Functional characterization of adenoviral/retroviral chimeric vectors and their use for efficient screening of retroviral producer cell lines

We have generated three different E1-deleted replication-defective adenovir al vectors expressing either Moloney murine leukemia virus (Mo-MuLV) Gag-Po l core particle proteins, gibbon ape leukemia virus (GALV) envelope glycopr oteins, or an MuLV-derived retroviral vector genome encoding mCD2 antigen, a murine cell surface marker easily detectable by flow cytometry. Each of t he three vectors was first characterized individually by infection of cells providing the complementary retroviral function(s) and able to induce the production of retroviral vectors with an efficiency similar to or higher th an that of FLY stable retroviral packaging cells [Cosset, F.-L., Takeuchi, Y., Battini, J.-L., Weiss, R.A., and Collins, M.K.L., (1995). J. Virol. 69, 7430-7436]. In small-scale pilot experiments, TE671 cells simultaneously c oinfected with the three adenoviral vectors efficiently released helper-fre e retroviral vectors in their supernatant, with titers greater than 10(6) i nfectious particles per milliliter by end-point titrations. Our results als o indicated that in contrast to retroviral vector-packageable RNAs, the ade novirus-mediated overexpression of both Gag-Pol and Env packaging functions had limited impact on retroviral titers. The primary mechanism suspected i s the premature intracellular cleavage of the Pr65(gag) precursor that we f ound in gag-pol-expressing cells, which in turn may impair the normal incor poration of high loads of functional Env. Last, the characterization of the adenoviral/retroviral chimeric vectors allowed the screening of various pr imate cells for retroviral production and we found that three hepatocyte-de rived cell lines were highly efficient in the assembly and release of infec tious retroviral particles.