1) The outermost protein of the retrovirus, the envelope, carries the primary determinant of viral tropism by virtue of its specific interaction with a cell surface receptor. We are studying the receptor for amphotropic murine leukemia virus (A-MLV) in order to increase our understanding of virus-receptor interactions and how they dictate the host range of a retrovirus. In addition, the A-MLV envelope is a component of the most commonly used retroviral vectors used in human gene therapy clinical trials. The receptors for A-MLV is a multiple membrane spanning phosphate supporters, and has been named Pit2, for this normal cellular function. We have used chimeric proteins followed by site-directed mutagenesis to analyze the structural role of the second extracellular domain of Pit2 in A-MLV receptor function. Most of the mutations made did not alter receptor function, suggesting that there is a lot of structural flexibility in this region which is tolerated. In contrast, a conservative amino acid substitution of serine to methionine in this region of a non-functional receptor or the converse substitution into a functional receptor reversed the ability of each to serve as an A-MLV receptor, suggesting a secondary structural determinant is involved in the virus receptor function of Pit2. 4) Phase I clinical trials involving the use of swine tissue have begun. However, pigs carry endogenous retroviruses called porcine endogenous retrovirus or PoEV. We have shown that activation of this normally latent retrovirus occurs in the course of mitogenic activation of pig primary blood mononuclear cells. The virus released from the activated cells is infectious for porcine and human cell lines. Preliminary molecular characterization of PoEV isolated from two breeds of pig, NIH mini pig and Yucatan pig demonstrate that, although similar to each other, they differ from a previously characterized pig retrovirus isolated from a pig kidney cell line, PK15, suggesting that there may be more than one locus in the pig genome capable of encoding infectious virus. These studies will help to elucidate the safety concerns regarding clinical issue of porcine cells, tissues, or organs and allow for development of diagnostic assays.
