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)


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• 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)




Retroviral infection process



(Flint et al., P

rinciples of Virology, 2000)




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*



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


• 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)


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)


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(S

luis-Crem

er, Arion and P

arniak, 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

N

N

NH2

O

O

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(F

lint 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(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)


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 Z

oller, Recom

binant DN

A, 1999)

THE END

reverse transcriptase: reversing the dogma ariel (אָרּיּאּלּ) grostern seminar for the guelph...

Documents

role of rt slide

structure of rt

antirt drugs rt

structure of unliganded

biology slide

p51 slide

antirt drugs resistance

dnadependent dna polymerase