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“What limit can be put to this power
(natural selection), acting during long
ages and rigidly scrutinising the
whole constitution, structure, and
habits of each creature,-favouring the
good and rejecting the bad.”
Charles Darwin Origin of the Species
Natural Selection
• All species produce more offspring than can possibly survive
and reproduce.
• Organisms differ in their ability to survive and reproduce,
in part due to differences in genotype.
• In every generation, those genotypes that promote survival
in the current environment are present in excess at the
reproductive age and therefore contribute disproportionately
to the offspring of the next generation.
• For natural selection to operate there must be heritable
variation for traits that are correlated with reproductive success.
Average Rates of Nucleotide Substitution
in Different Organisms
Organism/Genome
Plant chloroplast DNA
Mammalian nuclear DNA
Plant nuclear DNA
E. coli and Salmonella enterica bacteria
Drosophila nuclear DNA
Mammalian mitochondrial DNA
HIV-1
Substitution Rate
(per site, per year)
~ 1 x 10-9
3.5 x 10-9
~ 5 x 10-9
~5 x 10-9
1.5 x 10-8
5.7 x 10-8
6.6 x 10-3
The Degeneracy of the Genetic Code
Proline (4 synonymous codons)
CCT
CCC
CCA
CCG
Histidine (2 synonymous codons)
CAT
CAC
CAA - Gln
CAG - Gln
Measuring Selection Pressures in Genes
• Selection pressures can be measured by comparing
the relative rates (ratio) of synonymous (silent) (dS) and
nonsynonymous (amino acid changing) (dN) substitutions:
Seq 1:
Seq 2:
Ser
TCA
†
TCG
Ser
Met
ATG
*
ATA
Ile
Leu
TTA
†
CTA
Leu
Gly
GGG
†
GGT
Gly
Gly
GGA
**
ATA
Ile
† Synonymous substitution
* Nonsynonymous substitution
dN/dS < 1.0 = negative selection (functional constraint - most genes)
dN/dS ~ 1.0 = neutral evolution (pseudogenes)
dN/dS > 1.0 = positive selection
Genetic Variability of HIV
• HIV exhibits extremely high levels of genetic variation: the virus
evolves about 1 million times faster than human DNA (HIV-1 =
~10-3 subs/site/year, human DNA = 10-9 subs/site/year) .
• This is mainly due to:
– Extremely high mutation rate, particularly because the
replication enzyme reverse transcriptase that converts RNA to
DNA is very error prone
– High turnover of virus within infected individual ~1010 viruses
produced in each patient each day
– Rapid generation time (~2.6 days)
Phylogeny of global HIV-1
isolates + 197 strains
from the Congo
Genetic diversity at transmission
• Early analyses concentrated on the env gene
and revealed a low level of diversity in
seroconverters
• Ho D., Science, Leigh-Brown A., J. Virol
• So the infection was thought to be
established by a small “founder” set of
clones
• But that work did produce evidence of
greater diversity in gag, nef and pol than
env
Four Sexual Transmitters
Subtype A
D1
1.31
R1
D2
Subtype B
Gag p24
Neighbour-Joining
Tree
1.28
R2
D3
1.05
R3
D4
Donor
Recipient
Subtype
Reference sequences
1.0
Percent Divergence
1.02
R4
P32-Ia-M24
P32-Ia-M08
P32-Ia-J05
P32-Ia-M19
P32-Ia-M14
P32-Ia-M09
P32-Ia-J16
P32-Ia-J17
P32-Ia-M04
P32-Ia-J03
P32-Ia-M23
P32-Ia-M18
P32-Ia-M16
P32-Ia-M11
P32-Ia-M07
P32-Ia-M05
P32-Ia-M21
P32-Ia-M22
P32-Ia-M06
P32-Ia-M20
P32-Ia-M13
P32-Ia-J04
P32-Ia-J06
P32-Ia-M12
P32-Ia-M10
P32-Ia-M25
P32-Ia-J02
P32-Ia-J11
P32-Ia-J10
P32-Ia-J08
P32-Ia-J07
P32-Ia-J09
P32-Ia-J12
P32-Ia-J14
P32-Ia-M17
P32-Ia-M01
P32-Ia-M02
S32-I-A04
S32-Ia-AP11
S32-Ia-AP10
S32-Ia-AP06
S32-Ia-AP04
S32-I-AP12
S32-I-AP11
S32-I-AP07
S32-I-AP03
S32-I-AP01
S32-Ia-O10
S32-Ia-O05
S32-Ia-O02
S32-I-O11
S32-I-O10
S32-I-O08
S32-I-O07
S32-I-O06
S32-I-O05
S32-I-O04
S32-I-O03
S32-I-O01
S32-I-A18
S32-I-A17
S32-I-A15
S32-I-A14
S32-I-A13
S32-I-A12
S32-I-A11
S32-I-A08
S32-I-A06
S32-I-A05
S32-I-AP02
S32-I-AP06
S32-Ia-O07
S32-Ia-O12
S32-I-O02
S32-I-O09
S32-Ia-O01
S32-Ia-O04
S32-Ia-O03
S32-I-AP04
S32-Ia-AP12
S32-I-AP10
S32-I-AP08
S32-I-AP05
S32-I-AP09
S32-Ia-AP09
S32-Ia-AP08
S32-Ia-AP07
S32-Ia-AP03
S32-Ia-AP02
S32-Ia-AP05
0.01
p17-a-018.p47
p17-a-020.p47
p17-a-010.p47
p17-a-019.p47
p17-a-012.p47
p17-a-011.p47
p17-a-022.p47
p17-a-015.p47
p17-a-008.p47
p17-a-048.p47
p17-a-047.p47
p17-a-046.p47
p17-a-045.p47
p17-a-044.p47
p17-a-043.p47
p17-a-042.p47
p17-a-040.p47
p17-a-039.p47
p17-a-037.p47
p17-a-036.p47
p17-a-035.p47
p17-a-033.p47
p17-a-032.p47
p17-a-028.p47
p17-a-025.p47
p17-a-026.p47
p17-a-041.p47
p17-a-038.p47
p17-a-034.p47
p17-a-031.p47
p17-a-030.p47
p17-a-029.p47
p17-a-027.p47
p17-a-001.p47
p17-a-024.p47
p17-a-023.p47
p17-a-021.p47
p17-a-017.p47
p17-a-016.p47
p17-a-013.p47
p17-a-009.p47
p17-a-007.p47
p17-a-006.p47
p17-a-005.p47
p17-a-004.p47
p17-a-002.p47
p17-a-003.p47
p17-a-014.p47
DONOR
RECIPIENT
UNRELATED
SEROCONVERTER
Maximum Likelihood
phylogenetic tree
Prevalence of polymorphisms in HIV-1 protease and
RT at seroconversion and chronic infection
Proportion of polymorphisms
0.6
>1 year
Seroconversion
0.4
0.2
0
1
26
51
76
RT2
RT2 7
RT5 2
RT7 7
RT1 02
RT1 2 7
RT1 5 2
-0.2
-0.4
-0.6
Amino Acid (HIV-1 protease and RT)
RT1 7 7
RT2 02
RT2 2 7
Evading Cytotoxic T-Lymphocyte
Recognition May Enhance Viral
Fitness
Evading Cytotoxic T-Lymphocyte
Recognition May Enhance Viral
Fitness
Selection of escape mutants
A
replication
A1-A101
SELECTION
PRESSURE
e.g. A6
Some lead to increased
fitness - survive
e.g. A12
Some mutations lead to
reduced fitness - die off
Continued selection of escape
mutants
A6
replication
SELECTION
PRESSURE
A6.1-A6.101
e.g.A6.4
e.g. A6.9
Some lead to increased
Some mutations lead to reduced
fitness - survival
fitness - extinction
“Extinction has only separated
groups: it has by no means made
them: for if every form which has
ever been were suddenly to
reappear…all would blend together
by steps as fine as those between the
finest existing varieties”
Charles Darwin Origin of the Species
Viral Load
CTL Response
Do CTL Select for Escape Variants
During Acute Infection?
Weeks
Years
Rev
Tat
Vpx Tat
Gag
Vpr
Pol
700
1400
2100
Rev
Vif
2800
3500
4200
4900
5600
6300
Nef
Env
7000
7700
8400
9100
9800
10500
5 CTL Epitopes Accumulate
Variation at Different Rates During
Acute SIV Infection
Consensus
QGQYMNTPW
ARRHRILDIYL
IRFPKTFGW
GDYKLVEI
KRQQELLRL
Inoculum
.........
...........
.........
........
.........
.........
...........
T.Y......
........
.........
...H.....
...........
T.Y......
........
.........
.........
E..........
T.Y......
........
.........
......N..
E.......T..
T.Y..I...
........
.........
......N..
E.......M.F
T.Y..I...
........
.........
......N..
E.......K..
T.Y..I...
........
.........
....I.N..
E.......K..
..Y..I...
.....I.V
...H.....
......N..
E.......K..
T.Y..I...
.....I.V
...H.....
Epitope Variants Reduce CTL
Recognition
G
are
Qraphics
uickTim
needed
decom
e™
toand
see
pressor
athis picture.
100
Peptide bindingtoclassI
CTLrecognition
60
40
20
IRFPKTFGW
GDYKLVEI
QGQYMNTPW
KRQQELLRL
ARRHRILDIYL
0
Wild-type
%WildType
80
SIV Nef variation (yellow blocks)
clusters within epitopes defined by
MHC
Class
I
(green
blocks)
G
are
Qraphics
uickTim
needed
decom
e™
toand
see
pressor
athis picture.
C
G
are
Qraphics
uickTim
needed
decom
e™
toand
see
pressor
athis picture.
D
G
are
Qraphics
uickTim
needed
decom
e™
toand
see
pressor
athis picture.
A
Summary
• Antiretrovirus T lymphocytes recognise peptide
antigens dictated by, and bound to MHC Class I
molecules
• Genetic variants of the virus can alter or even
abolish immune recognition of infected target cells
• Viruses bearing these sequences have a survival
advantage
• Escape mutants grow out (at different rates) and
are positively selected
26 year old man admitted with aseptic
meningitis
•
•
•
•
•
•
Had unprotected anal sex on 20 Sept.1995
HIV Elisa negative on 10 October 1995
P24 gag antigen positive
Viral load >10 millions copies per ml
CTL assay 19 October 1995
Single nef response
HLA B8
NEF EPITOPE
FLKEKGGL
Intra-Host Evolution of HIV-1
SIHIGPGRAFYTTGE
SIPIGPGRAFYTTGQ
SIHIGPGGAFYTTGQ
SIHIGPGRAFYTTGD
SIPIGPGRAFYTTGD
GIHIGPGSAFYATGD
SIHIGPGRAFYTTGG
SIHIGPGRAVYTTGQ
GIHIGPGSAFYATGG
GIHIGPGRAVYTTEQ
RIHIGPGRAVYTTEQ
GIHIGPGSAFYATGR
RIYIGPGRAVYTTEQ
GIHIGPGSAVYATGG
RIYIGPGSAVYTTEQ
GIHIGPGSAFYATGG
RIGIGPGRSVYTAEQ
GIHIGPGSAVYATGD
GIHIGPGRAFYATGD
GIHIGPGRAVYTTGD
RIYIGPGRAVYTTDQ
Tip of the V3 loop (part of the
envelope protein of HIV-1)
- diversity in a single patient
• The HIV-1 envelope protein is under
very strong positive selection to help
the virus escape from the human
immune response (the V3 loop contains
epitopes for neutralising antibodies and
cytotoxic T-lymphocytes (CTLs).
• V3 loop dN/dS = 13.182
(Nielsen & Yang. Genetics 148, 929. 1998).
Does transmission of CTL escape viruses
impact on the global epidemic?
Questions
• Child can inherit a HLA Class I molecule which
has dictated a response in the mother
• The maternal immune response can select escape
mutants
Are these immune escape viruses detectable in the
mother?
Can the mother transmit these viruses?
Do these escape viruses propagate infection in the
child?
Father
Mother
Child
Father
Mother
B27
Child
B27
Father
B27
Mother
B27
Child
KK10 lies within a conserved region of p24 Gag
100%
50%
40%
20%
10%
5%
2%
1.3%
P I V Q N L Q G Q MV H Q A I S P R T L N A WV K V V E E K A F S P E V I P M F S A L S E G A T P Q D L N T M L N T V G G H Q A A M Q M L K E T I N EEAAEWDRLHPVHAGPIAPGQMREPRGSDIAGTTSTLQEQIGWMTNNPPIPVGEIYKRWIILGLNKIVRMYSPTSILDIKQGPKEPFRDYVDRFYKTLRAEQASQEVKNWMTETLLVQNANPDCKTILKALGPAATLEEMMTACQGVGGPGHKARVL
1
4567
11 14
25 2729
41 44
48
53
71
79
83 86
90 92
96 98
107 110 114
120 123
128
148
154
159
KRWIILGLNK
167 170
177
183 187
194
199
208
213 216 219 223
230
Structure of N terminal domain of p24:
Clustered mutations within B27 epitope,
necessary for escape
helix 7
Amino acid changes in the antigenic peptide
at the second position prevent binding to
HLA B27 molecules
100
% Maximum
80
KRWIILGLNK variants
60
KRWIILGLNK=R2L6
M
=R2M6
K M
=K2M6
K
=K2L6
T M
=T2M6
40
20
0
-9
-8
-7
-6
-5
peptide concentration (log molar)
-4
The HLA B27 Gag peptide is not recognized in
children of HLA B27-positive mothers
WHY?
2000
1925
1600
IFN-g
SFC/ 1200
million 800
PBMC
400
0
0
0
0
043-C
002-C 048-C 049-C
B27-ve mother B27+ve mothers
The HLA B27 bearing MOTHERS acquired an escape
virus and transmitted it to the CHILDREN
No P2 anchor
mutation
2000
1600
IFN-g
SFC/ 1200
million 800
PBMC
400
0
P2 anchor
mutation
shared with
mother
0
0
0
043-C
002-C 048-C 049-C
B27-ve mother B27+ve mothers
Father
Mother
B27
B27
RWIILGLNK
B27
- K- - -M- -
HIV IN PERTH, AUSTRALIA
Hypotheses
If CTL-driven selection operates on HIV-1 in populations
then selected mutations should:
• be within or close to CTL epitopes
• because of genetic restriction of immune responses,
mutations should be linked to particular HLA variants
• viral escape mutations would be evident as HLAassociated polymorphisms.
HLA alleles significantly associated
with HIV-RT polymorphisms
within published epitopes
putative epitopes
flanking residues
=Significant after correction
Moore et al (2002) Science 296, 1439
Natural selection by AIDS
Who wins the race?
• HIV-1 10 subs/site/year
-3
• Human genome (eg HLA)
-9
10 subs/site/year
“Nothing in biology makes sense,
except in the light of evolution”
Theodosius Dobzhansky 1973
Acknowledgments
•
•
•
•
•
•
•
•
•
•
Eddie Holmes
Paul Klenerman
David Watkins
Angela McLean
Simon Mallal
David O’Connor
David Price
Annette Oxenius
Andrew McMichael
Charles Bangham
•
•
•
•
•
•
•
•
Philippa Easterbrook
Jonathan Weber
Sarah Fidler
George Scullard
Anele Waters
Anne Edwards
Philip Goulder
The patients of The Chelsea and
Westminster Hospital; St Mary’s
Hospital; KwaZulu Natal