Gladstone Institute of Virology and Immunology, PO Box 419100, San Francisco, CA 94141-9100, USA1
Department of Medicine, School of Medicine, University of California San Francisco, San Francisco, CA 94141-9100, USA2
Author for correspondence: Mark Goldsmith (at Gladstone Institute of Virology and Immunology). Fax +1 415 695 1364. e-mail mgoldsmith{at}gladstone.ucsf.edu
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Abstract |
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Main text |
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EBO GP produced in an infected cell is cleaved intracellularly to produce membrane-bound GP2, a transmembrane protein that mediates membrane fusion, and associated GP1, an extracellular protein that presumably mediates virus attachment to a receptor at the cell surface (Volchkov et al., 1998a ). Free GP1 is also shed in a nonvirion-bound form and has been hypothesized to be pathogenic (Volchkov et al., 1998b
). MBG GP is processed similarly (Volchkov et al., 2000
). In addition, pre-translational processing of EBO GP results in the production of two EBO glycoprotein products, a shorter secreted product (sGP) derived from unedited mRNA transcripts and a polyprotein precursor to the longer membrane-bound form (GP1/GP2) encoded by edited transcripts (Sanchez et al., 1996
; Volchkov et al., 1995
). While GP1/GP2 mediates infection in a variety of cells, EBO sGP has been reported to bind to uninfected cells such as neutrophils (Yang et al., 1998
); the interpretation of these findings has been challenged (Maruyama et al., 1998
), and the precise functional relevance of EBO sGP in pathogenesis is unknown. In contrast, MBG GP transcripts apparently do not undergo such editing (Bukreyev et al., 1995
; Will et al., 1993
), and express exclusively the GP1/GP2 polyprotein which is cleaved and assembled into the full-length membrane-bound GP1/GP2 complex. These distinctions underscore the possibility that GP products from different filoviruses may induce different dysregulatory phenotypes in host cells.
In initial studies, genes encoding MBG GP and the Zaire (Z) subtype of EBO GP (provided by A. Sanchez, Centers for Disease Control and Prevention, Atlanta, GA, USA) were cloned into the mammalian expression vector pCMV4neo (Goldsmith et al., 1994 ) and separately co-transfected with pNL-Luc-E-R- (Connor et al., 1995
), the HIV-1 NL4-3 provirus carrying a luciferase reporter gene driven by the 5' LTR (provided by N. Landau, Salk Institute, La Jolla, CA, USA, via the AIDS Research and Reference Reagent Program) into 293T cells in order to produce pseudotype virus stocks as previously described (Chan et al., 2000
). To assess and compare production and function of MBG and EBO-Z GP in this system, Vero cells were challenged with Luc+ pseudotype viruses packaged by no GP, vesicular stomatitis virus (VSV) G protein (provided by J. Burns, University of California, San Diego, CA, USA), MBG GP or EBO-Z GP (Fig. 1A
), and luciferase expression was used to quantify virus entry as previously described (Chan et al., 2000
). Both MBG and EBO-Z pseudotypes infected Vero cells to comparable and significant levels, demonstrating that GP1/GP2 complexes encoded by both MBG and EBO-Z constructs were functionally competent for packaging virus and initiating target cell infections. Furthermore, MBG and EBO-Z pseudotype viruses generated from 293T transfections infected the same proportion of target cells at highest achievable titres (Chan et al., 2000
), indicating similar expression of functional GP in both preparations.
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To assess directly MBG and EBO-Z GP expression levels in 293T cells, SDSPAGE under reducing conditions and Western blotting were performed as previously described (Liu et al., 1997 ) on lysates [1% NP-40 lysis buffer containing 1x stock protease inhibitor cocktail set I (CalBiochem)] obtained from transfected samples. For detection, we used polyclonal guinea pig antisera (1:1000 dilution) raised against MBG (Musoke strain) or EBO-Z (Mayinga strain) virus, respectively (provided by A. Schmaljohn, United States Army Medical Research Institute for Infectious Diseases, Fort Detrick, MD, USA), and an HRP-conjugated goat anti-guinea pig IgG (H+L) secondary antibody (1:5000 dilution; Accurate Chemical and Scientific Corp.) (Fig. 1C
). Despite the presence of variable background bands, expression of both MBG and EBO-Z GP1/GP2 polyproteins was consistently and readily detected. As expected, sGP was undetectable since the cDNA used to express EBO-Z GP encoded only the edited full-length, membrane-bound GP and not the pre-edited cDNA encoding sGP (Xu et al., 1998
). In view of the comparable infectious titres of both pseudotype viruses generated from 293T transfections (Fig. 1A
) and the detectable expression of envelope glycoproteins of both viruses, the stark difference in detachment between EBO-Z and MBG GP transfected samples indicates that EBO-Z GP induces a distinct cellular dysregulatory effect compared with that of MBG.
To determine whether detachment of cells was caused specifically by cell-associated or released GP products, 293T cells were transfected with pEBO-Z-IRES2-GFP, an expression vector encoding EBO-Z GP upstream of an internal ribosome entry site (IRES) site separately driving translation of an enhanced green fluorescent protein (GFP) reporter gene. In this configuration, every transfected cell that produced EBO-Z GP products was marked by GFP expression. After 12 h, transfected cells were re-plated together with untransfected 293T cells at a 1:1 ratio, and detachment was measured at 24 h. If secreted GP products induce detachment in trans, a similar ratio of GFP-positive (transfected) and GFP-negative (untransfected) cells would be expected to be evident in the detached cell population as in the attached cell population. However, if cell-associated GP causes detachment in cis, only GFP-positive transfected cells should be present in the detached population. In fact, flow cytometry revealed that 25% of cells remaining attached were GFP-positive in this experiment (Fig. 2 A), while nearly all (95%) of released cells were GFP-positive. In other experiments, the proportion of attached cells that was GFP-positive varied with the specific mixture of input cells, but the GFP-positive proportion in the detached fraction was always nearly 100%. As a specificity control, 293T cells were similarly transfected with the parental vector pIRES2-GFP and re-plated with untransfected cells. No detachment was detected in these cultures, and 28% of adherent cells expressed GFP. These results clearly demonstrate that detachment of 293T cells is caused by cell-associated EBO-Z GP rather than shed GP products.
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Finally, we hypothesized that expression of EBO-Z GP results in cellular detachment as a consequence of modulating a specific intracellular signalling pathway(s). To identify such a signalling cascade in target cells, we screened a panel of phosphorylation inhibitors for the ability to block 293T cell detachment and found that the Ser/Thr kinase inhibitor 2-aminopurine (2-AP) potently inhibited detachment. When 2-AP (10 mM) was added to cultures 6 h after EBO-Z GP transfection, cellular detachment at 48 h was reduced by 77% as compared to untreated transfected controls (Fig. 3A). A Western blot using the guinea pig antisera raised against EBO-Z virus on separate lysates from control transfections and transfections treated with 2-AP confirmed equivalent expression of EBO-Z GP1/GP2 in both cultures (Fig. 3B
). Therefore, an as yet undefined Ser/Thr kinase activity induced by EBO-Z GP expression must be instrumental in mediating cellular detachment.
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Acknowledgments |
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Received 9 March 2000;
accepted 31 May 2000.