Questions asked:

• How Nef accomplishes the balance between the conflicting selective forces during a course of HIV-1 infection.

• Whether CTL responses can impose functional constraints in Nef.

a pathogenic factor

a dominant target by CTLs

Nef protein

database(n=443)

HLA-B35 negative(n=41)

HLA-B35 positive(n=28)

p<0.001

RPQVPLRPMTYRPQVPLRPMTFTPQVPLRPMTYothers

Autologous virus sequences in nefThis region elicits CTL

responses restricted by HLA-B35.

Interestingly, the TF double mutant is very rare.

T and F mutations are associated with HLA-B35+ patients.

early phase(n=14)

chronic phase(n=14)

RPQVPLRPMTF

TPQVPLRPMTY

The combination of TF mutations may impose functional constraints in

Nef

TF

Fwild

VY8-CTLs

RY11-CTLs

The TF mutations are rarely selected in combination

naturally.

T

The TF double mutant can escape from both

CTLs.

Fitness cost??

Primary CD4+ T cells were infected with the following viruses:

uninfected RPQVPLRPMTY Nef

HL

A-I

RPQVPLRPMTF TPQVPLRPMTY TPQVPLRPMTF

CD4

36.1 14.5 13.2 16.8 26.3 48.4

uninfected RPQVPLRPMTY Nef

HL

A-I

RPQVPLRPMTF TPQVPLRPMTY TPQVPLRPMTF

CD4

uninfected RPQVPLRPMTY Nef

HL

A-I

RPQVPLRPMTF TPQVPLRPMTY TPQVPLRPMTF

CD4

36.1 14.5 13.2 16.8 26.3 48.4

0 20 40 60 80 100 120 140 160 0 20 40 60 80 100 120

uninfected

ｗｔ , RPQVPLRPMTY

RPQVPLRPMTF

TPQVPLRPMTY

TPQVPLRPMTF

Nef

HLA class I CD4n=4 n=4

****

** ** p<0.001

The mutant Nefs are impaired in HLA class I down-regulation activity.

Gated in live p24 Ag+ subsets

0 3 6 9 12 15

0

50

100

150

200

250

RPQVPLRPMTY, wtRPQVPLRPMTFTPQVPLRPMTYTPQVPLRPMTF Nef

p24

Ag

(n

g/m

l)

Days post infection

HIV-1 replication kinetics in PBMC in vitro

The double mutant Nef is impaired in enhancement of virus replication

The TF double mutant decreased Nef’s activity in enhancement of viral replication, suggesting the selective disadvantage of the variant virus in vivo.

Day 0

3

infection to fresh PBMC

PHA activation

6

9

12 ....

collection for p24 ELISA

Conclusions

• A natural variability in a well-conserved PxxP region in Nef is associated with CTL responses.

• CTL escape could have severe consequences on the functionality of Nef, both for its role in immune evasion and intrinsic replicative potential of the virus.

• These results highlight CTL immunosurveillance as important modulators of Nef’s biological activity in the infected host during a course of HIV infection.