Our laboratory studies gammaretroviruses. These retroviruses are the simpler cousins of the human pathogen human immunodeficiency virus - they are less complex in their genetic structure and replication cycle. Study of gammaretroviruses is important to the regulation of biologics for several reasons: they have been used to create replication-defective vectors for gene therapy clinical trials, they are potential contaminants in several biological products, and they are likely to be present in xenotransplantation products, with the potential for transmission to recipients of these products. Our research has focused on the study of porcine endogenous retroviruses (PERV), an inherited genetic element found in the genome of all pigs. We have found that infectious virus can be isolated from pig plasma, as well as from activated pig blood cells. We have developed cell culture assays for detection of PERV, as well as sensitive and quantitative methods for detection of viral nucleic acids in cell culture, and animal and human tissue samples. Recently, we have been analyzing the viral and cellular determinants of infectivity in order to understand better the conditions where viral replication are likely to occur. More recently, our expertise with retroviruses has been extended to use the retroviral vector technology to study the determinants of entry on the envelope glycoprotein of Ebola virus. Gammaretroviruses in Biologics. Clinical trials using porcine tissues or cells are underway. Since all pigs carry in their genome porcine endogenous retroviruses (PERV), these xenotransplantation procedures carry the risk of exposure to and transmission of PERV. This year, we analyzed the functional elements of the long terminal repeats (LTR) of PERV. The LTRs are found at each end of the viral DNA and carry the cis-acting transcriptional regulatory elements. We were able to functionally identify several structural elements in the PERV LTR in this study: 1) the number of enhancer repeats influenced expression level in human 293 cells, but not other cell lines examined; 2) deletion of the putative "ATAA" box abrogates all expression activity; and 3) an element found in the 3' half of R and U5 suppresses transcriptional activity. In addition, we completed a study aimed toward evaluating the utility of the guinea pig as an animal model to study PERV replication in vivo. We exposed guinea pigs to PERV under three different conditions: 1) intra-peritoneal injection of cell-free or cell-associated PERV; 2) pre-treatment with allyl alcohol to induce hepatocyte proliferation; and 3) pre-treatment with Complete Freund's Adjuvant-complexed PERV. In all cases, we observed only limited evidence for viral infection which remained either at low levels for the duration of the experiment, or was transient. These results demonstrate that the guinea pig will not provide a suitable model to study PERV. Identification of Critical Amino Acid Residues on Filovirus Glycoproteins Required for Cell Entry. The viral protein responsible for initiating viral infection of filoviruses has been shown to be the glycoprotein (GP). We are using alanine-scanning mutagenesis to introduce mutations in cDNA encoding the envelope glycoprotein, GP1, for both the Marburg and Ebola strains of filovirus. Retroviral pseudotypes bearing control or mutated filovirus glycoproteins (GPs) are derived by transient transfection of 293T cells to study the effect of each mutation on viral tropism and entry. The goal of this study is to identify an epitope on the filovirus GP that is necessary to facilitate viral entry. Identification of such a domain may then allow for subsequent rational design of agents that may inhibit filovirus infection. Analysis to date has identified one region on Ebola virus GP that appears to be critical for infectivity, since all mutations introduced thus far in this domain render virus non-infectious. This project incorporates FY2002 projects 1Z01BM002004-09 and 1Z01BM002007-01.