Upload
lamcong
View
216
Download
0
Embed Size (px)
Citation preview
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,
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
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 2
4
RNA
5
Spontaneous
Programmed
Cytosine Uraci l
Cytosine Deamination
6
Contents
(2) Nucleic acid cytosine deaminases
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 3
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
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 4
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)
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 5
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
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 6
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
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 7
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
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 8
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
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 9
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
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 10
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
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 11
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
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 12
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
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 13
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
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 14
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
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 15
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
)
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 16
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
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 17
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
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 18
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
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 19
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