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    • To confirm and extend those findings of a differential inter

    2020-07-28

    To confirm and extend those findings of a differential interaction between hCrm1 and mCrm1 and Rev-RRE, we first attempted to use the yeast two and three hybrid systems, but failed to observe a detectable interaction between any of the Crm1s and Rev, in the presence or absence of HIV RRE RNA. We then turned to the luciferase complementation system in which protein interaction between amino and carboxy terminal firefly luciferase (FFLUC) fusion proteins results in detectable FFLUC activity by relative light units (RLUs) (Luker et al., 2004). Using this system we were able to detect a strong HIV Rev-Rev genetic interaction but not between Rev and any Crm1 fusion protein. Because the luciferase complementation system may only allow readout when each fusion partner is small enough to permit enzymatic catalysis and full-length Crm1 is greater than 100 kD in size, we next attempted the mammalian two-hybrid system, knowing that we could also separately transfect in HIV RRE RNA in plasmid form, driven by either RNAPII or III promoters. Based on these results, we tested the functionality of the Gal4DBD-Crm1 fusions in a functional assay, transfecting them into murine B78 Heparin sodium that had an integrated HIV reporter vector encoding both a truncated form of human cyclin T1 and blasticidin resistance (bsd) (Coskun et al., 2007), along with VSV G expression construct and an HIV vector encoding a truncated form of human cyclin T1 and eYFP. The B78 cells express murine Crm1, and this assay allows us to test the functionality of other Crm1 constructs (Fig. 2A). Human cyclin T1 is required since expression of both eYFP and bsd in the vector used are dependent upon the HIV long terminal repeat and the presence of Tat. As anticipated, the non-fused, full-length hCrm1 had the greatest activity in terms of infectious virus release, as measured by the number of blasticidin-resistant colonies on HOS cell targets (Fig. 2B). The Gal4DBD-hCrm1 fusion had ~25% of the activity of the non-fused hCrm1 but was significantly more active than the Gal4DBD-mCrm1 fusion and the Gal4DBD-hCrm1 411–412-414 mutant. As reported previously (Aligeti et al., 2014), the 2xNES-Rev-mCherry expression plasmid gave roughly 50% of the number of colonies, in the absence of hCrm1 (Fig. 2B). As anticipated, similar results were obtained using eYFP as a flow cytometric readout on the HOS targets (not shown). A related, cell-based approach was used to test the functionality of the various HIV Rev fusions, but in a trans-complementation assay using 293T producers transfected with both VSV G and a Rev-deficient HIV reporter vector encoding both FFLUC and bsd (Fig. 2C). As expected, the non-fused, full-length version of NL4-3 Rev had the greatest activity for both readouts (RLU and blasticidin-resistant colonies on susceptible HOS targets), RevM10-GFP fusion had very little to no activity, and the two VP16AD-Rev fusion constructs had between 25% and > 50% of the activity of the non-fused Rev (Fig. 2D and E). Of note, these constructs were all expressed in 293T cells, based upon immunoblotting (Fig. 3A). Based on these results, we decided to test both Gal4DBD-Crm1 fusions and the VP16AD-Rev fusions in the mammalian two hybrid system.