Studies on the host factors essential for HIV-1life cycle to develop novel anti-HIV drugs

人类免疫缺陷病毒1型（HIV-1）的复制依赖于宿主细胞因子，但只有有限数量的这种蛋白质被报道。通过表达克隆方法，我们建立并分析了293T细胞衍生的两个突变体细胞克隆C611和YD4，它们对HIV-1感染具有抗性，但允许小鼠白血病病毒（MLV）感染。定量聚合酶链反应（qPCR）研究表明，C611细胞在逆转录完成之前存在进入后阻断，而YD4细胞病毒DNA进入细胞核，但未能整合到细胞基因组中。这些基因块的遗传表型似乎是隐性的，因为突变体和亲本细胞之间的杂交恢复了对HIV-1感染的易感性。值得注意的是，这些缺陷没有已知的辅助因子补充，因此这些突变细胞有望用于鉴定HIV-1感染的新细胞辅助因子。除此之外，我们做了不同类型的实验，发现了3个额外的宿主因子，对HIV-1感染有积极或消极的作用。它们包括Arp2/3复合体，该复合体似乎对灵长类慢病毒和牛苗IMV感染的早期阶段很重要，以及两个阴性调节因子naf1和HEXIM1。对于CXCR4拮抗剂，我们已经通过体外研究完成了候选药物的筛选和选择，并采用小鼠模型进行了动物研究。其中，KRH-3955被认为是目前最好的，整个故事将被报道。

Replication of human immunodeficiency virus type 1 (HIV-1) depends on host cell factors, but only a limited number of such proteins have been reported. Through the use of expression cloning method, we established and analyzed two mutant human cell clones derived from 293T cells, C611 and YD4, which are resistant to HIV-1 infection, but permissive to murine leukemia virus (MLV) infection. Quantitative polymerase chain reaction (qPCR) studies revealed that C611 cells had a post-entry block prior to the completion of reverse transcription, whereas in YD4 cells viral DNA entered the nucleus, but failed to be integrated in the cellular genome. The genetic phenotype of these blocks appeared to be recessive, because hybridization between the mutant and parental cells restored the susceptibility to HIV-1 infection. Of note, the defects were not complemented by known cofactors, and hence these mutant cells are expected to be useful for identifying new cellular cofactors of HIV-1 infection.Beside this, we made different types of experiments and found 3 additional host factors, acting either positively or negatively for HIV-1 infection. They include the Arp2/3 complex which appears to be important for early step of primate lentivirus and vaccinia IMV infection, and two negative regulators,NAF-1 and HEXIM1.As to CXCR4 antagonists, we have completed the screening and selection of the candidates through in vitro studies and they have been subjected to animal studies using mice model. Amongst them, KRH-3955 is considered best at this moment and the whole story will be reported.