Cultures were infected at 3 h after G1
entry (Fig. ) with a replication-defective HIV-1 vector encoding the EGFP gene under control of the CMV-IE promoter-enhancer, pPCW-eGFP (4
). This vector was prepared by cotransfecting 293T cells with the packaging plasmids described previously (16
). This HIV-1 vector encodes the DNA flap, which was shown to be important for nuclear import (19
). The HIV-1 vector packaging system provides Gag and Pol proteins, Vpr, and the VSV-G surface protein to mediate cellular entry. As the vector genome is devoid of all replicative genes, no virus spread can occur within the colony. For preparation of vector stocks, virus-containing cell supernatants were passed through a 0.45-μm-pore-size filter to eliminate transfer of GFP-expressing producer cells during the infection. HeLa cells were infected by exposure to virus for 2 h. To interpret the GFP segregation patterns, it is important that the primary infected cell contain a single integrated provirus. Viral vector stocks were therefore diluted to the point where only 10 to 20% of colonies were GFP positive, corresponding to an effective multiplicity of infection (MOI) of less than 0.05 to 0.1 (correcting for two cells per colony at the time of infection). By Poisson distribution analyses, under these conditions the fraction of cells that experienced two integration events is negligible (0.0045 to 0.0012).
Integration and segregation of proviral DNA were monitored by following GFP reporter expression in daughters and granddaughters of the infected cell by using fluorescence microscopy. At a low MOI, only one of the two G1
cells per doublet will be infected in the vast majority of colonies (Fig. ). The uninfected bystander cell served as an internal control to monitor synchronous outgrowth of the colony and to confirm that there is no unusual cell-to-cell transmission of GFP. Due to outgrowth of one uninfected and one infected cell, SY provirus segregation would result in 50% GFP-positive cells per colony, while AS segregation would produce 25% GFP-positive cells (Fig. ). Under these experimental conditions, significant GFP expression was not detected after infection with an HIV-1 vector carrying an inactivating mutation in integrase (16
) (data not shown), confirming that the observed GFP segregation required vector DNA integration. We also confirmed that GFP readout directly correlated with integrated DNA by first passaging parallel infected cultures to eliminate unintegrated DNA, followed by sorting of GFP-positive and -negative cells and measurement of viral DNA by quantitative real-time PCR (data not shown). As demonstrated below, SY segregation was dependent on the infection time with respect to S phase, essentially ruling out the possibility that SY colonies could result from multiple integration events (i.e., in different chromosomes).
HIV-1 vector infection at 3 h into G1
resulted in 64% ± 19% GFP-positive SY colonies (Fig. and B). The remaining GFP-positive colonies scored as AS type, as expected (Fig. ). The high percentage of SY segregation is consistent with efficient integration into unreplicated host DNA and duplication to both sister chromatids when the host DNA replication fork passed through the newly integrated provirus. Integration into unreplicated host DNA requires that nuclear import of the preintegration complex occur prior to the end of S phase. We also infected HeLa cells synchronized 3 h post-entry into G1
with a similar murine leukemia virus (MLV)-based GFP vector (pLEGFP-C1; BD Biosciences) that was prepared by transfecting the AmphoPack-293 cell line (BD Biosciences). MLV is believed to be more dependent on mitosis for nuclear import (17
), and the percentage of SY segregants was lower, 19% ± 9% (Fig. ). However, as described below, this level of SY segregation is significantly above the background of the assay. We also note that the infection time (early in G1
) strongly favors detection of mitosis-independent integration into unreplicated DNA. These results indicate that, under these conditions, MLV may not be strictly dependent on mitosis for nuclear entry in cycling cells. Recently, we (12
) and others (11
) have demonstrated that an alpharetrovirus, avian sarcoma virus (ASV), can infect noncycling cells, implying the existence of a mitosis-independent nuclear import pathway for this virus. We found that early-G1
infection with an ASV-GFP vector (12
) resulted in a lower percentage of SY segregation (35% ± 7%) than was observed with the HIV-1 vector. However, the readout may be an underestimate of SY segregation due to rapid variegation of GFP expression within a subset of colonies (not apparent with HIV-1 or MLV vectors), which we confirmed to be due to gene silencing (unpublished data).
FIG. 3. Frequency of SY segregation after infection early in G1. (A) Percentage of SY GFP colony patterns after infection of HeLa cell doublets, 3 h into G1 with HIV-1 or MLV GFP vectors. Standard deviations (error bars) are shown. (B) Representative patterns (more ...)
The simplest interpretation of the segregation pattern observed after HIV-1 vector infection predicts that, as more of the host DNA is replicated, the probability of integration into unreplicated DNA will decrease. To test this prediction, HeLa cells were synchronized as described in the Fig. legend and were infected with the HIV-1 GFP vector at 13 and 16.5 h post-release into G1. As shown in Fig. , there was a decrease in the percentage of SY colonies after infection at 13 h (within S phase) compared to 3 h, with a concomitant increase in AS colonies, and these results support our interpretations of the segregation patterns. To provide biochemical support for our interpretations, we measured the timing of integration with the Alu-PCR method. To facilitate detection, scaled-up monolayer cultures were infected at a high MOI 3 h post-release into G1. HIV-1 DNA integration could be detected prior to the end of S phase, as expected (data not shown).
FIG. 4. Effect of infection time and cell cycle delay on frequencies of SY and AS segregation. (A) Effect of infection time on frequencies of SY and AS segregation. Cells were prepared by mitotic shake-off as described in the legend to Fig. and (more ...)
After infection at 16.5 h into the cell cycle (late S phase), SY colonies were observed only rarely (Fig. ), consistent with the depletion of unreplicated host DNA sites. This low percentage of SY colonies establishes the background of the assay.