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

Abstract

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 G 1 /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). T...

Inserting a Nuclear Targeting Signal into a Replication-Competent Moloney Murine Leukemia Virus Affects Viral Export and Is Not Sufficient for Cell Cycle-Independent Infection

“…Host factors and chromatin structure may also play an important role in cell cycle-independent viral entry, making the cell cycle regulation of murine retroviruses complex and intriguing. It has been reported that nuclear import of MoMuLV DNA mediated by an adenoviral protein was not sufficient for efficient retroviral transduction in cells arrested in the G 1 /S phase of the cell cycle (47). The requirement for passage through M phase has a direct impact on the type of cells that can be infected by murine-based retroviral vectors.…”

Use of synthetic vectors for neutralising antibody resistant delivery of replicating adenovirus DNA

“…19,22,26,27 To characterise the effect of TP on expression of transgenes encoded within the input virus DNA, we used DNA from an E1-deleted reporter virus expressing Green Fluorescence Protein (Ad GFP DNA) and compared GFP expression following transfection of cells using DOTAP/DNA with and without covalently linked TP. The experiment was performed in non-complementing A549 cells to prevent effects due to virus replication.…”

“…Although TP and pTP mediate different roles in the virus life cycle, it is feasible that the there are some overlapping activities. Lieber et al 26 also studied pTP and reported that the expression from a plasmid with a TP-binding site is enhanced 50-fold when used in cells transiently expressing pTP, concluding this was a result of improved transfer into the nucleus. These observations of a substantial effect of pTP contrast with the relatively minor activity reported in the studies here and demonstrate that the orientation, DNA context or mode of linkage of the pTP have an important influence on its biological activity.…”

High-Throughput, Library-Based Selection of a Murine Leukemia Virus Variant To Infect Nondividing Cells

“…The exact structure and composition of the PIC are undetermined, so any of these proteins could potentially be engineered to mediate nuclear import. Previous attempts to confer nuclear import capabilities to MLV have relied on rationally inserting nuclear localization signals (NLSs) into the MLV genome or substituting wild-type (WT) MLV domains with analogous HIV regions (11,35,48,51). Some of these modifications resulted in the nuclear localization of viral subcomponents but did not enable the successful infection of nondividing cells (35,48).…”

“…Previous attempts to confer nuclear import capabilities to MLV have relied on rationally inserting nuclear localization signals (NLSs) into the MLV genome or substituting wild-type (WT) MLV domains with analogous HIV regions (11,35,48,51). Some of these modifications resulted in the nuclear localization of viral subcomponents but did not enable the successful infection of nondividing cells (35,48). The insertion of a NLS into the MA protein of spleen necrosis virus, an avian retrovirus, was reported to mediate successful infection of arrested cells in vitro (43); however, recent attempts to duplicate this result suggest that the effect seen may be limited to the in vitro system utilized and that low titers could have possibly masked native WT ability (6,28).…”