Sundstrom Magnus, White Rebecca L, de Parseval Aymeric, Sastry K Jagannadha, Morris Garrett, Grant Chris K, Elder John H
Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.
J Virol. 2008 Sep;82(18):9134-42. doi: 10.1128/JVI.00394-08. Epub 2008 Jul 2.
Feline immunodeficiency virus (FIV) shares with T-cell tropic strains of human immunodeficiency virus type 1 (HIV-1) the use of the chemokine receptor CXCR4 for cellular entry. In order to map the interaction of the FIV envelope surface unit (SU) with CXCR4, full-length FIV SU-Fc as well as constructs with deletions of extended loop L2, V3, V4, or V5 were produced in stable CHO cell lines. Binding studies were performed using these proteins on 3201 cells (CXCR4(hi) CD134(-)), with or without the CXCR4 inhibitor AMD3100. The findings established that SU binding to CXCR4 specifically requires the V3 region of SU. Synthetic peptides spanning the V3 region as well as a panel of monoclonal antibodies (MAbs) to SU were used to further map the site of CXCR4 interaction. Both the SU V3-specific antibodies and the full-length V3 peptide potently blocked binding of SU to CXCR4 and virus entry. By using a set of nested peptides overlapping a region of SU specifically recognized by CD134-dependent neutralizing V3 MAbs, we showed that the neutralizing epitope and the region required for CXCR4 binding are within the same contiguous nine-amino-acid sequence of V3. Site-directed mutagenesis was used to reveal that serine 393 and tryptophan 394 at the predicted tip of V3 are required to facilitate entry into the target cell via CXCR4. Although the amino acid sequences are not identical between FIV and HIV, the ability of FIV to bind and utilize both feline and human CXCR4 makes the feline model an attractive venue for development of broad-based entry antagonists.
猫免疫缺陷病毒（FIV）与1型人类免疫缺陷病毒（HIV-1）的T细胞嗜性毒株一样，利用趋化因子受体CXCR4进入细胞。为了绘制FIV包膜表面单位（SU）与CXCR4的相互作用图谱，在稳定的CHO细胞系中制备了全长FIV SU-Fc以及缺失延伸环L2、V3、V4或V5的构建体。使用这些蛋白在3201细胞（CXCR4高表达、CD134阴性）上进行结合研究，有无CXCR4抑制剂AMD3100均可。研究结果表明，SU与CXCR4的结合特别需要SU的V3区域。使用跨越V3区域的合成肽以及一组针对SU的单克隆抗体（MAb）进一步绘制CXCR4相互作用位点。SU V3特异性抗体和全长V3肽均能有效阻断SU与CXCR4的结合以及病毒进入。通过使用一组与CD134依赖性中和V3 MAb特异性识别的SU区域重叠的嵌套肽，我们表明中和表位和CXCR4结合所需区域在V3的同一连续九氨基酸序列内。定点诱变用于揭示V3预测顶端的丝氨酸393和色氨酸394是通过CXCR4促进进入靶细胞所必需的。尽管FIV和HIV之间的氨基酸序列不同，但FIV结合并利用猫和人类CXCR4的能力使猫模型成为开发广泛的进入拮抗剂的有吸引力的场所。
