Nuclear Import of Moloney Murine Leukemia Virus DNA Mediated by Adenovirus Preterminal Protein Is Not Sufficient for Efficient Retroviral Transduction in Nondividing Cells

Moloney murine leukemia virus (MoMLV)-derived vectors require cell division for efficient transduction, which may be related to an inability of the viral DNA-protein complex to cross the nuclear membrane. In contrast, adenoviruses (Ad) can efficiently infect nondividing cells. This property may be due to the presence of multiple nuclear translocation signals in a number of Ad proteins, which are associated with the incoming viral genomes. Of particular interest is the Ad preterminal protein (pTP), which binds alone or in complex with the Ad polymerase to specific sequences in the Ad inverted terminal repeat. The goal of this study was to test whether coexpression of pTP with retroviral DNA carrying pTP-binding sites would facilitate nuclear import of the viral preintegration complex and transduction of quiescent cells. In preliminary experiments, we demonstrated that the karyophylic pTP can coimport plasmid DNA into the nuclei of growth-arrested cells. Retroviral transduction studies were performed with G1/S-arrested LTA cells or stationary-phase human primary fibroblasts. These studies demonstrated that pTP or pTP-Ad polymerase conferred nuclear import of retroviral DNA upon arrested cells when the retrovirus vector contained the corresponding binding motifs. However, pTP-mediated nuclear translocation of MoMLV DNA in nondividing cells was not sufficient for stable transduction. Additional cellular factors activated during S phase or DNA repair synthesis were required for efficient retroviral integration. Integration of viral DNA into the host chromosome is an essential step in the retrovirus life cycle. With their remarkably efficient ability to integrate, retrovirus vectors are an important tool in obtaining stable gene expression in vitro and in vivo (37, 65). However, the most commonly used and as yet best-characterized vectors based on mammalian C-type retroviruses cannot transduce quiescent cells, which often represent the targets for gene therapy approaches (20, 38, 43, 63). After entry of the retroviral core into the cytoplasm, the retroviral genome is reverse transcribed by using enzymatic activities associated with the incoming virion. The resulting double-stranded linear viral DNA is associated with viral proteins, forming a large nucleoprotein complex. To complete the retroviral life cycle, this preintegration complex must be translocated to the nucleus. The mechanisms of nuclear transport appear to differ among different retrovirus subfamilies and host cells (for a review, see reference 8). For Moloney murine leukemia virus (MoMLV)-derived vectors, it is believed that breakdown of the nuclear membrane during mitosis is necessary to allow access of the preintegration complex to chromosomal DNA. This is supported by two lines of evidence: (i) inhibitors, which delay the onset of mitosis, delay integration as well (3, 33, 38); and (ii) MoMLV proviruses segregate into only one daughter cell during the first mitotic division after infection, implying that integration occurs after DNA replication (19, 43). The MoMLV preintegration complex contains linear DNA associated with the CA, IN, and possibly RT and NC proteins (8). This complex with a sedimentation constant of ;160S is too large to pass through the nuclear membrane by