Moloney murine leukemia virus (M-MuLV) proviruses carrying integrase (IN) protein tagged either with a simian virus 40 (SV40) nuclear localization signal (NLS) or various antigenic epitopes were generated. Hexahistidine (His6), hemagluttinin (HA), or two consecutive HA sequences (2XHA) were fused to the C-terminus of IN as antigenic markers. These epitope-tagged IN proteins were stably expressed through multiple rounds of infection. The IN-His6, IN-HA, and IN-2XHA proteins, purified from virus, could be immunoprecipitated with antibodies against His6 and HA, respectively. An M-MuLV provirus encoding the SV40 large T antigen NLS fused to IN at the same position as the epitope tags was also passaged through cells. In contrast to the stability of the epitope tags, the SV40 NLS sequence was rapidly mutated by a frameshift mutation that introduced negatively charged amino acids into the basic NLS. The instability of the NLS suggests that the strong nuclear localization of the IN-SV40 NLS may have detrimental effects on virus assembly. These observations have implications for studying nuclear transport properties of M-MuLV and for engineering a murine-based retroviral vector for gene therapy. (C) 2000 Academic Press.
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