17th International HIV Drug Resistance Workshop 10-14 June 2008, Sitges, Spain

DEFINING THE STRUCTURAL AND FUNCTIONAL ROLES OF MUTATIONS IN GP120 ASSOCIATED WITH THE EMERGENCE OF HIV-1 CLINICAL RESISTANCE TO THE CCR5 ANTAGONIST VICRIVIROC

Antivir Ther. 2008; 13(Suppl. 3):A36 (abstract no. 34)

JA Howe, N Murgolo, Y Hou, L Ba, P Buontempo, J Strizki and R Ogert
Department of Biological Research – Virology, Schering-Plough Research Institute, Kenilworth, NJ, USA

BACKGROUND: Previous studies using heterologous chimeric envelopes showed that a C2-V5 domain from a vicriviroc (VCV)-resistant laboratory-adapted HIV-1 strain was sufficient to transfer resistance to a susceptible clone [1]. Here we used the same strategy to analyze the determinants of resistance in HIV env genes isolated from two patients that exhibited viral rebound in a VCV clinical trial.

METHODS: C2-V5 env domains obtained at baseline and at study discontinuation were cloned from two patient isolates into an ADA gp160 expression vector. Point mutations identified in the resistant sequence were reverted to baseline by site-directed mutagenesis. Single-cycle pseudovirus assays were used for VCV susceptibility studies and to measure relative viral infectivity. The baseline and resistant C2-V5 domains were also incorporated into a structural homology model in which gp120 is bound to an N-terminal binding domain of CCR5[2].

RESULTS: The HIV-1 env gene from one patient (number 91) contained six amino acid changes in the V3 loop and one change in the C4 region. The resistant clone from a second patient (number 8) contained a single change in the V3 loop and two changes in the C4 region. The chimera from patient 91 was completely resistant to VCV, whereas the clone from patient 8 displayed partial susceptibility. Mutation of residues F317L and G321R in the tip and the stem of the V3 loop region from patient 91 VCV-resistant env restored partial and complete susceptibility to VCV, respectively; whereas mutation of V3 loop residues N320D and K328E and the C4 amino acid change R429G significantly reduced pseudovirus infectivity but did not alter the resistant phenotype. Structural modelling results suggest that specific mutations identified in the V3 loop and C4 regions of the resistant clones may influence gp120 binding to the second extracellular loop or N-terminal domains of CCR5.

CONCLUSIONS: While no consistent pattern of resistance mutations to VCV has been identified, results from the two patients’ isolates suggest that mutations in the V3 loop and C4 region can be important determinants of the resistant phenotype. We identified mutations in the stem and tip region of the V3 loop that were associated with resistance level and other likely compensatory changes in the V3 stem, base and C4 region that affected viral infectivity. It will be necessary to analyze a larger number of clinical isolates in order to understand more fully the genotypic and phenotypic characteristics of VCV susceptibility.

REFERENCES:

1. Ogert RA, Wojcik L, Buontempo C, et al. Mapping resistance to the CCR5 co-receptor antagonist vicriviroc using heterologous chimeric HIV-1 envelope genes reveals key determinants in the C2-V5 domain of gp120. Virology. 2008 Apr 10;373(2):387-99.

2. Huang CC, Lam SN, Acharya P, et al. Structures of the CCR5 N terminus and of a tyrosine-sulfated antibody with HIV-1 gp120 and CD4. Science. 2007 Sep 28;317(5846):1930-4 doi: 10.1126/science.1145373.

2008-06-10
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