Intracellular proteolytic processing of bovine leukemia virus (BLV) envelope glycoprotein precursor (gp72) at the Cterminal end of the RVRR 268 J, site is an essential step for virus infectivity. Subtilisin/kexin-like convertases cleave proproteins at preferred RX(K/R)R J, sites, including those commonly found in viral envelope glycoprotein precursors. We first demonstrated that gp72 is processed into gp51/gp30 in both CV1 cells and the furin-deficient LoVo cells, leading us to compare the ability of mammalian convertases to cleave BLV gp72 in vitro. In contrast to the inability of the neuroendocrine PCI to cleave gp72, the convertases furin, PACE4, PC5-A and PC5-B, which process constitutively secreted precursors, can effectively cleave gp72 into gp51/gp30. N-terminal sequence analysis of the convertasegenerated gp30 demonstrated that cleavage occurs at the in vivoutilized RVRRiSPV site. Such furin-, PACE4-and PC5-mediated processing was completely inhibited by the oiiantitrypsin variant od-PDX. Mutagenesis of the gp72 cleavage site into RVRG-TPV resulted in complete abrogation of gp72 processing by endogenous CV-1 cells and by convertases in vitro. Since our in vitro data suggest a redundancy in the ability of the convertases to cleave gp72, RT-PCR analysis was used to define the convertases expressed in B-lymphocytes, representing one of the major targets of BLV infection. Our data revealed that only furin and the newly discovered PC7 mRNAs are expressed in Raji, B-Jab and LG2 cell lines.
PCR and R18 fluorescence dequenching assays have been combined to monitor the kinetics of fusion of bovine leukemia virus with target cells (CC81, OVK, or Raji). Antibodies raised against gp51 allow us to demonstrate that not only the hydrophobic N-terminal domain of the transmembrane glycoprotein gp30 but also specific domains of gp51 (amino acids 39 to 103) are involved in bovine leukemia virus-cell fusion.
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