HIV can modulate Gag NC/p1 cleavage and thereby affect PI susceptibility and viral replicative capacity Monique Nijhuis
HIV-1 protease • Essential for the production of infectious HIV-1 particles • Responsible for the ordered cleavage of two precursor proteins encoding structural proteins and viral enzymes
HIV-1 protease: ordered gag cleavage
HIV-1 protease inhibitor resistance Two protease inhibitor resistance mechanisms: 1. Mutations in the viral protease itself Reduce affinity of the viral protease for the inhibitor 2. Mutations in the substrate of the viral protease (NC/p1 CS) Enhanced substrate cleavage
Gag NC/p1 substrate based PI resistance Gag Gag p1 p1 NC MA CA p6 p17 p17 p24 p24 p7 p7 TFP PR RT INT p2 p2 Pol Pol NC p1 NC p1 428 429 430 431 432 433 434 435 436 437 E R Q A N F L G K I 431V - - - V - - - - - - - - - - - - - - - V 437V - - - - - - - - - T 437T - - - - - - - - E T 436E+437T Nijhuis et al, PLOS Medicine 2007
Increased NC/p1 cleavage of mutant Conc. RO033-4649 (nM) NC NC p6 p6 NC NC increase in overall Gag cleavage (CA/MA-antibody)
Phenotypic susceptibility of NC/p1 mutants - - why don’t we see these NC/p1 CS changes that often? Nijhuis et al, PLOS Medicine 2007
Effect of NC/p1 changes on viral replicative capacity 10000000 1000000 HXB2 100000 pg p24/ml 431V 437V 437T 10000 436E+437T 1000 100 0 1 2 3 4 5 6 7 9 10 13 Days - reduced replication capacity be repaired?
Three evolutionary pathways for the double NC/p1 mutant NC p1 433 438 428 429 430 431 432 434 435 436 437 W E R Q A N F L G K I HXB2 GAG AGA CAG GCT AAT TTT TTA GGG AAG ATC TGG - - - - - - - - - E T HXB2 436E+437T --- --- --- --- --- --- --- --- --- G-- -C- - - - - - - - - - - T Pathway 1 -C- --- --- --- --- --- --- --- --- --- --- - - - - - - - - R E T --- Pathway 2 --- --- --- --- --- --- --- A-- G-- -C-- R - - - - - - - - E T Pathway 3 --- --- --- --- --- --- --- G-- -C- A-- ---
Effect of NC/p1 changes on viral replicative capacity 10000000 1000000 HXB2 100000 pg p24/ml 436E+437T -436E +435R 10000 +438R 1000 100 0 1 2 3 4 5 6 7 8 9 11 12 13 Days
Effect of evolution on Gag cleavage analyzed by quantitative immunoblotting CAPSID MATRIX Ratio p17/sum of p17reactive 90 60 Ratio p24/sum of p24 80 50 reactive products 70 HXB2 HXB2 60 40 products 436E + 437T 436E + 437T 50 -436E 30 -436E 40 + 438R + 438R 20 30 + 435R + 435R 20 10 10 0 0 50 nM 100 nM 50 nM 100 nM Concentration RO033-4649 Concentration RO033-4649
Effect of observed evolution on PI susceptibility
Modulation of Gag cleavage in relation to PI resistance Viral optimum Hyper Resistant Susceptible? Replicative Capacity Gag cleavage efficiency
Acknowledgements University Medical Center Utrecht, The Netherlands Noortje van Maarseveen, Pauline Schipper, Dorien de Jong Charles Boucher European Commission Grant QKL 2-CT-2001-02360 Grant LSHP-CT-2007-037693
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