Transcript Human Cytomegalovirus Modulation of Host Cell Functions

Reverse Transcriptase:
reversing the dogma
Ariel (‫ )אָרּיּאּלּ‬Grostern
Seminar 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)
(ICTV database)
HIV
Overview of Reverse Transcriptase (i)
The family of Retroviruses
Retroviral infection process
RNA-> DNA: the role of RT
Structure/Function of RT
Retroviral Infection Process
(Flint et al., Principles of Virology, 2000)
Overview of Reverse Transcriptase (i)
The family of Retroviruses
Retroviral Infection Process
RNA -> DNA: the role of RT
Structure/Function of RT
RNA -> DNA: the role of RT
+ve strand
-ve strand
(Gotte, Li and Wainberg, 1999)
RNA -> DNA: the role of RT
Summary of role of RT:
*RNA-dependent DNA polymerase*
*DNA-dependent DNA polymerase*
*RNase H activity*
*Strand displacement*
Overview of Reverse Transcriptase (i)
The family of Retroviruses
Retroviral Infection Process
RNA-> DNA: the role of RT
Structure/Function of RT
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
p66
Active sites:
DNA polymerase
- located at the 6- 10- 9
sheet of palm subdomain
p55
Structure/Function of RT
- YMDD motif (all
polymerases)
- catalytic residues:
D110*, D185*, D186
* Bind Mg2+
(Coffin, Hughes and Varmus, Retroviruses, 1997)
Active sites:
DNA polymerase
Structure/Function of RT
Active sites:
DNA polymerase
Finger
(Huang et al., 1998)
Fancy version
palm
thumb
Structure/Function of RT
p66
Active sites: RNase H
- Located in RNase H
domain
p55
Structure/Function of RT
Active sites:
RNase H
(Saraianos et al, 2001)
-
Catalytic residues: D443, E478,
D498
-
Binding of divalent cation (not yet
known if Mg2+ or Mn2+)
-
Restoration of 3’-OH at cut
Structure/Function of RT
DNA binding: Fingers, palm and thumb of
(Coffin, Hughes and Varmus, Retroviruses, 1997)
p66 form binding channel
Blue: fingers
Red: palm
Green: thumb
Gray/white:DNA
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
dNTP
iii) Nucleophilic attack of -phosphate of dNTP by 3’-OH
of “primer,” facilitated by bound Mg2+
iv) PPi released
v) Ribonucleoside cleaved from 3’ end of template at
RNase H active site, facilitated by bound metal ion
vi) Movement along template (?) and repeat i-vi
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
Inhibition of RT: anti-RT drugs
Classes:
1. Nucleoside Analogs
2. Non-nucleoside RT inhibitors
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
Inhibition of RT: anti-RT drugs
Structures of Nucleoside Analogs
(Sluis-Cremer, Arion and Parniak, 2000)
Inhibition of RT: anti-RT drugs
Mode of Action of Nucleoside Analogs
O
O
CH3
HN
HN
O
CH3
O
O
O
HO
P
O
O
N3
O
O
P
O
O
O
N3
P
O
O
O
O
N3
3’
5’
NH2
O
O
O
P
N
O
N
O
HN
O
O
O
O
PPi
O
O
O
CH3
HN
O
O
O
P
O HN
O
O
O
O
CH3
P
O
O
P
O
O
O
P
O
O
O
O
N3
N3
5’
CH3
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
Inhibition of RT: anti-RT drugs
Structures of Non-nucleoside RT Inhibitors
(Flint et al., Principles of Virology, 2000)
Inhibition of RT: anti-RT drugs
RT with bound nevirapine
nevirapine
p66
p51
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
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 Drugs
Nucleoside analog resistance:
mutated residues
(Sluis-Cremer, Arion and Parniak, 2000)
Resistance to RT Drugs
Nucleoside analog resistance:
Pyrophospholytic cleavage
(Sluis-Cremer, Arion 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 biology
RT-PCR
(Watson, Gilman, Witkowski and Zoller, Recombinant DNA, 1999)
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