In Vitro and in vivo Studies of Murine Polytropic Retrovirus Infections: a Dissertation

Abstract

Murine leukemia viruses (MuLV) are retroviruses that play important roles in the study of oncogenes, integration, transcriptional regulation and gene therapy. Mink cell focus-inducing (MCF) viruses are polytropic MuLVs that by definition infect cells from a wide variety of species. Their ability to infect human cells and their utility as gene therapy vectors were not well characterized. To address this issue, primary and immortalized human cells were tested for their ability to be infected by MCF packaged defective vectors as well as replication competent MCF virus. A new packaging cell line, called MPAC, was created to package defective retroviral vectors in virus particles with envelope proteins derived from a Moloney mink cell focus-inducing (Mo-MCF) virus. The cellular tropism of MPAC-packaged retroviral vectors was the same as replication competent MCF viruses. Testing various established cell lines showed some human cell lines could be infected with MPAC-packaged vectors while others cannot. In addition, I show that some human cells fully support MCF virus replication while others either partially or fully restrict MCF virus replication. This indicates that some human cells express a protein on their surface that acts as a receptor for MCF viruses and allows MCF viral entry. In addition, the human cells that express a receptor for MCF viral entry did not show any further block to viral replication. An important determinant in the pathogenic phenotype of MCF 247 has been mapped to the enhancer region of the retroviral long terminal repeat (LTR). Recombination of endogenous genetic elements with the 3' portion of envoccurs and incorporates unique LTR sequences. Most strongly pathogenic MCF viruses have a duplication of the enhancer element found in the LTR. AKR mice are an inbred strain of mice that develop spontaneous T-cell lymphomas between 6 and 12 months of age. 12-25 % of MCF induced early lymphomas of AKR mice show MCF viral integration's near c-myc in an opposite transcriptional orientation. A replication competent MCF virus containing a bacterial amber suppressor tRNA gene (supF) was used to investigate the changes in the enhancer region following injection of MCF containing one enhancer in the LTR. Newborn AKR mice were injected with the supF tagged replication competent virus and observed for signs of leukemia development (ruffled fur, lethargy, and tumor development). When these signs were detected, the animals were sacrificed and DNA was prepared from the isolated tumors. Thirty-one tumors DNA were analyzed for the presence of supF tagged virus and rearrangement of the c-myc locus. Nine supF tagged proviral LTRs integrated near c-myc from four animals were PCR amplified, sequenced, and/or cloned. All of the enhancer elements analyzed were derived from proviruses that integrated in a reverse orientation with respect to c-myc locus. Two of the isolated enhancer elements contained only a few base changes whereas the majority contained duplications of different sizes that encompassed different transcription factor binding sites. The duplicated enhancer regions contained duplications from 82-134 bp in length. One tumor contained a proviral enhancer with only 5 bp changes relative to the injected virus. This suggests that the enhancers need only a few specific base changes relative to the injected virus to accelerate leukemogenesis. The other three tumors contained proviral enhancers with various size duplications and additional transcription factor binding sites. These data suggest that the injected virus is not pathogenic unless the enhancer region is altered. One proviral integration site encompassing a duplicated enhancer region and 139 bp of the c-myc gene locus was PCR amplified, cloned and sequenced. A search of the current transcription factor database (Transfac 3.3) showed no known transcription factor binding site sequences were created at the junction of the enhancer duplications. The common motif of LVb, core NF-1, and GRE transcription factor binding sites, described by Golemis at al (57), was conserved throughout the isolated enhancers. Most of the enhancer elements contained additional NF-кB and/or GRE sites in close proximity to the conserved LVb-core region. These results support the hypothesis that additional NF-кB and/or GRE binding sites cooperatively interact with the conserved GRE-NF-1-LVb-core motif in c-myc induced leukemogenesis. In addition, two unique families of enhancer duplications were identified. The two families contained enhancers isolated from different tumors that displayed sequence homology and transcription factor binding site organization unique to each group.