1 Introduction Alphavirus Togaviridae Strauss and Strauss, 1994 Strauss and Strauss, 1994 Liljestrom and Garoff, 1991; Smerdou and Liljestrom, 1999 Glasgow et al., 1997, 1998; Scallan et al., 1997 Fazakerley et al., 2002; Lundstrom et al., 2003, 2001; Perri et al., 2000 bcl-2 Ashe and Berry, 2003; Cory and Adams, 2002; Tsujimoto and Shimizu, 2000 c Kluck et al., 1997; Yang et al., 1997 Nencioni et al., 2003; Rodgers et al., 1997 Levine et al., 1993; Lundstrom et al., 1997; Mastrangelo et al., 2000; Scallan et al., 1997 Levine et al., 1996 Grandgirard et al., 1998; Murphy et al., 2001 Liljestrom and Garoff, 1991 bcl-2 Dorokhov et al., 2002 c 2 Materials and methods 2.1 Plasmid construction Liljestrom and Garoff, 1991 Ivanov et al., 1997 Hertz and Huang, 1992 Fig. 1 Heteractis crispa HcRed HcRed Fig. 1 Pac Karlsson and Liljestrom, 2003 2.2 Cells and viruses 2 Liljestrom et al., 1991 2.3 Transfection and collection of virus-like particles (VLPs) Liljestrom and Garoff, 1991 Karlsson and Liljestrom (2003) Smerdou and Liljestrom (1999) 2.4 Metabolic labelling 35 35 2.5 Immunoblot analysis BHK-21 cells were infected and samples collected as described above. After SDS-PAGE, proteins were transferred to a nitrocellulose membrane, probed with rabbit polyclonal antisera against SFV nsP1, EGFP (in-house), HcRed (Clontech), with a mouse monoclonal antibody against Bcl-2 (Santa Cruz Biotechnology, Inc.) or with a mouse monoclonal antibody against beta-actin (C4) (Cruz Biotechnology, Inc.) and visualized by ECL immunoblot detection kit (Amersham Life Science). 2.6 Analysis of bicistronic SFV vector cytotoxicity in BHK-21 cells Garmashova et al., 2006 6 2 3 2.7 Immunofluorescence microscopy c c 2.8 Analysis of the viability of AT3 cells infected with SFV VLPs 6 3 Results 3.1 Expression of d1EGFP and Bcl-2 using bicistronic SFV vectors Fig. 2 Fig. 2 Fig. 2 Fig. 2 Fig. 2 Fig. 2 Fig. 2 Fig. 2 Fig. 2 Fig. 2 Thus, vectors containing IRES elements expressed Bcl-2 earlier and to higher levels. Presence of a duplicated subgenomic promoter or IRES element following the Bcl-2 encoding sequence did not have any major effect on the time-course of infection, however, differences in expression levels of the first marker protein, d1EGFP, were observed. 3.2 Effects of infections by recombinant SFV VLPs on host cell protein synthesis Fig. 3 Fig. 3 Fig. 3 Fig. 3 Fig. 2 3.3 Survival of cells infected with Bcl-2 expressing bicistronic replicons Glasgow et al., 1997, 1998; Scallan et al., 1997 Lundstrom et al., 1997; Scallan et al., 1997 Fig. 4 Fig. 4 Fig. 4 Fig. 4 Fig. 4 3.4 c c Kluck et al., 1997; Yang et al., 1997 Scaffidi et al., 1998 c c Fig. 5 c Fig. 5 c Fig. 5 c c Fig. 5 c c c c Fig. 6 c Fig. 6 Fig. 5 c Fig. 6 c 3.5 Bcl-2 expression does not protect SFV-infected AT3-neo cells against cell death c Cepko et al., 1984 Levine et al., 1993 c c Fig. 7 c Fig. 8 Fig. 8 c Fig. 9 Fig. 9 4 Discussion Glasgow et al., 1997; Griffin, 2005; Li and Stollar, 2004 Li and Stollar, 2004; Nava et al., 1998 Levine et al., 1993; Lundstrom et al., 1997; Mastrangelo et al., 2000; Scallan et al., 1997 Lepik et al., 2003 Reed, 1994; Reed et al., 1991 Fig. 2 Fig. 2 Fig. 3 bcl-2 Tsujimoto and Croce, 1986 Cheng et al., 1997; Kirsch et al., 1999 c Kirsch et al., 1999 Grandgirard et al., 1998 c bcl-2 HcRed Fig. 2 Fig. 3 Fig. 4 bcl-2 Fig. 2 c Figs. 5 and 6 Figs. 7 and 8 Fig. 8 c Nava et al., 1998; Li and Stollar, 2004 bcl- Scallan et al., 1997 Bcl-2 Fig. 9 Bick et al., 2003