It has been observed widely that productive infection and/or transduction by oncoretroviruses (e.g., MuLV and ASV) requires host cell division. Both S phase (20
) and mitosis (44
) have been implicated as being critical for propagation of these viruses. It is proposed that late G1
or S phase provides a supportive environment for efficient reverse transcription for ASV (20
) and MuLV (40
), as well as HIV-1 (22
), while nuclear membrane breakdown during mitosis allows access to host chromosomes for MuLV DNA integration (44
). Early studies suggested that reverse transcription and integration of ASV could occur during S phase (20
) and that passage through mitosis was required to activate the integrated viral DNA for expression and particle production (20
). In assessing the role of the cell cycle in retroviral replication, there is an important distinction between productive infection and transduction. For the analysis of retroviral vectors, which are normally replication deficient, only the efficiency of transduction is usually considered. Our results indicate that mitosis is not essential for transduction by ASV; however, other aspects of the cell cycle may be required for productive infection.
In the present study, two methods were used to assess the requirement for mitosis for efficient nuclear entry and integration of ASV DNA. First, we show that nuclear forms of ASV DNA (containing LTR junctions) can be detected in both avian and mammalian cells that have been arrested by different methods to prevent mitosis. The amounts of these nuclear forms in arrested cells are comparable to those in dividing cells (Fig. ). The relatively high levels of DNA import, compared to cycling cells, argues against passive import of the viral DNA into the nucleus. Similarly, it is unlikely that a subpopulation of dividing cells could account for these results; this is especially true for γ-irradiated HeLa cells in which G2 arrest is typically uniform (Fig. ). These results suggest that ASV uses an active nuclear import mechanism, likely via the nuclear pore. We were also able to detect a lesser amount of MuLV DNA in the nuclei of γ-irradiated HeLa cells, suggesting that it may also be imported actively, albeit at a lower efficiency than ASV. The second method that we used to monitor nuclear import of ASV DNA was through transduction of a GFP reporter. Efficient GFP expression was detected in cells arrested with γ-irradiation, nocodazole, mitomycin C, or aphidicolin (Fig. through 6). We also monitored GFP reporter gene expression in individual arrested cells to rule out the possibility that ASV-mediated transduction was the result of leaky cell division (Fig. ). As GFP expression was not observed after infection with an IN-deficient vector, we conclude that reporter expression requires integration in this system. Long-term expression (ca. 2 weeks) of GFP in mitomycin C-arrested cells also indicated that integration had likely occurred in arrested cells (data not shown).
Although mitomycin C-arrested chicken DF-1 cells could be transduced by ASV (as indicated by expression of the CMV-driven GFP reporter), these cells were unable to support detectable release of infectious particles. The mechanism of this restriction remains to be investigated but is consistent with the earlier report indicating that passage through mitosis was required for productive infection (20
HIV-1 can transduce (and productively infect) certain nondividing cells. HIV-1 encodes multiple determinants that mediate active nuclear import of the viral DNA (8
), and it is believed that this allows HIV-1 to bypass the requirement for mitosis in nondividing cells. However, cell-cycle-specific components (2
) likely play an important role in the efficiency of HIV reverse transcription (and possibly integration). Notably, HIV-1 nuclear import determinants appear to be necessary but not sufficient for infection of all nondividing cells (11
). For example, HIV-1 reverse transcription is compromised in quiescent (G0
) T cells, likely due in part to the limitations in deoxyribonucleotide precursor pools and the transition to late G1
is required for completion of reverse transcription (24
). Increasing the efficiency of reverse transcription in quiescent cells by boosting deoxyribonucleotide pools appears to be insufficient for transduction, indicating that other cell-cycle-specific components are limiting (23
). HIV-1 is able to transduce terminally differentiated (presumably G0
) cells such as neurons and macrophages; however, deoxyribonucleotide pools (2
) or cell activation (22
), respectively, can influence the efficiency of transduction. In contrast to limitations in quiescent cells, HIV-1 can transduce efficiently cycle-arrested cells (e.g., G2
-arrested HeLa cells) (4
). We note a similarity in that ASV reverse transcription was initially shown to be blocked in quiescent cells (20
), while here we demonstrate efficient transduction of cycle-arrested cells. It is possible that for both ASV and HIV-1, the ability to infect terminally differentiated (G0
) cells may be limited by threshold levels of cellular components.
Our results indicate that ASV DNA import and integration can occur during interphase and are not dependent on mitosis. To prevent mitosis, cell cycle arrest was induced by γ-irradiation, mitomycin C, nocodazole, or aphidicolin. For successful transduction in cycle-arrested cells, the cell cycle phase in which the arrest occurs must be able to support early events (reverse transcription, DNA import, and integration). As mentioned, cellular components enriched during S phase (e.g., deoxyribonucleotide precursors) are important to support efficient retroviral reverse transcription (15
). Hydroxyurea treatment prevents entry into S phase by depleting deoxyribonucleotide precursor pools via inhibition of ribonucleotide reductase (33
). We observed that ASV reverse transcription and transduction (Fig. ) were highly compromised in hydroxyurea-arrested cells, as expected. This observation is therefore consistent with a dependency on S phase.
Aphidicolin also blocks entry into S phase by inhibiting DNA polymerase α. However, when asynchronous cells are treated, cells that are in S phase at the time of aphidicolin addition are arrested within S phase (21
). In such cells, deoxyribonucleotide precursor levels are apparently preserved (42
), and thus cells in S phase at the time of arrest may be able to support reverse transcription. Therefore, the ability of ASV to transduce aphidicolin-arrested cells (Fig. ) is not at odds with a dependency on S phase. However, the efficiency of transduction of aphidicolin-arrested cells was reduced compared to that of cycling cells (Fig. ). Early studies (19
) indicated that aphidicolin inhibited circularization of ASV DNA as well as DNA integration, independently of cell cycle arrest. Recent studies have shown that the nonhomologous-end-joining pathway may be involved in the final repair of the retroviral integration intermediate (9
), as well as circularization of unintegrated linear viral DNA (32
). Aphidicolin may inhibit the nonhomologous-end-joining pathway (41
), which is required for efficient retroviral transduction. Thus, it is possible that aphidicolin can have direct effects on transduction efficiency by inhibiting DNA repair at the integration site, although this remains to be investigated.
Cells arrested in mitosis with the microtubule inhibitor nocodazole could also support efficient transduction by ASV. The ability to transduce nocodazole-arrested cells also does not rule out a role for S phase, as DNA synthesis can reinitiate without cytokinesis after long-term exposure to microtubule inhibitors (Fig. ) (7
). We also observed that G2
-arrested HeLa cells could be transduced efficiently by ASV, indicating that this cell cycle phase supported all early steps in viral replication, as is the case for HIV-1. It is possible that the required S-phase components (e.g., deoxyribonucleotide precursors and factors) remain in sufficient amounts to support transduction of G2
-arrested cells. Taken together, our results appear to rule out a requirement for mitosis and support the idea that S phase is important for early events in ASV DNA replication.
The requirement for mitosis was also analyzed by monitoring ASV transduction (via GFP expression) in individual synchronized cells in real time. Synchronized HeLa cells infected during G1 were found to express GFP prior to mitosis, indicating that active nuclear import can occur during interphase in dividing cells (Fig. ). These results again indicate that nuclear import of ASV DNA is not limited to cells undergoing mitosis; rather, our results suggest an active import process whereby trafficking of the preintegration complex to the nucleus is not temporally or physically restricted to mitosis in dividing cells. As mitosis only comprises a small percentage of the cell cycle (<10%), such a mitosis-independent pathway would suggest a more efficient import process in dividing cells than is generally believed to exist. However, we note that these results do not exclude nuclear capture of ASV DNA after mitosis.
Lastly, we measured transduction in terminally differentiated cells that are withdrawn from the cell cycle (G0
). We observed that mouse hippocampal neuron explants could be transduced efficiently by ASV (Fig. ). Paradoxically, it was previously shown that ASV reverse transcription is deficient in quiescent chicken embryo fibroblasts arrested in G0
). It has been generally believed that terminally differentiated cells cannot reenter the cell cycle. Recent studies indicate that neurons have the potential to reenter the cell cycle and that reentry into S phase (without cytokinesis) is associated with neuronal cell death (54
). It is possible that a subset of explanted postmitotic neurons maintain proliferative potential and that they are able to support early events in ASV infection. Transduction of neurons by HIV-1 vectors appears to be limited by deoxyribonucleotide levels (2
); the fresh embryonic neuron explants described here may provide a threshold level to support ASV reverse transcription.
The results presented here support the idea that ASV encodes determinants that mediate nuclear import of viral DNA. A likely candidate for at least one of these determinants is the ASV IN (25
). ASV IN encodes a functionally defined, noncanonical NLS. Single amino acid substitutions in the NLS cause a significant delay in viral replication without compromising IN biochemical activity in vitro (26
). Multiple substitutions in the NLS are lethal for viral replication (unpublished observations). Using a permeabilized-cell assay, we have partially characterized the import pathway utilized by the ASV IN NLS. The pathway involves the nuclear pore but is apparently novel in terms of energy requirements and receptors (Andrake et al., unpublished data). We are currently investigating whether this pathway is required for ASV transduction of arrested cells. Recent studies indicate that amino acid substitutions in a putative HIV-1 IN NLS affect HIV-1 (3
The relative efficiencies of transduction by HIV-1-, MuLV-, and ASV-based vectors were recently compared in aphidicolin-arrested human cells (18
). The transduction efficiency of the ASV vector was found to be greater than that of the MuLV vector. However, it is well-established that transduction by ASV vectors in differentiated adult mouse tissues is inefficient; therefore, the utility of such vectors is thought to be limited (28
). The variables that influence ASV transduction in such tissues are still unknown. It is important to consider that the efficiency of transduction (as measured by reporter expression) is determined by the efficiency of a number of individual steps, including reverse transcription, nuclear import, integration, reporter gene expression, and silencing of the integrated DNA. It has been generally believed that the basis of observed transduction differences between lentiviral (i.e., HIV-1) and oncoretroviral (MuLV) vectors relates to nuclear import functions. Our results indicate that assumptions regarding a mitosis-dependent import of ASV DNA are incorrect. We are currently investigating the biochemical pathway of DNA import and the role of the IN-NLS. Further study of the cell and tissue variables that affect retroviral transduction efficiencies should provide insights of significant value in the development and use of these vectors for laboratory and possibly gene therapy purposes.