Reverse Transcriptase: reversing the dogma Ariel (אָרּיּאּלּ) Grostern Seminar for the Guelph...
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Transcript of Reverse Transcriptase: reversing the dogma Ariel (אָרּיּאּלּ) Grostern Seminar for the Guelph...
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
(ICT
V 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., P
rinciples 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 W
ainberg, 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 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
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,
Hug
hes
and
Var
mus
, Ret
rovi
ruse
s, 1
997)
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(S
luis-Crem
er, Arion and P
arniak, 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
N
N
NH2
O
O
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(F
lint 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(S
luis-Crem
er, Arion and P
arniak, 2000)
Resistance to RT DrugsNucleoside analog resistance:
Pyrophospholytic cleavage(S
luis-Crem
er, Arion and P
arniak, 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 Z
oller, Recom
binant DN
A, 1999)
THE END