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CXCR4 antagonist-induced coreceptor switch from X4 to R5 phenotype in vitro determined by a single amino acid substitution in the V3 region of human immunodeficiency virus type 1 gp120. Maeda Y, Yusa K, Harada S. Kumamoto University, Graduate School of Medical Sciences,
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CXCR4 antagonist-induced coreceptor switchfrom X4 to R5 phenotype in vitrodetermined by a single amino acid substitution in the V3 region of human immunodeficiency virus type 1 gp120 Maeda Y, Yusa K, Harada S. Kumamoto University, Graduate School of Medical Sciences, Department of Medical Virology, Kumamoto, Japan
Coreceptor switch of HIV-1 in vivo • CCR5-using viruses (called R5 viruses) are associated with transmission between human, and are predominant throughout the course of infection. • CXCR4-using viruses emerge at late stage of infection, and are associated with accelerated disease progression.
Coreceptor switch of HIV-1 in vitro • The coreceptor switch has not been reproduced in vitro under selective pressure by coreceptor inhibitors although the target cells express both coreceptors.
Characteristics of R5X4 viruses • Most of CXCR4-using viruses retain to use CCR5 called R5X4 viruses. • Replication of R5X4 virusesis blocked by CXCR4 inhibitors alone in the cells expressing both CCR5 and CXCR4.
Sensitivity of various strains of HIV-1 to coreceptor inhibitors Numbers represent mean EC50 values (nM) of TAK-779 and T140 in indicated cells
In vitro study of coreceptor switch • Cell: PM1/CCR5 • Virus: 89.6 strain (R5X4 variant) • Inhibitor: T140 (CXCR4 antagonist)
Sensitivity of T140-escape mutant passaged in PM1/CCR5 determined by MAGI/CCR5
V3 region amino acid sequences from 89.6-infected cells passaged and selected in T140 wild 89.6 CTRPNNNTRRRLSIGPGRAFYARRNIIGDIRQAHC T140(-) ................................... 9/9 T140(+) ..........S........................ 8/12 ................................... 3/12 ........................S.......... 1/12 308
SU TM Afl II Nhe I β-actin promoter Rabbit –β-globin poly A V1 V2 V3 V4 V5 308 89.6AN wild CTRPNNNTRRRLSIGPGRAFYARRNIIGDIRQAHC 89.6AN R308S ..........S........................ Construction of 89.6 envelope expression vector carrying Afl II and Nhe I Produce luciferase-reporter HIV with Envs Check sensitivity to coreceptor inhibitors
The substitution R308S conferred total resistance to T140 in NP2/CD4/CCR5/CXCR4 cells
The substitution R308S conferred cross-resistance to AMD3100in NP2/CD4/CCR5/CXCR4 cells
The substitution R308S conferred increased sensitivity to TAK-779in NP2/CD4/CCR5/CXCR4 cells
Coreceptor usage of various Envs in NP2/CD4 cell line expressing single coreceptor
The sensitivities of various Envs to coreceptor inhibitors in single coreceptor expressing cells Numbers represent mean EC50 values (nM) of coreceptor inhibitors in indicated cells. NA: not applicable
Conclusion (I) • A single amino acid substitution (R308S) in the 89.6 Env conferred total resistance to T140 and AMD3100 but increased sensitivity to TAK-779 in the infection of cells expressing both coreceptors.
Conclusion (II) • The mutation R308S was correlated with increased affinity of the Env for CCR5. • The 89.6 strain carrying R308S is likely to be reverted from X4 to intermediate or transitional phenotype close to R5 viruses.
Conclusion (III) • The use of R5X4 viruses is important to induce coreceptor switch in vitro.