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    • Co transfection of T cells with Gal DBD hCrm and

    2020-07-31

    Co-transfection of 293T lumiracoxib mg with Gal4DBD-hCrm1 and VP16AD-NL43Rev plasmids along with the Gal4-FFLUC reporter led to an increase in RLU which was greater compared to when either plasmid was transfected alone or when VP16AD-RevM10 plasmid was used (Fig. 3B and C). The amount of RLU was significantly higher when Gal4DBD-hCrm1 was transfected compared to Gal4DBD-mCrm1, and the increase in RLU was observed in the presence and absence of co-transfected HIV RRE (Fig. 3B and C), consistent with the Rev-hCrm1 interaction being independent of the RRE. This may be due to the abundance of cellular RNA to which Rev can bind in a non-specific manner, which would largely be absent in the biochemical in vitro experiments described above (Fig. 1). To confirm that the observed higher level of RLU observed in the presence of Gal4DBD-hCrm1 and VP16AD-Rev was specific, increasing amounts of non-fused, full-length HA-hCrm1 or HA-mCrm1expression plasmid was transfected into 293T cells, and the RLU decreased significantly only in the presence of increasing amounts of hCrm1, not empty vector or mCrm1 (Fig. 4A, B). We next turned our attention to other lentiviral Revs, including those of FIV, HIV-2, and EIAV, each of whose function is thought to be dependent on Crm1. Because these Revs are all functional in 293T and not murine cells, with the possible exception of EIAV Rev, we compared the interaction effects of hCrm1 vs mCrm1. In each case the VP16AD-Rev fusion was detected by immunoblotting, although for uncertain reasons the HIV-2 Rev fusion was expressed at lower levels (Fig. 5A). For FIV and HIV-2 Rev fusions with VP16AD, co-transfection with Gal4DBD-hCrm1 resulted in significantly higher RLUs compared to co-transfection with Gal4DBD-mCrm1, in the presence and absence of FIV or HIV-2 RRE, respectively (Fig. 5B and C). On the other hand, for EIAV Rev fusion we never observed a significant increase in RLU when using Gal4DBD-hCrm1 compared to Gal4DBD-mCrm1, in the presence or absence of EIAV RRE (Fig. 5B and C). This is consistent with our prior failure to achieve higher infectious EIAV release from murine cells in the presence of hCrm1 compared to mCrm1 (Elinav et al., 2012). We also tested the Gal4DBD-hCrm1-411-412–414 mutant in the above assays. By immunoblotting we confirmed equal expression of that fusion protein compared to Gal4DBD-hCrm1 and Gal4DBD-mCrm1 in 293T cells (Fig. 6A). For all lentivirus Rev fusions tested, there was no increase in RLU in the presence of Gal4DBD-hCrm1-411-412–414 fusion compared to Gal4DBD-hCrm1; in fact typically compared to Gal4DBD-mCrm1 there was a decrease in RLU (Fig. 6B and C). With regards to HIV, HIV-2, and FIV Rev, the presence or absence of the corresponding lentiviral RRE appeared to have little impact on the fold-effect observed with the hCrm1 fusion (Fig. 6B and C). Again, in the presence or absence of EIAV RRE, for EIAV Rev there was no difference in the fold-effect seen, irrespective of Gal4DBD-Crm1 used (Fig. 6C).