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Foamy viruses (FVs; also known as spumaviruses or spumaretroviruses) were once classified as one of the genera of the Retroviridae. However, they possess different replication and gene regulation strategies from other retroviruses and consequently these characteristics led to the creation of a distinct viral subfamily known as the Spumaretrovinae by the International Committee on Virus Taxonomy in 2002. In fact, their replication may bridge the gap between retroviruses and hepadnaviruses (1). However, they share no commonalities in their pathology, because FVs have not been found to cause any apparent diseases (5, 7). So research concerned with their distinct gene regulation strategies and interaction with host cells has generated much interest. Furthermore, their distinct characteristics may contribute to the construction of gene vector based on FVs and benefit work on disease therapy.
Tas, the transactivator of FVs, plays a key role in viral replication and gene regulation by binding with LTR and IP (4). Recently, it has been reported that the interaction of Tas and some cellular proteins, such as the promyelocytic leukemia protein, could affect both viral gene expression and infectivity (6). The 249aa Borf1 (or BTas, the Tas equivalent of PFV) is encoded by the first open reading frame (ORF). Previously, we had demonstrated that Borf1 was the transactivator of BFV (2) and could regulate viral gene expression by binding to LTR and IP (3, 8). To further investigate its structural localization and function, we raised a hightiter anti-Borf1 serum against the recombinant soluble Borf1 protein. Western blot analysis and immnofluoresence assay showed that this antiserum could recognize the eukaryotic Borf1 with high specificity. Also, the nuclear and cytoplasmic localization of Borf1 was confirmed in HeLa cells that were transfected with Borf1. Furthermore, with this specific antibody, we showed the nuclear and cytoplasmic localization of Borf1 in Fetal Bovine Lung (FBL) cells that were infected by BFV. These results further confirmed our findings about the transactivator role of Brof1 in BFV infection, and provided an effective way to monitor the activities of Borf1 with this specific antibody, as well as a way to further investigate the molecular mechanism of this key process in infection.
Subcellular Localization Analysis of Bovine Foamy Virus Borf1 Protein
- Received Date: 23 July 2007
- Accepted Date: 20 December 2007
Abstract: The Borf1 protein is encoded by an immediate-early gene of the bovine foamy virus (BFV) and plays a key role in the viral life cycle. Borf1 is a DNA binding protein which can transactivate both the long terminal repeat (LTR) and the internal promoter (IP) of BFV by specifically binding to the transactivation responsive element (TRE). To analyze the subcellular localization of Borf1 during the BFV life cycle, this gene was cloned into a prokaryotic expression vector and expressed in a soluble form. After the purification and immunization, we raised the mouse anti-Borf1 serum with a high titer based on ELISA results. Western blot analysis showed that the antiserum could specifically recognize the Borf1 protein that was expressed in 293T cells. With this specific serum, we revealed the nuclear and cytoplasmic localization of Borf1 in HeLa cells that was transfected with Borf1. Moreover, the immuno-fluorescence assay also showed that the localization of Borf1 during the infection and transfection of BFV was identical.