Accumulation and intranuclear distribution of unintegrated human immunodeficiency virus type 1 DNA

Abstract

The RNA genome of human immunodeficiency virus type 1 (HIV-1) is converted into DNA after infection in order to integrate into the host cell DNA. However, a large number of these reverse-transcribed genomes remain unintegrated in the nucleus of infected cells. Currently, there are no data available about the intranuclear distribution pattern of unintegrated HIV-1 DNA in relation to nuclear structures as observed on the single-cell level. In the present study, we investigated the intranuclear fate of unintegrated viral DNA in cell lines expressing CD4 and coreceptors (HOS-CD4.CCR5 and U373-MAGI-CXCR4(CEM)) infected with HIV-1 (strain 89.6). We used a novel approach to distinguish in situ unintegrated from integrated viral DNA by performing fluorescent in situ hybridization on cells in which stress-induced chromosome condensation had been induced, a procedure that contracts chromosomes independent of the cell cycle. Cells infected for 15 h accumulated large amounts of HIV-1 DNA which was located between the condensed chromosome strands, allowing the identification of this viral DNA as unintegrated. In contrast, in HeLa/LAV, a cell line carrying integrated HIV-1 genomes, the great majority of viral DNA colocalized with the cellular DNA. We show that unintegrated HIV-1 DNA does not evenly distribute within the host cell nucleus but tends to aggregate into clusters containing many copies of the viral genomes. The formation of these DNA clusters was independent of viral DNA replication and thus appeared to result solely from multiple infections. The DNA aggregates remained in the nuclei of infected cells for at least 25 h after the infection was stopped. The emergence of transcription sites, which most likely denote sites of the integrated provirus, lagged clearly behind the accumulation of viral DNA. These transcription foci could not be linked to unintegrated DNA molecules, suggesting that this DNA type is unable to transcribe, at least at levels comparable to those of integrated DNA. Neither unintegrated HIV-1 DNA nor transcription foci nor integrated DNA was observed to associate with nuclear domain 10 (ND10), a nuclear structure known to represent the site where several DNA viruses replicate and transcribe. Also, HIV-1 does not modify ND10 at early or late times of infection. There was no specific association of HIV-1 transcripts with splicing factor SC35 domains, in contrast to what has been reported for a number of both cellular and viral genes. Surprisingly, unintegrated HIV-1 DNA was found to accumulate within or in close association with SC35 domains, demonstrating a specific distribution of the viral DNA within the host cell nucleus. Taken together, our results demonstrate that unintegrated proviral HIV-1 DNA does not randomly localize within infected cells but preferentially aggregates in the nucleus within SC35 domains.

FIG. 1

FISH and immunostaining of HIV-1-infected cells. Labeled components are denoted in their respective color in each panel. In some images (A, B, F, K, and L), Hoechst 33258 staining of cellular DNA is shown in blue to visualize nuclei. Images C through E and G through J were acquired by confocal microscopy, whereas images A, B, F, K, and L represent nonconfocal micrographs. (A) HOS-CD4.CCR5 cell after 15 h of infection with HIV-1 and hybridization with an HIV-1-specific probe to detect viral DNA. The nucleus displays numerous HIV-1 DNA signals of various sizes. (B) In HeLa/LAV, a permanently HIV-1-infected cell line that carries integrated viral genomes, individual HIV-1 DNA signals of uniform size are detectable by FISH (arrows). (C) Infected HOS-CD4.CCR5 cell that underwent the SICC procedure to condense interphase chromosomes and was probed for HIV-1 DNA. Viral DNA can be found almost exclusively between the contracted chromosomes stained by propidium iodide. (D) HeLa/LAV cell processed as described in the legend for panel C. In contrast to DNA in infected HOS-CD4.CCR5 cells, most viral DNA in HeLa/LAV cells localizes on the condensed chromosomes (arrow). Note that the HIV-1 signal is present as a double dot, indicating the presence of an integrated viral genome in both sister chromatids after S phase. (E) Visualization of HIV-1 transcripts in an infected HOS-CD4.CCR5 cell in which SICC was induced. Viral RNA foci denote the sites where HIV-1 transcripts are released from the integrated proviral DNA into the interchromosomal space. The insert shows at higher magnification that these RNA foci actually emerge from the chromosomal DNA. Note that for detection of transcripts, cells are not treated with RNase, resulting in additional staining of RNA by propidium iodide as seen in part of the cytoplasm. (F) Accumulation of HIV-1 DNA in the nucleus of a HOS-CD4.CCR5 cell upon multiple infection. Unintegrated HIV-1 DNA aggregates into clusters as revealed by signals of different sizes. (G) Infected HOS-CD4.CCR5 cell probed for HIV-1 RNA and immunostained with antibodies against the ND10 protein Sp100. HIV-1 transcripts are concentrated into foci (arrows) which are not associated with ND10. (H) Infected HOS-CD4.CCR5 cell probed for HIV-1 DNA and stained with antibodies against Sp100 to visualize ND10. DNA signals are not associated with ND10. (I) Visualization of HIV-1 RNA by FISH and splicing factor SC35 by immunostaining of an infected HOS-CD4.CCR5 cell. RNA foci and SC35 domains are not specifically associated with each other but show a random nuclear distribution. The right cell is uninfected. (J) Infected HOS-CD4.CCR5 cell stained for HIV-1 DNA by FISH and SC35 by immunofluorescence. HIV-1 DNA associates with SC35 domains. (K) U373-MAGI-CXCR4_CEM cells infected for 15 h and probed for HIV-1 DNA. Virtually all cells contain HIV-1 DNA. (L) The same experiment described in the legend to panel K was performed, but cells were probed for HIV-1 RNA. Only 13% of the cells display signals specific for HIV-1 RNA (arrow).

FIG. 2

Sizes of hybridization signals representing integrated DNA (in HeLa/LAV cells) and unintegrated DNA (in HOS-CD4.CCR5 cells infected for 15 h). The sizes of 150 randomly chosen signals in each cell line were quantified by counting the number of pixels of each signal (see Materials and Methods). Integrated DNA is detected as more or less uniform signals of small sizes (two to seven pixels), whereas unintegrated DNA comprises a wide variety of sizes, indicating its tendency to aggregate into clusters.

FIG. 3

U373-MAGI-CXCR4_CEM cells were infected with HIV-1 for 15 h and then further incubated in virus-free medium. Cells were fixed 6, 15, and 40 h after starting the infection and probed for HIV-1 DNA or RNA. Six hours after infection, viral DNA, but no RNA, was visible in 15% of the cells. After 15 h, virtually all cells contained viral DNA whereas RNA was detectable in only 13% of the cells. Forty hours after infection, the number of cells containing HIV-1 transcripts increased to 95%. Viral DNA remained detectable in all cells after 40 h even though the HIV-1-containing supernatant had been removed earlier at 15 h postinfection.