Population Level Analysis of HIV1 Hypermutation and its Relationship with APOBEC3G

1
Population Level Analysis of HIV-1 Hypermutation
and its Relationship with APOBEC3G Vif Genetic
Variation
Craig Pace, Jean Keller, David Nolan, Ian James,
Silvana Gaudieri, Corey Moore, Simon Mallal
2
HIV-1 G?A Hypermutation

• Inordinate number of guanidine ? adenine
• substitutions in proviral DNA
• Mediated by the host-encoded cytidine
• deaminases APOBEC3G and APOBEC3F1,2
• Counteracted by viral-encoded vif 3

1Mangeat et al, Nature, 2003 2Zhang et al,
Nature, 2003 3Sheehy et al, Nat Med, 2003
3
APOBEC3G 3F target cytosines in specific
sequence contexts
minus strand
plus strand
TAGG
TGAG
TGGG
TAAG
GA
AA
Bishop et al, Curr Biol, 2003
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Hypermutation - WA HIV Cohort Study
AIM
To describe G?A hypermutation and its clinical
significance in a large clade B infected cohort
and the influence of APOBEC3G and vif genetic
variation
Patients

• 136 antiretroviral-naïve, male (88) Caucasian
  (84) pts
• near full-length clade B proviral sequences
  (mean 7,013 1,932 bp/pt)
• 10,626 G ? A substitutions

Measures of hypermutation

• G?A substitutions all other substitutions (HM
  score)
• G?A substitutions A?G substitutions
• G?A substitutions consensus G

5
12 patients (8.8) were considered outliers in
their hypermutation score
20
0.80
1
0.70
15
0.60
0.50
3
Frequency
10
5
0.40
8
10
12
0.30
5
0.20
0.10
0
0.10
0.20
0.30
0.40
0.50
0.60
0.70
0.80
Hypermutation score
Hypermutation score
Pace et al, Nat Immunol, in review
6
G?A substitutions in HM sequences occurred
preferentially in the GG context
GA
50.00
GC

GG
GT
40.00
30.00
Mean ()
20.00


10.00
0.00
Non-HM
HM
Denotes significant difference (Plt0.05) in
context frequency between HM and non-HM
sequences red lines indicate nucleotide
frequency in consensus sequence
Pace et al, Nat Immunol, in review
7
APOBEC3G, rather than APOBEC3F, was the dominant
contributor to clade B HIV-1 G?A hypermutation in
vivo
non-HM HM P
value APOBEC3G GG 21.9 (7.0) 43.5 (13.8)
lt0.001 TG 22.5 (5.2) 30.7 (8.8) 0.001 GnG 17.9
(5.0) 30.5 (10.0)
lt0.001 TGG 6.5 (3.9) 17.1 (7.0)
lt0.001 APOBEC3F GA 37.2 (8.3) 42.0
(14.9) 0.320
values expressed as mean (SD). P values
represent non-parametric P values.
Pace et al, Nat Immunol, in review
8
APOBEC3F-mediated G?A hypermutation was evident
in 2 (1.5) patients
100
Non-HM
HM
80
60
GA ()
40
20
0
0
10
20
30
40
50
60
GG ()
Pace et al, Nat Immunol, in review
9
Hypermutation in vivo was universally associated
with defective vif
Def vif absence of start codon and/or presence
of in-frame stop codon(s)
upstream of W174.
(TAGA)
ATG
HM
W21
M1
I (ATAG)
(TAGA)
(TAGT)
HM
W21
M29
W38
M8
M1
(TAGA)
ATG
(TAAG)
HM
W21
M1
W70
M29
I (ATAG)
(TAGG), (TAAG)
HM
M8
M1
W70
(TAGG)
ATG
HM
M1
W70
(TGAA)
ATG
HM
W89
M1
(TAGG)
ATG
N-HM
W174
M1
Ochsenbauer et al, J. Gen Virol. 1996 Wang et
al, Microbes Infect. 2005 Pace et al, Nat
Immunol, in review
10
Hypermutation in vivo was associated with
glutamic acid61
D61E (GAT/GAC ? GAA/GAG) R92K
(AGA/AGG ? AAA/AAG) R113N (GAC/GAT ?
AAC/AAT)
represents significant difference (Plt0.05) in
amino acid frequency between HM and non-HM
patients
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Contribution of APOBEC3G Genetic Variation to
Hypermutation
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Pace et al, Nat Immunol, in review
nl novel SNP - synonymous SNP -
non-synonymous SNP ND not determined
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APOBEC3G-mediated hypermutation was associated
with the APOBEC3G 6892 C allele
6,892 C T non-HM 69
(30) 161 3G-HM 11 (55) 9
P0.042
6,892 CC CT TT non-HM 9 (8)
51 55 3G-HM 3 (30) 5 2
P0.056
Pace et al, Nat Immunol, in review
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but was not associated with the APOBEC3G 625
allele
625 C T non-HM
178 (57) 52 3G-HM 15 (75) 5
P0.784
625 CC CT TT non-HM
66 (57) 46 3 3G-HM 6
(60) 3 1
P1.00
Pace et al, Nat Immunol, in review
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Hypermutation was associated with significant
reductions in plasma viremia
P0.001
P0.057
7.00
6.00
logvl
5.00
4.00
3.00
1
2.00
-VE
VE
-VE
VE
-VE
VE
-VE
VE
-VE
VE
HM
HLA_B57
HLA_B27
HLA_B58
CCR5_32
Pace et al, Nat Immunol, in review
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Conclusions

• APOBEC3G and -3F-mediated G?A hypermutation was
  evident in 7.4 and 1.5 of patients,
  respectively.
• HM was associated with significant reductions in
  viral load (0.7 log10 copies/mL), independent of
  CD4 count and HLA-B and CCR5 alleles.
• HM was universally associated with defective vif
  and statistically associated with vif glutamic
  acid61 and the APOBEC3G 6892 C allele.

Future Analysis

• Analysis of mixed bases - do Rs (A and G) occur
  preferentially in an APOBEC3G or -3F context?

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Acknowledgements

• Co-authors Jean Keller, David Nolan, Ian James,
  Silvana Gaudieri, Corey Moore Simon
  Mallal
• WA HIV Cohort participants
• DCIBG CCIBS staff - HLA CCR5 data
• - viral load data
• - full-length HIV
  sequencing data

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Allele frequencies of 10-15 can be detected by
sequencing pooled DNA
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