reverse transcriptase: reversing the dogma

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)


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




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)



Retroviral infection process



(Flint et al., Principles of Virology, 2000)




RNA -> DNA: the role of RT


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


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


• 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


• Domains: – Fingers– Palm– Thumb– Connection– RNAse H*

*in p66 only, not p51


Active sites: DNA polymerase

- located at the 6- 10- 9 sheet of palm subdomain

p66

p55


Active sites:DNA polymerase

- YMDD motif (all polymerases)

- catalytic residues: D110*, D185*, D186

* Bind Mg2+

(Coffin, H

ughes and Varm

us, Retroviruses, 1997)


Active sites:DNA polymerase

Fancy version

(Huang et al., 1998)

Finger

palm

thumb


Active sites: RNase H

- Located in RNase H domain

p66

p55


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)


dNTP binding: Fingers subdomain bends to

trap incoming dNTP


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


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


Structures of Nucleoside Analogs

Inhibition of RT: anti-RT drugs(Sluis-C

remer, A

rion and Parniak, 2000)

Mode of Action of Nucleoside Analogs


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


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


p51

p66nevirapine

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)


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


Resistance to RT DrugsNucleoside analog resistance:

Pyrophospholytic cleavage(Sluis-C

remer, A



Non-nucleoside analog resistance mechanism:

• Mutations alter hydrophobic pocket

– esp. single exchange at position 181 in p66

RT: its other role in biologyRT-PCR

(Watson, G

ilman, W

itkowski and Zoller, R

ecombinant D

NA

, 1999)

THE END

reverse transcriptase: reversing the dogma

Documents

dnadependent dna polymerase

rna dna

a1992 structure of rt

a1991 structure of rt

structure of unliganded

role of rtstructurefunction

rnadependent dna polymerase

antirt drugsrt