RETROVIRUSES AND THEIR RECEPTORS

涉及使用猪组织的第一阶段临床试验已经开始。然而，猪携带内源性逆转录病毒，称为猪内源性逆转录病毒或PERV。我们先前已经证明，这种正常潜伏的逆转录病毒在猪外周血单个核细胞(PBMC)暴露于有丝分裂物质后激活。从激活的细胞中释放的病毒对猪和人类细胞系具有传染性。我们现在已经对该病毒分离株的外膜进行了克隆和测序，发现它是A类外膜，以前被定义为人类嗜性外膜。此外，我们还调查了一些人类来源的细胞和细胞系，以确定病毒的趋向性。重要的是，我们发现在所利用的体外条件下，人PBMC对PERV感染具有抵抗力。这一结果对制定指南以监测猪异种移植受者是否有PERV感染的证据具有重要意义。我们还研究了一系列非人类细胞系，作为开发允许感染的合理动物模型的第一步。我们发现，水貂和猫科动物来源的细胞系支持有效的病毒感染，而来自其他被研究物种的细胞系不支持。最后，我们已经开始将我们为研究PERV而开发的方法应用于猪血浆衍生产品的分析。在对猪第VIII因子(商标为Hyate C)的研究中，我们能够在最终产物中检测到PERV序列和PERV逆转录酶活性。然而，传染性研究没有提供任何证据表明存在传染性病毒。因此，CBER能够就接受者面临的风险做出明智的决定，并允许Hyate C继续留在市场上。逆转录病毒最外层的蛋白，即包膜，通过与细胞表面受体的特异性相互作用，携带病毒嗜性的主要决定因素。我们正在研究目前在人类基因治疗临床试验中用作逆转录病毒载体的C型逆转录病毒作为模型。这些研究将增加我们对病毒-受体相互作用以及它们如何决定逆转录病毒宿主范围的理解。今年，我们与伊拉·伯克沃尔合作，从两性小鼠白血病病毒中分离出重组包膜糖蛋白。这种试剂将被用来开发一种结合分析，以阐明与病毒附着和病毒进入有关的受体区域。此外，我们与Maribeth Eiden合作研究了一种长臂猿白血病病毒株，这种病毒不能感染中国仓鼠细胞系，即使细胞表达这种病毒的功能受体。这一发现表明，C型逆转录病毒可能还需要一个“辅助受体”才能进入细胞，这可能是逆转录病毒进入的一个统一特征。

I. Phase I clinical trials involving the use of swine tissues have begun. However, pigs carry endogenous retroviruses, called porcine endogenous retrovirus or PERV. We have previously shown that activation of this normally latent retrovirus occurs after exposure of pig primary peripheral blood mononuclear cells (PBMC) to mitogenic agents. The virus released from the activated cells is infectious for porcine and human cell lines. We have now cloned and sequenced the envelope of this virus isolate and found that is a class A envelope, previously defined as a human-tropic envelope. In addition, we surveyed a number of human-derived cells and cell lines to determine the tropism of the virus. Importantly, we found that human PBMC were resistant to infection by PERV under the in vitro conditions utilized. This result has implications for developing guidelines to monitor recipients of porcine xenografts for evidence of PERV infection. We also examined a range of non-human cell lines as an initial step developing a rational animal model that is permissive to infection. We found that mink and feline-derived cell lines support productive viral infection, while cell lines derived from other species examined did not. Finally, we have begun applying our methods developed to study PERV towards analysis of porcine plasma-derived products. In a study of porcine factor VIII, trade name Hyate C, we were able to detect PERV sequences and PERV reverse transcriptase activity in the final product. However, infectivity studies did not provide any evidence for presence of infectious virus. Hence, CBER was able to make an informed decision regarding the risk to recipients, and allowed Hyate C to remain on the market. II. The outermost protein of the retrovirus, the envelope, carries the primary determinant of viral tropism through its specific interaction with a cell surface receptor. We are studying type C retroviruses that are currently used as retroviral vectors in human gene therapy clinical trials as models. These studies will increase our understanding of virus-receptor interactions and how they dictate the host range of a retrovirus. This year, in collaboration with Ira Berkower, we were able to isolate recombinant envelope glycoprotein from amphotropic murine leukemia virus. This reagent will be used to develop a binding assay to elucidate regions of the receptor involved in virus attachment vs. viral entry. In addition, we collaborated with Maribeth Eiden to study a strain of gibbon ape leukemia virus that cannot infect a Chinese hamster cell line, even though the cells express functional receptors for this virus. This finding suggests that type C retroviruses may also require a "coreceptor" for entry into cells, which may be a unifying feature of retroviral entry.