Reverse Transcriptase (i)
Structure/Function of RT
Retroviral Infection Process
RNA-> DNA: the role of RT
The family of Retroviruses
Structure/Function of RT
Efforts to determine structure of RT:1990 – first crystal reported by Unge et al – too poor for
x-ray diffraction1991 – RNase H domain structure determined at 2.4 A1991 – Structure of RT with bound Fab and DNA/DNA
strand at 3.5 A1992 – Structure of RT with bound inhibitor at 3.5 A1994 – First structure of unliganded RT, at 3.2 A
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
Structure/Function of RT
• Domains: – Fingers– Palm– Thumb– Connection– RNAse H*
*in p66 only, not p51
Structure/Function of RT
Active sites: DNA polymerase
- located at the 6- 10- 9 sheet of palm subdomain
p66
p55
Structure/Function of RT
Active sites:DNA polymerase
- YMDD motif (all polymerases)
- catalytic residues: D110*, D185*, D186
* Bind Mg2+
(Coffin, H
ughes and Varm
us, Retroviruses, 1997)
Structure/Function of RT
Active sites:DNA polymerase
Fancy version
(Huang et al., 1998)
Finger
palm
thumb
Structure/Function of RT
Active sites: RNase H
- Located in RNase H domain
p66
p55
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)
Structure/Function of RT DNA binding: Fingers, palm and thumb of
p66 form binding channel
Blue: fingersRed: palmGreen: thumbGray/white:DNA
(Cof
fin, H
ughe
s and
Var
mus
, Ret
rovi
ruse
s, 19
97)
Structure/Function of RT
dNTP binding: Fingers subdomain bends to
trap incoming dNTP
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
Structure/Function of RT (ii)Model of activity (steps):i) 18 nucleotides bind in central channel of RT, with 3’-
OH at polymerase active site (p66)ii) Open fingers allow dNTP entrance, then close to capture
dNTPiii) Nucleophilic attack of -phosphate of dNTP by 3’-OH
of “primer,” facilitated by bound Mg2+
iv) PPi releasedv) Ribonucleoside cleaved from 3’ end of template at
RNase H active site, facilitated by bound metal ionvi) Movement along template (?) and repeat i-vi
Overview of Reverse Transcriptase (ii)
Structure/Function of RT
Resistance to anti-RT drugs
RT: its other role in biology
Inhibition of RT: anti-RT drugs
Inhibition of RT: anti-RT drugs
Classes:
1. Nucleoside Analogs
2. Non-nucleoside RT inhibitors
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
Inhibition of RT: anti-RT drugs
Structures of Nucleoside Analogs
Inhibition of RT: anti-RT drugs(Sluis-C
remer, A
rion and Parniak, 2000)
Mode of Action of Nucleoside Analogs
Inhibition of RT: anti-RT drugs
O
CH3
N3
OHO
NH
O
O
CH3
N3
O
NH
OOP
O
O
O
CH3
N3
O
NH
OOP
O
O
OP
O
O
O
O
CH3
N3
O
NH
OOP
O
O
OP
O
O
OP
O
O
O
O
OO
O
CH3
N3
O
NH
NN
NH2
OO
O
O P O
O
O P O
PPi
5’
5’
3’
O
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
Structures of Non-nucleoside RT Inhibitors
Inhibition of RT: anti-RT drugs(Flint et al., Principles of V
irology, 2000)
RT with bound nevirapine
Inhibition of RT: anti-RT drugs
p51
p66nevirapine
Overview of Reverse Transcriptase (ii)
Structure/Function of RT
Inhibition of RT: anti-RT drugs
RT: its other role in biology
Resistance to anti-RT drugs
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)
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
Resistance to RT DrugsNucleoside analog resistance:
mutated residues(Sluis-C
remer, A
rion and Parniak, 2000)
Resistance to RT DrugsNucleoside analog resistance:
Pyrophospholytic cleavage(Sluis-C
remer, A
rion and Parniak, 2000)
Resistance to RT Drugs
Non-nucleoside analog resistance mechanism:
• Mutations alter hydrophobic pocket
– esp. single exchange at position 181 in p66
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
RT: its other role in biologyRT-PCR
(Watson, G
ilman, W
itkowski and Zoller, R
ecombinant D
NA
, 1999)
THE END