CD4 and CD8 Molecules of Non-human Primates; Molecular Cloning and Their Roles in SIV Infection

为了阐明SIV与猴的CD4和CD8分子之间的相互作用，我们尝试了分子克隆并将其与人类的克隆进行了比较。用Gubler-Hoffmann方法从食蟹猴胸腺细胞中合成的c DNA片段插入到lambdaZAP噬菌体载体系统中，构建食蟹猴胸腺细胞c DNA文库，并与聚合酶链式反应扩增的猴CD 4和CD 8基因片段进行空斑杂交。结果表明，猴CD4基因编码区由1377个核苷酸组成，与人CD4基因具有较高的同源性(95%)。氨基酸序列比对表明，在信号肽和跨膜区都只有一个氨基酸被取代，而在细胞质中没有差异。氨基酸的取代集中在胞外Ig样V1结构域的CDR-1区域，这表明该区域可能的构象变化对HIV结合具有重要意义。我们用猴、人及其嵌合的CD4基因构建了哺乳动物表达载体，并建立了在细胞表面表达各自CD分子的Hela转化子。用合体形成法检测细胞对HIV和SIV的敏感性，用聚合酶链式反应检测前病毒。HIV不仅可以感染表达人V1区的转化子，还可以感染猴V1区，提示猴CD8分子可能是SIV和HIV的细胞受体。猴CD8基因编码区由708个残基组成，与人CD8有很高的同源性(95%)。推导的氨基酸序列比对表明，该替换均匀地分布在整个编码区，与猴子的CD4不同。以猴CD8基因为模板，通过体外翻译的方法获得了预期的26 KDa分子。

To elucidate the interaction between SIV and monkey CD4 and CD8 molecules, we attempted molecular cloning and to compare those with human counterparts. Cynomolgus monkey thymocyte cDNA library was constructed in a lambdaZAP phage vector system by inserting cDNA synthesized from thymocyte mRNA with Gubler-Hoffmann method, and screened by plaque hybridization with PCR-generated putative monkey CD4 and CD8 gene fragments. The coding region of monkey CD4 gene was revealed to consist of 1377 nucleoside acids and show high homology (95%) to human CD4 gene. Alignments of deduced amino acid sequences revealed that only one amino acid was substituted in both signal peptide and transmembrane domains, and that there was no difference in its cytoplasmic domain. The substitution of amino acids was concentrated on CDR-1 region of extracytoplasmic Ig-like V1 domain, suggesting the possible conformational change in this region reported to be important for HIV binding. We constructed mammalian expression vectors with monkey, human and their chimeric CD4 genes and established Hela transformants expressing respective CD molecules on the cell surface. The susceptibility of these cells to HIV and SIV was examined by synthcium formation and by detection of provirus with PCR method. HIV could infect not only transformant expressing human V1 domain but also monkey V1 domain suggesting that monkey CD4 molecule might serve as the cellular receptor to both SIV and HIV.The coding region of monkey CD8 gene was revealed to consist of 708 residues and also show high homology (95%) to human CD8. Alignments of deduced amino acid sequences displayed that the substitution was distributed evenly in the entire coding region, differing from monkey CD4. Expected 26KDa molecule was produced by in vitro translation method with monkey CD8 gene.