The interferon-induced transmembrane proteins (IFITMs) are a family of highly related antiviral factors that affect numerous viruses at two steps: in target cells by sequestering incoming viruses in endosomes and in producing cells by leading to the production of virions that package IFITMs and exhibit decreased infectivity. While most studies have focused on the former, little is known about the regulation of the negative imprinting of virion particles infectivity by IFITMs and about its relationship with target cell protection. Using a panel of IFITM3 mutants against HIV-1, we have explored these issues as well as others related to the biology of IFITM3, in particular virion packaging, stability, the relation to CD63/MVB, the modulation of cholesterol levels and the relationship between negative imprinting of virions and target cell protection. The results we have obtained exclude a role for cholesterol and indicate that CD63 accumulation does not directly relate to an antiviral behavior. We have defined regions that modulate the two antiviral properties of IFITM3, as well as novel domains that modulate the protein stability and that, in so doing, influence the extent of its packaging into virions. The results we have obtained however indicate that, even in the context of an IFITM-usceptible virus, IFITM3 packaging is not sufficient for the negative imprinting. Lastly, while most mutations concomitantly affect target cell protection and negative imprinting, a region in the CTD exhibits a differential behavior, potentially highlighting the regulatory role that this domain may play on the two antiviral activities of IFITM3. [ABSTRACT FROM AUTHOR]