title: Differential effects of C-terminal molecular tagged integrase on replication competent moloney murine leukemia virus.
abstract: 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 (His(6)), 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-His(6), IN-HA, and IN-2XHA proteins, purified from virus, could be immunoprecipitated with antibodies against His(6) 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.
authors.affiliation: Department of Biochemistry, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, New Jersey 08854, USA.
authors.forename: J A
authors.initials: JA
authors.lastname: Seamon
authors.forename: M
authors.initials: M
authors.lastname: Adams
authors.forename: S
authors.initials: S
authors.lastname: Sengupta
authors.forename: M J
authors.initials: MJ
authors.lastname: Roth
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002453:Cell Cycle; D002460:Cell Line; D003001:Cloning, Molecular; D004279:DNA, Viral / Q000032:analysis / Q000235:genetics; D019426:Integrases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D008979:Moloney murine leukemia virus / Q000201:enzymology* / Q000235:genetics / Q000502:physiology*; D019913:Nuclear Localization Signals; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013539:Simian virus 40; D014162:Transfection; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014779:Virus Replication
chemical_list: D004279:DNA, Viral; D019913:Nuclear Localization Signals; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D019426:Integrases