Structural and Functional Analysis of HIV-1 Coreceptors: Roles of Charged Residues and Posttranslational Modifications on Coreceptor Activity
Abstract
CXCR4 and CCR5 are chemokine receptors and are coreceptors for human immunodeficiency virus (HIV-1). Host cells must express CD4 and a coreceptor for optimal HIV-1 entry. The delineation of the critical regions involved in the interactions within the Env-CD4-coreceptor complex has been under intensive investigation. To define these regions we have employed an alanine-scanning mutagenesis strategy of the extracellular domains of CXCR4 coupled with a highly sensitive reporter-gene assay for HIV-1 Env-mediated membrane fusion. Using a panel of 47 different CXCR4 mutations, we have identified several charged residues that appear important for coreceptor activity for X4 Envs: mutations E15A and E32A in the N-terminus, D97A in extracellular loop (ecl)-1, and R188A in ecl-2 impaired coreceptor activity for X4 and R5X4 Envs. Mutation to alanine of one of the six tyrosines present in CXCR4, Y7, decreased coreceptor function. In addition, alanine substitution of any of the four extracellular cysteines alone resulted in conformational changes of varying degrees, while paired cysteine deletions could partially retain structure. Our data supports the notion that all four cysteines are involved in disulfide bond formation. We have also identified substitutions which greatly enhance or convert CXCR4 s coreceptor activity to support R5 Env-mediated fusion: N11A, R30A, D187A, and D193A. Mutation of the aspartic acid at position 187 had the greatest effect on tropism. Mutation of an analogous serine in CCR5 to aspartic acid reduced CCR5 coreceptor activity with R5X4 and some R5 isolates. Mutation of the N11, a putative glycosylation site, had the second greatest effect on CXCR4 tropism. We determined that this site has a large carbohydrate moiety that is responsible for preventing CXCR4 from supporting R5 Env-mediated membrane fusion. Our data suggests the presence of conserv
Document Details
- Document Type
- Technical Report
- Publication Date
- Jan 01, 2000
- Accession Number
- ADA421435
Entities
People
- Donald J. Chabot
Organizations
- Uniformed Services University of the Health Sciences