Intersubtype differences in the effect of a rare p24 Gag mutation on viral replicative

fitness

Denis Chopera

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.

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]

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

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

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

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

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

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

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

Effect of M250I mutation on p24 helix-6

-M250I destabilizes capsid helix 6 in subtype B viruses

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

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

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