For a better understanding of this observation, we further analyzed the part of ASAP on VLP production in different cell lines. Machupo disease (MACV), Tacaribe disease (TCRV), Latino disease (LATV), Pichinde disease (PICV), and Lassa disease (LASV) in six different cell lines: HEK293T, Huh-7, A549, Vero76, BHK-21, and NIH3T3 cells. JUNV, MACV, and LASV Z proteins efficiently produced VLPs in all tested cell lines, while the efficiencies of VLP production from the additional arenavirus Z proteins were cell type-dependent. The contribution of the L-domain(s) within Z protein to VLP production also highly depended within the cell type. These results suggested that every arenavirus offers its own particle-production mechanism, which is different among the cell types. < 0.05; ??< 0.01; ???< 0.001, BMS 599626 (AC480) were considered statistically significant. In all of the graphs, data are demonstrated as the mean and standard deviation of four self-employed experiments. Results Z-Mediated VLP Production of Arenaviruses in Six Different Cell Lines The sole manifestation of arenavirus Z proteins can induce VLP production in cells (Perez et al., 2003; Strecker et al., 2003; Urata et al., 2006). First, we examined whether Z proteins of JUNV, MACV, TCRV, LATV, PICV, and LASV can create and launch VLPs from cells. In this study, we used six BMS 599626 (AC480) cell lines from different origins, HEK293T, Huh-7, A549, Vero76, BHK-21, and NIH3T3 cells. At 48 or 76 hpt of Z manifestation plasmids, tradition supernatants comprising VLPs and cell lysates were prepared and analyzed BMS 599626 (AC480) by WB to detect FLAG-tagged Z proteins using an anti-FLAG antibody. As demonstrated in Number 3 and Table 1, JUNV, MACV, and LASV Z efficiently produced VLPs in all six cell lines. PICV Z produced significantly lower amounts of VLP in HEK293T (17%), A549 (7%), and BHK-21 (9%) cells, and slightly lower amounts of VLP in Huh-7 (49%) and NIH3T3 (46%) cells, compared to JUNV Z. The ratios of TCRV Z-mediated VLP production in A549, Vero76 and BHK-21 cells were 64%, 64%, and 56%, respectively, when compared to JUNV Z. LATV Z manifestation efficiently produced VLP, relative to JUNV Z manifestation in Huh-7 and Vero76 cells, while those in HEK293T and NIH3T3 cells were slightly lower relative to JUNV Z (64% and 56%, respectively). VLP production in LATV Z A549 and BHK-21 cells was significantly lower relative to JUNV Z (25% and 11%, respectively). Taken collectively, these data display that the effectiveness of Z-mediated VLP production of TCRV, PICV, and LATV is definitely cell-type dependent. Open in a separate window Number 3 Arenavirus Z-mediated VLP production. HEK293T Rabbit Polyclonal to IRX2 cells (A), Huh-7 cells (B), A549 cells (C), Vero76 cells (D), BHK-21 cells (E), and NIH3T3 cells (F) were transfected with manifestation plasmids for JUNV, MACV, TCRV, LATV, PICV, or LASV Z-FLAG. At 48 h post-transfection (hpt) (ACC,E) or 72 hpt (D,F), VLPs and whole-cell lysates were collected and analyzed by western blot (WB). In all experiments, actin served as a loading control. VLP production by JUNV Z-WT was arranged at 1.0 while a standard, and the data shown are averages and standard deviations of four indie experiments (ideal panels). *< 0.05; **< 0.01; ***< 0.001. TABLE 1 The effectiveness of arenavirus Z-mediated VLP production. < 0.001. TABLE 2 The contribution of late (L)-website on arenavirus Z-mediated VLP production. < 0.01, ***< 0.001. (iii) Part of the ASAP Sequence in TCRV Z-Mediated VLP Production Most NW arenavirus Z proteins possess a PT/SAP motif, in the form of an L-domain at their C-terminus. However, TCRV Z only possesses an ASAP motif, much like PT/SAP, at its C-terminus (Number 2C). Previous studies have shown the ASAP sequence does not contribute to Z-mediated VLP production in 293T cells (Urata et al., 2009; Groseth et al., 2010). For a better understanding of this observation, we further analyzed the part of ASAP on VLP production in different cell lines. WT and Mut (ASAP AAAA) Z, having a C-terminal HA tag, were recognized using an anti-HA antibody. TCRV Z-Mut exhibited only a slight reduction in VLP production compared to Z-WT in HEK293T, A549, Vero76, and NIH3T3 cells (12%, 29%, 19%, and 4% reduction, respectively) (Numbers 6A,C,D,F and Table 2). In contrast, VLP production mediated by Z-Mut was significantly reduced compared to that by Z-WT in Huh-7 and BHK-21 cells (76% and 81% reduction, respectively) (Numbers 6B,E and Table 2). These results strongly suggest that the ASAP sequence, within TCRV Z, functions as an L-domain in some cell lines. Open in a separate window Number 6 Role of the ASAP motif in TCRV Z-mediated VLP production: (A) HEK293T cells were.