Human immunodeficiency virus type 1 DNA nuclear import and integration are mitosis independent in cycling cells

Abstract

An essential step in human immunodeficiency virus type 1 (HIV-1) replication is the movement of the viral preintegration complex from the cytoplasm into the nucleus. The pathway(s) and timing for HIV-1 DNA nuclear entry in cycling cells have not been established. Here, we show that if cycling cells are infected before S phase, viral DNA can be integrated prior to passage of the host DNA replication fork through the integration site, as indicated by stable inheritance in both daughter cells. We conclude that efficient nuclear entry can occur independently of mitotic nuclear disassembly in cycling cells.

FIG. 1.

Experimental design and interpretation. (A) Diagram showing the outcome of retroviral DNA integration into unreplicated (top) or replicated host DNA (bottom) in cells progressing through the cell cycle. Host DNA is depicted as a single acrocentric chromosome. Integration into replicated DNA is depicted during S but could occur during G₂ or post-mitosis. If mitosis is required for nuclear entry and integration, only AS segregation would be observed. See text for a further description. (B) Experimental design. Mitotic cells (M) were prepared by shake-off. After entry into G₁, cells were infected with HIV-1, ASV, or MLV GFP vectors. Pedigrees show outgrowth of the colony after synchronous cell division. Open and closed circles indicate uninfected and infected cells, respectively. Diagram shows predicted outcomes if integration occurs in unreplicated (top) or replicated (bottom) host DNA, resulting in SY or AS segregation, respectively.

FIG. 2.

Synchronization of HeLa cells and delineation of S phase. (A) Mitotic HeLa cells were prepared by brief nocodazole treatment, shake-off, and replating. Phase-contrast micrographs of cells after replating and attachment (top, 0.5 h after plating) or after G₁ entry (bottom, 3 h after plating) are shown. Different fields are shown in the two micrographs. Cell doublets that are flat, clearly indicating G₁ entry (e.g., two colonies on the right), were typically marked prior to infection such that subsequent GFP segregation could be attributed to infection of synchronized cells. (B) HeLa cells were prepared by mitotic shake-off as in panel A and were analyzed for DNA content by FACScan analysis with standard techniques. Series on the left shows entry into S phase, and series on the right shows exit from S phase (from separate synchronization experiments). Time, in hours post-mitotic shake-off, is indicated to the right of each graph.

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 of GFP expression after infection of G₁ cell doublets with the HIV-1 and MLV GFP vectors and subsequent synchronous outgrowth. Examples of AS and SY patterns are 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. 2 and were infected with the HIV-1 GFP vector at 13 or 16.5 h post-M phase, as indicated at the top. As a comparison, data for the 3-h time point are reproduced from Fig. 3A. The timing of cell cycle stages (determined in Fig. 2B) is shown below the graph. Colonies were scored according to SY, AS, or AS* patterns, and the results are expressed as a percentage of total informative GFP colonies for each time point. The sum of columns at each time point is 100%. Standard deviations (error bars) are shown for the most prominent colony type. (B) Effect of cell cycle delay on frequencies of SY and AS segregation. Diagram of experimental design is shown. Cells were infected at 3 or 7 h after G₁ entry (open arrows) and were treated with aphidicolin (APH) for 18 h and released (filled arrow) or were left untreated as a control. The transient drug-induced G₁/S arrest is indicated (X). (C) Results of experiment diagrammed in panel B. Quantitation of SY segregation as a percentage of total of informative GFP-positive colonies is shown.