Intersubtype differences in the effect of a rare p24 Gag mutation on viral replicative fitness
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Intersubtype differences in the effect of a rare p24 Gag mutation on viral replicative
fitness
Denis Chopera
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Background• Certain immune-driven mutations that occur in
conserved regions of the HIV-1proteome often affect viral replication [Brockman et. al., 2010; Crawford et. al., 2007; Miura et. al., 2009]
• Rare mutations occurring in p24 Gag were identified in elite controllers in subtype B infections and these have a severe fitness cost to viral replication [Miura et. al., 2009]
• The extent to which these mutations affect viral fitness in other HIV-1 subtypes has not been explored.
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Study Objectives• This study examined the impact of a rare Gag
mutation, M250I, on subtype B and C viral replicative fitness
• Rationale: -M250I is one of the mutations identified in elite controllers in subtype B [Miura et. al., 2009]-The mutation is negatively associated with a common HLA-B*57 escape mutation, T242N [Martinez- Picado et. al., 2006;
Chopera et. al., 2011]
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Elite Controllers Chronic0.0
0.2
0.4
0.6
0.8
1.0
5 of 69 5 of 803(7.2%) (0.6%)p=0.0005
Freq
uenc
y
B58_ST non B58_ST B58_ST non B58_ST0.0
0.2
0.4
0.6
0.8
1.0250M250I
Elite Controllers Chronic5 of 26 0 of 43 3 of 83 2 of 710(19.2%) (0%) (3.6%) (0.026%)p=0.0059 p=0.0095
Freq
uenc
y
Distribution of M250I by cohort and HLA in subtype B
-M250I associated with HLA-B58 supertype and is enriched in elite controllers in subtype B infections
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M250I+ (N=5) M250I- (n=788)0.4
0.6
0.8
1.0
1.2
1.4
1.6p=0.06
Gag
-Pro
teas
ere
plic
atio
n ca
paci
ty
2 3 4 51
2
4
8
16
32
64 W T (NL4-3)M250IT242N
Days Post Infection
Fol
d-in
crea
se in
infe
cted
cel
ls
M250I and subtype B replication
-M250I impairs viral replication in vitro
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Acute Chronic0.0
0.2
0.4
0.6
0.8
1.0M250I250
8 of 53(15%)p=ns
41of 405(11%)
Freq
uenc
y
Distribution of M250I by cohort in subtype C
-M250I more common in subtype C viruses
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M250I+ (N=41) M250I- (N=364)0.2
0.4
0.6
0.8
1.0
p=0.95
Gag
-Pro
teas
ere
plic
atio
n ca
paci
ty
2 3 4 51
2
4
8
16
32
64 W TM250IT242N
Days Post Infection
Fol
d-in
crea
se in
infe
cted
cel
ls
A B
Subtype C replication capacity
-Impairs viral replication in site-directed mutants but not in patient-derived isolates
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Why is the M250I mutation more common in subtype C infections
and why is it not associated with a fitness cost in plasma-derived
viruses?????Secondary mutations have been shown to fully or
partially restore viral fitness incurred by CTL escape mutations [Brockman et. al., 2007; Crawford et. al., 2007; Schneidewind et. al., 2007]
We applied phylogentically-corrected methods to detect amino acids that co-vary with the M250I mutation
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Codon 250 Covarying codon Association TT TF FT FF Total p-value q-value250M 252G Negative 55 317 34 7 413 7.4E-09 8.0E-06250M 260D Positive 264 108 4 37 413 1.9E-08 8.0E-06250M 252S Positive 220 153 2 38 413 2.7E-07 7.0E-05250M 247M Negative 0 372 4 36 412 2.5E-05 5.0E-03250M 256V Positive 192 180 3 38 413 3.3E-05 5.0E-03250M 207D Positive 38 334 0 41 413 2.2E-04 3.0E-02250M 260E Negative 107 265 37 4 413 7.5E-04 8.0E-02250I 252G Positive 33 6 55 318 412 1.4E-08 8.0E-06250I 260D Negative 4 36 264 110 414 1.4E-07 4.0E-05250I 252S Negative 2 37 220 154 413 5.4E-07 1.0E-04250I 256V Negative 3 37 192 181 413 5.0E-05 7.0E-03250I 247M Positive 4 35 0 374 413 5.9E-05 8.0E-03250I 207D Negative 0 39 38 335 412 3.1E-04 3.0E-02250I 260E Positive 36 4 109 265 414 8.2E-04 8.0E-02
TT - Number of sequences with both codon 250 residue and covarying residueTF - Number of sequences with codon 250 residue but without covarying residueFT - Number of sequences without codon 250 residue but with covarying residueFF - Number of sequences with neither codon 250 residue nor covarying residue
Covariation between codon 250 and other Gag sites in the chronic subtype C cohort
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2 4 6 8 10 120
20
40
60
80
100
120WT
T242N
M250I
IGE
NIGE
Subtype C site-directed mutants
Days postinfection
Fold
incr
ease
in v
iral l
oad
0 1 20.2
0.4
0.6
0.8
p=ns
Subtype C patient-derived recombinant viruses
Number of compensatory mutationsG
ag-P
rote
ase
repl
icat
ion
capa
city
WT
T242N
M250I
G248A
/M25
0I
T242N
/M25
0I0.4
0.5
0.6
0.7
0.8
0.9
1.0
1.1
Subtype B site-directed mutants
Slo
pe o
f vira
l spr
ead
M250I compensation in subtype C and B viruses
-There is compensation of M250I mutation in subtype C but not subtype B viruses
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Effect of M250I mutation on p24 helix-6
-M250I destabilizes capsid helix 6 in subtype B viruses
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Summary• In subtype B infections, the M250I mutation is
very rare and enriched in elite controllers expressing HLA-B58 supertype
• The mutation is associated with a reduction in viral replication in subtype B viruses
• M250I is not associated with a fitness cost in patient-derived (subtype C) isolates
• In subtype C, the associated mutations, S252G and D260E partially restore viral replication
• M250I destabilizes helix 6 of the capsid and this effect is more pronounced in subtype B
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Conclusion
• Understanding the selection mechanism for the M250I mutation may be useful for HIV vaccine immunogen design
• Vaccine designing strategies need to take into consideration the inter-subtype differences in the specific mutations selected by under immune pressure and their relative impact on viral fitness
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Acknowledgements
Dr. Zabrina BrummeDr. Mark BrockmanEric MartinLaura Cotton
Dr. Carolyn WilliamsonDr. Clive GrayDr. Zenda WoodmanDr. Darren MartinDr. Nobubelo NganduDr. Roman Ntale
Dr. Salim Abdool-KarimDr. Koleka MlisanaCAPRISA 002 Study Team
Dr. Jonathan Carlson
Dr. Thumbi Ndung’uDr. Jaclyn Mann (Wright)
Dr. Richard Harrigan
Dr. Bruce WalkerDr. Toshiyuki MiuraDr. Florencia Pereyra