Partners AIDS Research Center Massachusetts General Hospital HLA-B27-Associated Escape Mutations Dramatically Reduce Viral Fitness of HIV-1 Arne Schneidewind Partners AIDS Research Center Massachusetts General Hospital Boston

Background • HLA-B27 is a “protective” MHC class I allele in HIV infection - Genetic effect observed late in disease (Gao et al, Nat Med., 2005) - immunodominant CD8 response targets KK10 in p24 capsid - CTL escape mutations in KK10 occur very late in disease • Studies have illustrated that drug resistance mutations and CD8 escape mutations can impact viral replication Hypothesis The effect of CD8 escape mutations on viral fitness may contribute to immune control associated with HLA-B27

Goals • Examine the impact of CD8 escape mutations in B27- KK10 (KRWIILGLNK) on HIV-1 replicative fitness • Assess the ability of compensatory mutations to rescue the fitness defects of a CD8 escape mutation • Explore mechanisms for the late escape from this immunodominant CD8 T cell response

Viral Escape in B27-KK10 • Various CD8 escape and compensatory mutations in KK10 have been previously described : Goulder et al Nature Med 1997; Kelleher et al JEM 2001; Goulder et al Nature 2001; Feeney et al JVI 2004; Ammaranond et al AIDS Res Hum Retroviruses 2005 Upstream mutation 90 aa

Viral Escape in B27-KK10 • Various CD8 escape and compensatory mutations in KK10 have been previously described : Goulder et al Nature Med 1997; Kelleher et al JEM 2001; Goulder et al Nature 2001; Feeney et al JVI 2004; Ammaranond et al AIDS Res Hum Retroviruses 2005 Upstream mutation 90 aa • L268M occurs early after infection and is a weak CD8 escape mutation

Viral Escape in B27-KK10 • Various CD8 escape and compensatory mutations in KK10 have been previously described : Goulder et al Nature Med 1997; Kelleher et al JEM 2001; Goulder et al Nature 2001; Feeney et al JVI 2004; Ammaranond et al AIDS Res Hum Retroviruses 2005 Upstream mutation 90 aa • Mutation R264K at the anchor position P2 diminishes binding to HLA-B27 and represents the predominant CD8 escape mutation in KK10

Viral Escape in B27-KK10 • Various CD8 escape and compensatory mutations in KK10 have been previously described : Goulder et al Nature Med 1997; Kelleher et al JEM 2001; Goulder et al Nature 2001; Feeney et al JVI 2004; Ammaranond et al AIDS Res Hum Retroviruses 2005 Upstream mutation 90 aa • An upstream compensatory mutation (S173A) has been proposed - Kelleher / Brander (manuscript in preparation)

Compensatory Mutation lies in Proximity to Escape Mutations N-term p24 capsid

Compensatory Mutation lies in Proximity to Escape Mutations N-term p24 capsid R264K L268M

Compensatory Mutation lies in Proximity to Escape Mutations N-term p24 capsid S173A

Escape Mutation R264K Reduces Infectivity - 12% - 96% Replace with powerpoint title Xhrs post infection Assay to measure p24

Escape Mutation R264K Reduces Replicative Capacity can we split this into 2 figures? First with WT, LM, RK, SA 2nd adding in RKLM, SARKLM Let’s talk about the insert too... Need to highlight SA reduction too

Escape Mutation R264K Reduces Replicative Capacity can we split this into 2 figures? First with WT, LM, RK, SA 2nd adding in RKLM, SARKLM Let’s talk about the insert too... Need to highlight SA reduction too

Escape Mutation R264K Reduces Replicative Capacity can we split this into 2 figures? First with WT, LM, RK, SA 2nd adding in RKLM, SARKLM Let’s talk about the insert too... Need to highlight SA reduction too

Escape Mutation R264K Reduces Replicative Capacity can we split this into 2 figures? First with WT, LM, RK, SA 2nd adding in RKLM, SARKLM Let’s talk about the insert too... Need to highlight SA reduction too

Reduces p24 Production on PBMCs Escape Mutation R264K Reduces p24 Production on PBMCs

Escape Mutation R264K doesn’t alter Release of p24

Escape Mutation R264K doesn’t alter Cleavage of Gag WT RK SARKLM WT RK SARKLM p24  p55  p24  supernatant intracellular

Conclusions CD8 escape mutation R264K exhibits a dramatic defect in infectivity and replicative capacity Upstream mutation S173A in helix 2 effectively compensates for the fitness defect of R264K • The dramatic fitness cost of R264K, and the requirement for a simultaneous compensatory mutation, may explain the late escape from this CD8 response R264K variant seems to be blocked at an early step of the replicative cycle Add in the ability of other upstream compensatory mutations to rescue other KK10 mutations (data not shown)

This project was supported by NIH Grant R21 AI067078 Acknowledgment Mark A. Brockman Sylvie Le Gall Rahma I. Adam Matthew P. Lahaie Christian Brander Tony Kelleher Bruce D. Walker Todd M. Allen This project was supported by NIH Grant R21 AI067078