No abstract

Ebola virus causes an acute hemorrhagic fever that is associated with high morbidity and mortality. The viral glycoprotein is thought to contribute to pathogenesis, though precise mechanisms are unknown. Cellular pathogenesis can be modeled in vitro by expression of the Ebola viral glycoprotein (GP) in cells, which causes dramatic morphological changes, including cell rounding and surface protein down-regulation. These effects are known to be dependent on the presence of a highly glycosylated region of the glycoprotein, the mucin domain. Here we show that the mucin domain from the highly pathogenic Zaire subtype of Ebola virus is sufficient to cause characteristic cytopathology when expressed in the context of a foreign glycoprotein. Similarly to full length Ebola GP, expression of the mucin domain causes rounding, detachment from the extracellular matrix, and the down-regulation of cell surface levels of β1 integrin and major histocompatibility complex class 1. These effects were not seen when the mucin domain was expressed in the context of a glycophosphatidylinositol-anchored isoform of the foreign glycoprotein. In contrast to earlier analysis of full length Ebola glycoproteins, chimeras carrying the mucin domains from the Zaire and Reston strains appear to cause similar levels of down-modulation and cell detachment. Cytopathology associated with Ebola glycoprotein expression does not occur when GP expression is restricted to the endoplasmic reticulum. In contrast to a previously published report, our results demonstrate that GP-induced surface protein down-regulation is not mediated through a dynamin-dependent pathway. Overall, these results support a model in which the mucin domain of Ebola GP acts at the cell surface to induce protein down modulation and cytopathic effects.

Gene transfer systems based on lentiviruses have emerged as promising gene delivery vehicles for human gene therapy due to their ability to efficiently transduce nondividing target cells. Both primate and nonprimate lentiviruses have been used for construction of lentiviral vectors. An early generation of gene transfer system based on bovine immunodeficiency virus (BIV) has been developed (R. D. Berkowitz, H. Ilves, W. Y. Lin, K. Eckert, A. Coward, S. Tamaki, G. Veres, and I. Plavec, 2001, J. Virol. 75, 3371-3382). In this study, we mapped the BIV Rev response element (RRE) to 312 bp of the Env coding region. Furthermore, we compared transduction efficiencies of vectors containing different portions of the BIV Gag coding region and found that the first 104 bp of gag contains a functional part of the BIV packaging signal. These findings enabled the generation of a minimal BIV-based lentiviral vector. The minimal transfer vector construct consists of a self-inactivating long terminal repeats (LTR), minimal packaging sequence, putative central polypurine tract, minimal RRE, an internal promoter driving the gene of interest, and a woodchuck hepatitis posttranscriptional regulatory element. In addition, we constructed a BIV packaging construct containing gag/pol, minimal Rev/RRE, and the accessory gene vpy. The regulatory gene tat and the accessory genes vif and vpw have been inactivated or truncated. The current system has significantly reduced regions of homologies between the transfer vector and the packaging constructs. The vectors generated from this system achieved a titer of greater than 1 x 10(6) transducing units per milliliter and are fully functional as indicated by their ability to efficiently transduce both dividing and nondividing cells. These modifications should provide improved safety features for the BIV-based gene transfer system.

Lentivirus-based gene transfer systems have demonstrated their utility in mediating gene transfer to dividing and nondividing cells both in vitro and in vivo. An early-generation gene transfer system developed from bovine immunodeficiency virus (BIV) has been described (Berkowitz et al., J. Virol. 2001;75:3371-3382). In this paper, we describe the development of second-generation (three-plasmid) and third-generation (four-plasmid) BIV-based systems. All accessory genes (vif, vpw, vpy, and tmx) and the regulatory gene tat were deleted or largely truncated from the packaging construct. Furthermore, we split the packaging function into two constructs by expressing Rev in a separate plasmid. Together with our minimal BIV transfer vector construct and a vesicular stomatitis virus G glycoprotein-expressing plasmid, the BIV vectors were generated. The vectors produced by the three- and four-plasmid systems had titers greater than 1 x 10(6) transducing units per milliliter and were fully functional as indicated by their ability to efficiently transduce both dividing and nondividing cells. These results suggest that the accessory genes vif, vpw, vpy, and tmx are dispensable for functional BIV vector development. The modifications made to the packaging constructs improve the safety profile of the vector system. Finally, BIV vectors provide an alternative to human immunodeficiency virus-based gene transfer systems.