innate immunity to retroelements by human aid/apobec3 ... · apobec expression construct transform...

Innate Immunity to Retroelements

by Human AID/APOBEC3 Proteins

Prof. Reuben Harris

The screen versions of these slides have full details of copyright and acknowledgements 1

1Searle Scholars Program

Innate Immunity to Retroelementsby Human AID/APOBEC3 Proteins

IMV

Prof. Reuben HarrisUniversity of Minnesota,

[email protected]

2

Contents

(1) Nucleic acid basics

(2) Nucleic acid cytosine deaminases

(3) Retrovirus restriction (e.g., HIV, MLV)

(4) Endogenous retroelement restriction (e.g., Ty1)

(5) Non-LTR retroelement restriction (e.g., L1)

(6) A unifying hypothesis

3

Contents

(1) Nucleic acid basics



Prof. Reuben Harris


4

RNA

5

Spontaneous

Programmed

Cytosine Uraci l

Cytosine Deamination

6

Contents

(2) Nucleic acid cytosine deaminases



Prof. Reuben Harris


7

Human AID/APOBEC Family Members

HXE - X24-28 - PCX2-4C

Zn2+

A1, AID, A3C * ca. 200

A3F/G * * 373/384

8

Cartoon

of AID & APOBEC

Family

Members in Vertebrates

Harris & Liddament, 2004, Nat.Rev.Immunol. ;Koonin & colleagues 2005, Cell Cycle A4

9

Are All AID/APOBEC Family Members

DNA Mutators?

Vector

orAPOBECExpression

Construct

Transform E. coli

Amp+

Pick individual colonies

Grow overnight

Rif+

Amp+

Viable cell count

RifR colonies

Mutation frequency& local preferences

Evidence from an E. coli-based mutation assay



Prof. Reuben Harris


10

Effects of Expressing Some APOBECs

in E. coliMutation Frequency

(UDG+)1

8

18

20

530

10

120

120

260

2600

Mutation Frequency(UDG-)

Harris et al. , 2002, Mol Cell

80

11

Hottest Spots for Deamination

in E. coli ApoB

APOBEC1 G T C T CC T C C G

AID

APOBEC3C G T C T CC G C A C

APOBEC3GC C C T G

C G C A CT A C G A

5’ -2-1 m +1 +2 3’

5’G G G A C

APOB mRNA editing& ???

Function

Ab diversification

? ? ?

? ? ?

12

Contents

(3) Retrovirus restriction (e.g., HIV, MLV)



Prof. Reuben Harris


13

Human Cells Contain

an Anti-Retroviral Factor

HIVPermissive

Non-permissive

∆VIF

APOBEC3G

Sheehy et al. , 2002, Nature

14

Model for HIV Restriction by APOBEC3G

Harris et al.,2003, Cell

Genomic (+) RNA

Genomic (+) RNA

RTase (1-st strand cDNA synthesis)

1-st strand (-) DNA

RNAse H

APOBEC3G

15

A GFP-Based Assay

for Retroviral Infectivity

Enter

GRNA

DNA C

A3GDNA U

UDNA A

G/C → A/THypermutation

Deaminate

Integrate

TDNA A

LTR LTRGFP+ Helper Plasmids (Gag/Pol/Env) +/- A3G or Control

Bud

APOBEC3G

Assemble



Prof. Reuben Harris


16

APOBEC3G Inhibits Retroviral Infection

17

Retroviral Restriction by APOBEC3G

and -3F

Liddament et al., 2004, Curr Biol;

18

Strand-Specific Retroviral Hypermutation

5’ G A (5’ TC)A3F

472

A3G 5’ G G (5’ CC)

e.g., Harris et al., 2003, Cell; Mangeat et al., 2003, Nature; Zhang et al., 2003, Nature;Wiegand et al., 2004, EMBO J; Liddament et al., 2004, Curr Biol



Prof. Reuben Harris


19

Cumulative Effect of A3F- and A3G-

Dependent Retroviral Hypermutations

APOBEC3G

Retroviral

Hypermutationin vivo

APOBEC3F

+

Janini et al., (2001) J Virol 75, 7973; Li et al., (1991) J Virol 65, 3973

20

APOBEC3F and APOBEC3G Are Broadly (Co-)Expressed

Liddament et al.,2004 Curr Biol

21

HIV Vif Counteracts A3G > A3F



Prof. Reuben Harris


22

Mechanism of APOBEC3G Inhibition

by HIV-1 Vif

Yu, Gabuzda and other groupsHarris & Liddament (2004) Nat.Rev.Immunol

23

Model for Retroelement Restriction

Cullen, Hance, Harris, Landau, Malim, Trono & Others (2003-present)

24

Summary 3

APOBEC3F & -3G are potent retroviral restrictorswith distinct dinucleotide deamination preferences

� Are tandemly encoded with strong DNA level similarities (coding & non-coding)

� Co-expressed broadly

� Interact by immunoprecipitation (Wiegand et al. , 2004, EMBO J.)

� Have differential Vif resistances

Account for patient-derived hypermutation patterns



Prof. Reuben Harris


25

Contents

(4) Endogenous retroelement restriction (e.g., Ty1)

26

Human APOBEC Family Members

*

A3F/G * * 373/384

C -> U deamination

27

Alternative APOBEC3 Functions?

Harris & Liddament, 2004,Nat.Rev.Immunol.

Vif+/-

Vif+/-

Vif -

Genomic com

plexity (“junk”)

Positive

selection



Prof. Reuben Harris


28

Might Some APOBECs Function

to Restrict the Mobility of Endogenous Retroelements?

LTR GAG POL ENV LTR

“ACCESSORIES” (e.g., VIF)

LTR GAG POL LTR

LTR retrotransposons: e.g., HERV, copia, Ty1

Retroviruses: e.g., HIV-1, MuLV

29

Can APOBEC3 Inhibit Yeast Ty1?

APOBEC3G

Ty1

A. Schumacher, UMND. Nissley, NCI, Frederick

30

Ty1 Retrotransposition Assay

Curcio et al., 1991, Nissley et al. , 1998



Prof. Reuben Harris


31

APOBEC3G and APOBEC3F

Inhibit Ty1 Retrotransposition

HIS3

Schumacher et al. , 2005, PNAS

32

APOBEC3G & -3F Restrict Ty1 by a cDNA

Cytosine Deaminase Mechanism

HIS3

To To

A3G A3FFrom From

33

Human APOBEC3 Proteins Inhibit Ty1

and a Variety of Other Retroelements

Dutko et al., 2005, Curr Biol (Ty)Esnault et al. , 2005, Nature (MusD/IAP)Schumacher et al. , 2005, PNAS (Ty)Other studies since …

APOBEC3G

Ty1



Prof. Reuben Harris


34

Summary 4:

MacDuff & Harris, 2006, Curr Biol

Human A3 proteins constitute a powerful innate defense that protects mammals from LTR-type retroelements

35

Contents

(5) Non-LTR retroelement restriction (e.g., L1)

36

Human APOBEC Family Members

*

* *

C -> U deamination



Prof. Reuben Harris


37

Differential Localization of Human A3s

Live HeLa cellsScale bar by GFP cell is 10 µµµµ mStenglein & Harris, 2006, JBC

A3B-GFP

+/-αααα -HIV -

A3DE-GFP

αααα -HIV

++

A3F-GFP

αααα -HIV +++

A3G-GFP

αααα -HIV -

GFP

αααα -HIV

38Adapted from Bannert and Kurth, 2004, PNAS and Ostertag and Kazazian, 2001, Ann Rev Genet

HERV (~9.2 kb)

Endogenous Transposable Elements (45%)

DNA Transposons (3%)e.g., Sleeping Beauty

‘Cut and Paste’ Transposition

Endogenous Retroelements (42%)‘Copy and Paste’ Transposition

RNA intermediate

Non-LTR Retrotransposons(34%)

e.g., LINEs (L1, 17%)e.g., SINEs (Alu, 11%)

LTR Retrotransposons(8%)

e.g., HERVs

Retroviruses(Exogenous

Retroelements)

39cytoplasm

nucleoplasm

LTR REe.g., Ty1

L1

HIV

Replication Strategies

of L1 & LTR-Retroelements



Prof. Reuben Harris


40

LINEs Replicate via Target-Primed Reverse Transcription (TPRT)

in the Nucleus

| | | | | | | | | A A A | | | | | |

Genomic integration site

ORF2

(EN/RT)

LINE mRNA

ORF1

41

L1s Destabilize Genomes & Cause Disease

• Gene insertion

• Inter (intra) chromosomal recombination

• Trans-mobi lization

Ostertag and Kazazian, 2001, Ann Rev Genet

42

L1 Transposition Assay

Ostertag et al. , 2000, NARImage from Turelli et al. , 2004, JBC



Prof. Reuben Harris


43

293-APO-HA

Can A3B Inhibit L1 Retrotransposition?

Monitor L1 transposition

by flow cytometry

Stenglein & Harris, 2006, JBC

Transfect

Select with puromycin

44

A3B & A3F Inhibit L1 Retrotransposition

0

5

10

15

20

25

30

0 3 6 9 12 15 18 21

Days Post Transfection

VEC

A3DA3G

A3F

A3B

GFP P

osi

tive

Cells

(%

)

45

A3B & A3F Inhibit L1 Retrotransposition

0.0

0.4

0.8

1.2

1.6

Vector A3B A3D A3F A3G

Ret

rotr

ansp

osit

ion

Rat

e (

% /

day

)



Prof. Reuben Harris


46

HIV Restriction by HA-Tagged Constructs

0.0

0.2

0.4

0.6

0.8

1.0

1.2

Vector A3B A3D A3F A3G

Rela

tive

HIV

Rest

ricti

on

47

A3B Deaminase Activity* Is Not Required

for L1 Retrotransposition

0

5

10

15

20

25

30

0 3 6 9 12 15 18 21

Days Post Transfection

GF

P Po

siti

ve C

ells

(%

)

VEC

A3B*A3B

48

Less Retrotransposed L1 DNA

Accumulates in the Presence of A3B & A3F

intron

360 bp

Non-specific

L1-GFP



Prof. Reuben Harris


49

Model for how A3F & A3B inhibit L1

transposition?

cytoplasmnucleoplasm

LINE-1TPRT

A3G

A3DA3F

A3B

50

Residues 65-190 of A3F & A3B

Are 96% Identical (120/125)

51

A3F Appears Strongly Expressed

in Human Testeswww.proteinatlas.org



Prof. Reuben Harris


52Adapted from Bannert and Kurth, 2004, PNAS and Ostertag and Kazazian, 2001, Ann Rev Genet

Endogenous Transposable Elements (45%)

DNA Transposons (3%)e.g., Sleeping Beauty

‘Cut and Paste’ Transposition

Endogenous Retroelements (42%)‘Copy and Paste’ Transposition

RNA intermediate

Non-LTR Retrotransposons(34%)L1, Alu

LTR Retrotransposons

(8%))

e.g., HERVs

(Ty1, IAP, MusD)

Retroviruses(Exogenous

Retroelements)(HIV, SIV, MLV, EIAV,

FIV, FV, HTLV, etc.)

HERV (~9.2 kb)

A3

53

Summary 5

� The human double DNA cytosine deaminase domain

proteins locate to the cytoplasm (A3D,F,G)

or the nucleoplasm (A3B)

� A3F and A3B, despite locating to different

subcellular compartments,

inhibit LINE-1 retrotransposition

(likely SINEs such as Alu elements also)

� A3F appears highly expressed in the human testes

54

Contents

(6) A unifying hypothesis



Prof. Reuben Harris


55

(I) APOBEC3 proteins, and other family members such as AID, are cellular sentinels that function in a programmed manner to inhibit retroelement replication

(I) A3 expression can restrict many different retroelements(II) Strong positive selection(III) A3’s & AID shared a common AID-like ancestor(IV) Retroelements are nearly ubiquitous

Some AID proteins may still possess retroelement restriction abilities, whereas A3’s likely lost the ability to diversify antibody genes

Hypothesis:

Key Prediction:

(II) These proteins can also be recruited for more specific roles (e.g., apoB mRNA editing by APOBEC1, Ab gene diversificat ion by AID, ???)

Some Evidence:

56

Current Lab MembersDonna MacDuff*

Natalia MartemyanovaApril Schumacher*Dr. Keisuke Shindo

Mark Stenglein*Jon Strong

Yoftahe AbebeJohn Albin

Dr. William Brown*Guylaine HachéStefán JónssonRebecca LaRue

Dr. Michael Neuberger*,

MRC Lab of Molecular Biology, Cambridge, U.K.

Drs. Michael Malim*, Kate Bishop* & colleaguesKings College London, U.K.

Dr. Vala AndresdottirUniversity of Iceland

Dr. Dwight Nissley*, NCI, Frederick, MD

Past Lab MembersAmy Briggs

Jennie DoeblerDr. Mark Liddament*

Eeson Rajendra

Searle Scholars Program

IMV

57

innate immunity to retroelements by human aid/apobec3 ... · apobec expression construct transform...

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