DEVELOPMENT OF MONOCLONAL ANTIBODIES FOR CHARACTERIZATION OF THE BOVINE IMMUNODEFICIENCY VIRUS TMX PROTEIN

Abstract

The bovine immunodeficiency virus (BIV) is a lentivirus that is morphologically, immunologically, and genetically resembled the human and simian immunodeficiency viruses, HIV and SIV, respectively (Gonda, et al. 1987, Garvey, et al. 1990). The BIV genome organization is similar to that of HIV in that it contains the gag, pol , and env structural genes and six putative nonstructural regulatory genes (vif, tat, rev, vpu, vpy, and tmx). The BIV genome does not contain a nef (negative factor) as HIV, but a novel transcript, tmx, has been found in BIV-infected cells that is related to Env proteins. Tmx (p18ͭ ͫ ͯ ) is encoded by a small, singly spliced viral mRNA, whose coding sequences are derived from the 3' end of the genome. Based on its relative location in the genome, BIV tmx is thought to be an analogue of the HIV nef gene (Gonda, et al. 1994). A panel of mouse monoclonal antibodies (MAbs) were generated using GST-Tmx fusion proteins as the immunogen. Two MAbs to BIV p18ͭ ͫ ͯ were selected and designated as Tmx 1F8 and 2D12. They were characterized by enzyme-linked immunosorbent assay (ELISA), western blotting, two-antibody sandwich ELISA, indirect immunofluorescence assay (IFA), and postembedding immunoelectron microscopy (IEM) localization assay. Both MAbs are kappa (K) light chain IgG1 and were reactive to purified native and denatured GST-Tmx fusion proteins as well as Tmx present in lysed BIV virions. However, the MAbs did not detect Tmx in BIV-infected cell lysates by western blotting. Using IFA, BIV Tmx was localized to the perinuclear region and cytoplasm of BIV-infected cells. Differential fixation as applied to preserve cell surface membrane proteins in immunolocalization by IFA, but no cell surface-associated Tmx proteins were detected. Using IEM, Tmx was localized in BIV virions, but no cell surface Tmx proteins were detected. The location of BIV Tmx in both BIV-infected cells and virions was confirmed using rabbit polyclonal antibody (PAb) made to a synthetic peptide (TMP-4) derived from the C-terminus of the Tmx protein. The biological function of HIV Nef is thought to be both a positive and negative regulator, but its functions as a positive or negative effector for the virus replication is not clearly understood (de Ronde et al. 1992; Miller et al. 1994; Spina et al. 1994; Hammes et al. 1989; Kim et al. 1989). Nevertheless, Nef is essential for HIV replication and pathogenesis, particularly Nef-induced CD4 down-modulation (Rhee S. and J. Marsh 1994). There is a reason to believe that there are some similarities between BIV Tmx and HIV Nef based on the similarity of their gene location and the BIV Tmx is located in the same region of the cell as HIV Nef. More studies on BIV Tmx are needed to determine its relationship to HIV Nef and the role of Tmx and Nef in lentivirus pathogenesis. The development of MAbs in this study provides a tool for future analysis of the BIV Tmx protein that may provide additional information.