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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 2013 Mar 19;110(14):5636–5641. doi: 10.1073/pnas.1216254110

Single-Cell Imaging of HIV-1 Provirus (SCIP)

Cristina Di Primio a, Valentina Quercioli a, Awatef Allouch b, Rik Gijsbers c, Frauke Christ c, Zeger Debyser c, Daniele Arosio d, Anna Cereseto e,1
PMCID: PMC3619299  PMID: 23513220

Abstract

Recent advances in fluorescence microscopy provided tools for the investigation and the analysis of the viral replication steps in the cellular context. In the HIV field, the current visualization systems successfully achieve the fluorescent labeling of the viral envelope and proteins, but not the genome. Here, we developed a system able to visualize the proviral DNA of HIV-1 through immunofluorescence detection of repair foci for DNA double-strand breaks specifically induced in the viral genome by the heterologous expression of the I-SceI endonuclease. The system for Single-Cell Imaging of HIV-1 Provirus, named SCIP, provides the possibility to individually track integrated-viral DNA within the nuclei of infected cells. In particular, SCIP allowed us to perform a topological analysis of integrated viral DNA revealing that HIV-1 preferentially integrates in the chromatin localized at the periphery of the nuclei.

Keywords: yH2AX, retargeting, CBX-LEDGF

Technical developments in imaging-based techniques have greatly improved our understanding of HIV–host cell interactions. HIV-1 virions labeled with fluorophores were pivotal in shedding light onto multiple aspects of the virus–host interplay during all steps of HIV-1 replication cycle (1–13). Nevertheless, few optical approaches have been so far developed to visualize viral particles within the nuclear compartment (14, 15), which limits our comprehension of the interaction between HIV-1 and the nuclear architecture. Moreover, the existing detection tools are based on the visualization of the viral protein complexes or envelope but not of the viral DNA with the only exception of the fluorescence in situ hybridization (FISH) technique. Even though FISH is a powerful technique, it is not very sensitive for HIV-1 detection and moreover disrupts the native architecture of the nuclear compartment as it requires harsh denaturation conditions. In addition, this technique does not allow the discrimination between integrated and nonintegrated viral DNA (16, 17). Here we describe a fluorescent approach to visualize HIV-1 DNA in the nuclear compartment of infected cells. We exploited a site-specific genome engineering technique that represents one of the most promising approaches to detect specific genome regions in modified organism (18) allowing for their spatial localization in the cell (19, 20). This technique couples endogenous repair pathways, induced by rare cutting endonuclease, with immunofluorescence analysis. Rare cutting endonucleases, such as the yeast-homing endonuclease I-SceI, specifically cuts target sequences that cannot be found in the mammalian genome. By engineering DNA to contain the I-SceI cleavage site, it is thus possible to induce endogenous repair mechanism for double-strand breaks (DSBs) at specific genomic positions. DSB repair leads to the formation of distinct subnuclear structures that are generally referred to as “foci” (21). The first sensor of the DSB is the histone H2AX, which becomes massively phosphorylated at serine 139 (γ-H2AX). Foci of DNA repair are thus visible through immunofluorescence by using specific γ-H2AX antibodies. Here, by inserting an I-SceI site in the HIV-1 genome, we show that individual proviral DNA can be efficiently detected at the level of a single infected cell [Single-Cell Imaging of HIV-1 Provirus (SCIP)]. The power of SCIP is the temporal and spatial analysis of HIV-1 in individual nuclei of infected cells, a great benefit over currently used techniques. A 3D topological analysis, performed by SCIP, demonstrated that integrated viral DNA localizes at the periphery of the nuclei revealing important insights in the nuclear biology of HIV-1.

Results

Construction of the HIV–I-SceI Reporter System and Viral DNA Cleavage Validation.

DNA DSB leads to the phosphorylation of H2AX molecules and the formation of γ-H2AX foci, which can be visualized by immunofluorescence (IF) technique. The ability of the rare cutting endonuclease I-SceI to induce specific DSB was exploited to detect HIV-1 DNA in infected cells. In specific, we engineered HIV-1 viral particles by inserting an 18-bp target site for the endonuclease I-SceI into viral transfer vectors (pHR-CMVGFP-I-SceI, pHR-CMVΔGFP-I-SceI; Fig. 1A); the cleavage activity of the enzyme on viral DNA was then verified by recombinant I-SceI digestion (Fig. S1).

Fig. 1.

Fig. 1.

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Detection of γ-H2AX foci associated with HIV-1 DNA in U2OS cells. (A) Schematic representation of viral constructs and mechanism of γ-H2AX foci formation. The plasmid pHR-CMVGFP was modified by inserting the I-SceI target site to produce either the HIV-CMVGFP-I-SceI virions expressing GFP in infected cells or the HIV-CMVΔGFP-I-SceI GFP deleted. The endonuclease I-SceI (blue spot) exogenously expressed by transfecting infected cells cleaves the I-SceI site producing DNA DSBs. H2AX molecules at the DSB site become phosphorylated forming γ-H2AX foci (red spots) detectable by immunofluorescence. (B) U2OS-6 and U2OS-8 stable cell clones containing pHR-CMVGFP-I-SceI and expressing or not expressing the I-SceI endonuclease (pCBASce) by transient transfection were immunostained with anti–γ-H2AX antibodies: γ-H2AX foci (in red) viral GFP expression (in green). Scale bars, 10 μm. (C) Chromatin immunoprecipitation (ChIP) analysis of viral DNA associated with γ-H2AX. (Upper) Schematic representation of the positions of the primers used for real-time PCR quantification. (Lower) The γ-H2AX ChIP analysis in the U2OS-8 cell clone expressing or not expressing the I-SceI endonuclease. Relative enrichment represents the enrichment of γ-H2AX compared with an IgG control (normalized with a PCR internal control to a locus other than the viral DNA). Error bars represent SDs from at least two independent experiments.

The system was initially evaluated in U2OS clones obtained by stable transfection of the transfer vector (pHR-CMVGFP-I-SceI) and subsequent clonal selection. Representative stable cell clones, U2OS-6 and U2OS-8, containing an average of 6 and 22 integrated DNA copies, respectively, as quantified by quantitative PCR (qPCR), were transfected with the I-SceI endonuclease encoding plasmid (pCBASce) and immunostained with specific anti–γ-H2AX antibodies to detect nuclear foci by confocal microscopy. Fig. 1B shows that γ-H2AX foci are clearly detectable in nuclei of U2OS-6 and U2OS-8 cells expressing the I-SceI endonuclease, although few background signals, likely generated by spontaneous DNA strand breaks, are detected in nontransfected cells. Moreover, cells positive for γ-H2AX foci were also expressing the viral reporter gene (GFP; Fig. 1B). Quantification analysis of the γ-H2AX foci revealed that the total number of γ-H2AX foci per nucleus closely correlates with the integrated viral DNA copies measured by qPCR (Fig. S2 A and B).

To further prove that the γ-H2AX foci detected by IF correspond to the viral DNA containing the I-SceI site we performed chromatin immunoprecipitation experiments (ChIP) using γ-H2AX antibodies. Chromatin immunoprecipitates from U2OS-8 cells expressing or not expressing the I-SceI endonuclease (Fig. S2C) were amplified by real-time PCR with primer pairs covering the immediate proximity of the I-SceI site (primer pairs A and B) and with primers targeting the viral DNA (MH531-532) (22) (Fig. 1C). We observed an enrichment of γ-H2AX (10–30-fold) with primers specific for the I-SceI region and viral sequences; no enrichment was obtained in control samples (Fig. 1C).

Similar results were obtained using stable clones infected with HIV-1 particles containing the I-SceI restriction site (HIV-CMVGFP-I-SceI) and pseudotyped with the vesicular stomatitis virus G (VSV-G) envelope (Fig. S2 D and E). Taken together, our data proved that the I-SceI target site–mediated modification of the HIV-I genome allows visualizing viral DNA at the nuclear level.

Visualization of Viral DNA in Infected Cells.

The HIV-I-SceI reporter system was then evaluated in the context of viral infection using HIV-CMVGFP-I-SceI viral particles pseudotyped with the VSV-G envelope, in U2OS cells transfected with the pCBASce plasmid. Cells were immunostained with anti–γ-H2AX antibodies at 24 and 48 h postinfection and analyzed by 3D confocal microscopy. As shown in Fig. 2A, γ-H2AX foci were detected in infected cells expressing both the GFP reporter gene and the I-SceI endonuclease (Fig. 2A, Top), and background signals could be detected in infected (GFP positive) cells in the absence of the I-SceI endonuclease (Fig. 2A, Middle). When infecting cells with or without I-SceI endonuclease with the control viral vector HIV-CMVGFP, only background signals derived from spontaneously formed DSBs was observed (Fig. S3).

Fig. 2.

Fig. 2.

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Visualization of viral DNA in infected cells. (A) Detection of γ-H2AX foci and GFP in cells expressing (Top) or lacking (Middle) the exogenous I-SceI endonuclease. Cells were infected by integration competent virus or by integration defective virus (D116A) (Bottom). Scale bars, 10 μm. (B) Quantification of γ-H2AX foci number at 24 and 48 hpi in cells infected with 4 RTU of pHR-CMVGFP-I-SceI or pHR-CMVGFP-I-SceID116A (means ±SEM from three independent experiments after background subtraction). At least 150 cells per experiment have been analyzed; [Pwt vs. D116A = 1.332e-15 at 24 hpi, Pwt vs. D116A = 4.692e-13 at 48 hpi, Kolmogorov–Smirnov test (KS test)]. (See Materials and Methods for background correction.) (C) Quantification of the number of γ-H2AX foci at 48 h and 13 d postinfection. Cells were infected with 0.004 RTU (white bars), 0.4 RTU (gray bars), or 4 RTU (black bars) (means ± SEM from at least two independent experiments after background subtraction); at least 200 cells per experiment have been analyzed. (D) Quantification of γ-H2AX foci number per cell with respect to percentage of GFP positive cells at the three different RTU after background subtraction; (P0.04RTU vs. bg = 0.04542, P0.4RTU vs. bg = 2.7e-5, P4RTU vs. bg = 2.9e-7, KS test).

Because, in the early replication steps, the viral DNA species exist as nonintegrated or integrated proviral DNA, we investigated the ability of the HIV-I-SceI visualization system to detect the two different forms. To this aim we infected cells with an integration defective HIV-1 virus (HIV-CMV-GFP-I-SceID116A) bearing the D116A mutation in the integrase active site causing a catalytic defect in integrase. Following infection this mutant virus produces either linear or circularized (2-LTR) nonintegrated DNA. Results in Fig. 2A, Bottom show that even though infected cells produce GFP from nonintegrated forms, no γ-H2AX foci could be detected. To better describe this observation the foci formation in cells infected with HIV-CMV-GFP-I-SceI wild-type or integration-defective (D116A) viruses was quantified. The quantification analysis was set up by comparing three approaches in cells infected with increasing amounts of HIV-CMVGFP-I-SceI. The first approach, 2D quantification, consisted in counting the number of γ-H2AX foci in the optical section taken in the center of each cell nucleus (Fig. S4A). The second approach measured the total γ-H2AX foci fluorescence intensity in the central optical section of each nucleus (Fig. S4B). The third approach, 3D quantification, consisted in the automated counting of the number of γ-H2AX foci within the entire nuclear volume and was based on the nuclear 3D reconstruction from the Z stacks, by using dedicated imaging software (Imaris BITPLANE Scientific Software, ImageJ NIH). These three approaches showed analogous fold increase at increasing viral titer values (Fig. S4B), thus proving the validity HIV-I-SceI reporter system to detect HIV-1 genomes in infected cells. In the further study we applied the 2D quantification approach. We observed that in noninfected control cells the background number of γ-H2AX foci varied among individual experiments (Fig. S4C). For this reason a rigorous procedure was set up for the evaluation of the background number of foci. Background number of foci was quantified in noninfected cells expressing the I-SceI enzyme and subtracted in each individual experiment from the number of foci detected in cells infected with the HIV-CMVGFP-I-SceI (exemplified in Fig. S4 C–E and Materials and Methods).

The foci quantification in cells infected with HIV-CMV-GFP-I-SceI wild-type or integration-defective (D116A) viruses was performed at 24 and 48 h postinfection. We observed that cells infected with wild-type virus were positive for γ-H2AX foci at both time points, but only background values of γ-H2AX foci were detected by using the HIV-CMV-I-SceID116A virus (Fig. 2B and Fig. S5). Therefore, we can conclude that the HIV-I-SceI system specifically detects HIV-1 DNA integrated into the host genome of individual infected cells; accordingly this method was named Single-Cell Imaging of HIV-1 Provirus.

To further explore the correlation of γ-H2AX foci formation with amounts of viral genomes in time and to establish the lowest multiplicity of infection that can be used in SCIP, cells were infected with increasing amounts of HIV-CMVGFP-I-SceI (0.04, 0.4, and 4 RT units (RTU) as measured in ref. 23) and analyzed at 48 h and 13 d postinfection. To avoid continuous foci formation at both time points the I-SceI endonuclease was transfected at 24 h before immunostaining. The number of foci per nucleus detected at both 48 h and 13 d increased consistently with increased viral titers, thus indicating their correlation with the amounts of integrated DNA and their stability from the time of infection (Fig. 2C and Fig. S5). The analysis performed by measuring the number of foci above background in cells positive for GFP, revealed that as few as 15% of infected cells (GFP positive) can be detected through SCIP (Fig. 2D). However, to obtain maximum sensitivity the following experiments were performed using 4 RTU leading to almost 95% infectivity (Fig. 2D).

Finally, to verify the specificity of the γ-H2AX foci signals detected in HIV-CMVGFP-I-SceI infected cells, coimmunostaining was performed with Rad51 or 53BP1 antibodies recognizing DNA repair focus hallmark proteins. Signal colocalization was observed between γ-H2AX and Rad51 or 53BP1 foci, thus confirming the specificity of the γ-H2AX foci (Fig. S6A).

These results demonstrate that SCIP is a technique to simultaneously analyze HIV-1 expression and integration in individual cells. This single-cell investigation tool, as opposed to the conventional bulk approaches, is relevant to monitor the expression/integration profiles within a population of infected cells.

SCIP Analysis after Inhibition of Retroviral Replication.

To further test the SCIP technique as a tool to analyze HIV-1 proviral DNA at single-cell level, infections were performed in different conditions, where viral replication was inhibited by cofactor knockdown or antivirals.

Transportin-SR2 (TRN-SR2, TNPO3) is a cellular import factor of SR-rich proteins that is involved in HIV-1 entry in the nuclei of infected cells (24–31). In fact, after TRN-SR2 knockdown HIV-1 nuclear entry is inhibited and, consequently, the amount of integrated DNA is reduced. Cells depleted for TRN-SR2 (using siRNA-SR2) and expressing the I-SceI endonuclease were infected with the HIV-I-ΔGFP-SceI virus (4 RTU) and quantitatively analyzed for the amounts of γ-H2AX foci. As shown in Fig. 3 A and B and Fig. S5, the number of γ-H2AX foci dropped to background levels in TRN-SR2 knockdown cells thus indicating a lack of viral integration. Because alterations of TRN-SR2 levels may interfere with H2AX phosphorylation, the formation of foci was verified in TRN-SR2 knockdown cells treated with neocarzinostatin (NCS), a strong inducer of DSBs foci formation. In these conditions H2AX molecules remain phosphorylated proving normal foci formation after depletion of TRN-SR2 (Fig. S6B).

Fig. 3.

Fig. 3.

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SCIP analysis in transportin-SR2 knockdown cells and in anti-retroviral drug treated cells. (A) Immunofluorescence detection of γ-H2AX foci (yellow spots) in cells transfected with fluorescent siRNA (blue spots) targeting TRN-SR2 mRNA (siRNA-SR2) or a mismatch control siRNA (siRNA-MM) and infected with 4RTU of HIV-CMVΔGFP-I-SceI. HIV-CMVΔGFP-I-SceI virus was used to avoid the crosstalk between fluorophores. Nuclear middle z stacks are shown. Scale bars, 10 μm. (B) Quantification of γ-H2AX foci in cells transfected by siRNA-MM (black bars) and cells transfected by siRNA-SR2 (white bars) after background subtraction; (p MM+ISceI vs. SR2+ISceI = 7.946e-12, KS test). (C) Immunofluorescence detection of γ-H2AX foci (red spots) in infected cells (4 RTU) either untreated (ctrl) or treated with antiretroviral drugs as indicated. (Right) The γ-H2AX foci signal merged with the GFP signal. (D) Quantification of γ-H2AX foci in infected cells either untreated (ctrl-black bar) and in or treated as indicated (white bars) (means ±SEM from at least two independent experiments after background subtraction). Drugs concentrations are indicated. (Put vs. 100nM AZT = 9.555e-05, Put vs. 1uM = 4.485e-07, Put vs. 10uM = 4.384e-08, Put vs. 2uM nev = 0.0003, Put vs. 10uM nev = 8.572e-13, Put vs. 100nM ral = 3.466e-11, Put vs. 500nM ral < 2.2e-16, Put vs. 1uM ral < 2.2e-16, Put vs. 100nM CX = 0.0004, Put vs. 1uM CX = 3.046e-13, Put vs. 10uM CX = 6.122e-10, KS test). (See SI Materials and Methods for background correction.)

To further test SCIP as an efficient tool for the analysis of HIV replication steps up to integration, infections were performed with the HIV-CMVGFP-I-SceI vector (4 RTU) in the presence of reverse transcriptase inhibitors (AZT and nevirapine) or an integrase inhibitor (raltegravir) used in antiretroviral therapy. In addition, a recently developed antiviral compound, the LEDGIN CX05045, which inhibits HIV-1 integration by disrupting the binding between integrase and LEDGF/p75, was also used (32, 33).

The quantitative analysis of these experiments demonstrates that both reverse transcriptase and integrase inhibitors decreased the number γ-H2AX of foci (Fig. 3 C, Middle and D; and Fig. S5). The distinct activity of these drugs in HIV-1 replication is indicated by the differential expression of GFP. In fact, GFP expression was greatly reduced in cells treated with reverse transcriptase inhibitors (nevirapine and AZT) as reported both by microscopy (Fig. 3C, Left) and by FACS analysis (Fig. S7) at 36 h postinfection. However, cells treated with integrase inhibitors (raltegravir and CX05045) show unaltered levels of GFP expression (Fig. 3C, Left; and Fig. S7) produced by nonintegrated viral DNA.

Therefore, SCIP has the power to discriminate whether the absence of integration following infection (no γ-H2AX foci) is generated by specific inhibition of viral integration (GFP positive cells) or by upstream viral impairments (GFP negative cells).

Integrated Viral DNA Localizes at the Nuclear Periphery.

HIV-1 preintegration complexes (PICs) preferentially localize at the nuclear periphery, suggesting that integration may preferentially occur at the border of the nuclear compartment (14). To verify this hypothesis we analyzed the localization of the integration spots through SCIP. After measurement of the individual nuclear radius and the γ-H2AX foci radial positions, the γ-H2AX foci distances from the nuclear lamin were established. The analysis in U2OS cells revealed that 48 h postinfection in U2OS cells, 55% of the integrated viral DNA positioned within 1.5 μm of distance from the lamin (Fig. 4 A and B, black bars). The same analysis was performed in CEMss T cells, a lymphoid cell line, thus more physiologically relevant in HIV-1 infection. We observed that 62% of HIV-1 proviruses localized in the periphery of the nucleus (Fig. 4 C and D) and were mostly detected within 0.5 μm distance from the lamin. Therefore, compared with U2OS, cells the peripheral positioning was even more pronounced. The viral arrangement along the inner surface of the nuclear lamin of the CEMss-infected cells clearly emerged from the z sectioning of the infected cells (Fig. 4E).

Fig. 4.

Fig. 4.

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Analysis of the proviral DNA nuclear localization. (A) Immunofluorescence images of γ-H2AX foci (in red) and nuclear lamin (in blue) from U2OS cells infected with 4 RTU of HIV-CMVGFP-I-SceI. Nuclear middle z stacks are shown. Scale bars, 10 μm. (B) Distribution of the distances of γ-H2AX foci from the nuclear lamin. Black bars represent the frequency counts of γ-H2AX foci in cells infected with HIV-CMVGFP-I-SceI, red bars represent the frequency counts of γ-H2AX foci induced by NCS (P = 3.273e-10, KS test). (C) Immunofluorescence images of γ-H2AX foci (in red) and nuclear lamin (in blue) from CEMss T cells infected with 4 RTU of HIV-CMVGFP-I-SceI. Nuclear middle z stacks are shown. Scale bars, 10 μm. (D) Distribution of the distances of γ-H2AX foci from the nuclear lamin. Black bars represent the frequency counts of γ-H2AX foci in cells infected with HIV-CMVGFP-I-SceI, red bars represent the frequency counts of γ-H2AX foci induced by NCS (P = 0.0004, KS test). (E) Z sectioning of an infected CEMss nucleus. Scale bars, 10 μm.

To verify that no bias was introduced by the SCIP approach on the nuclear topology, the analysis was also performed in U2OS and CEMss cells treated with NCS to induce random DNA DSBs. As shown in Fig. 4 B–D (red bars) NCS-induced γ-H2AX foci do not show any preferential nuclear localization by distributing into the whole nuclear compartment. Finally, γ-H2AX foci generated by spontaneous DSBs in U2OS and CEMss cells shows no preferential distribution within the nucleus in the absence of infection (Fig. S8 A and B).

To investigate over time the behavior of the HIV-1 provirus in the nuclear compartment, the same topological analysis was also performed 13 d postinfection. HIV-1 proviruses distribution changed remarkably over the explored time interval (Fig. 5A). Moreover, according to the classical nuclear topology analysis where the nucleus can be divided into three concentric zones of equal surface area (34, 35), the outcome of the SCIP analysis indicated a positioning of HIV-1 proviruses at the nuclear periphery: at 48 h postinfection, the majority of the proviral DNA localized in the outer rim of the nuclear compartment and at day 13 the virus was randomly distributed in the three nuclear zones defined (Fig. 5B).

Fig. 5.

Fig. 5.

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Analysis of the proviral DNA nuclear localization at different time points after infection. (A) Distribution of distances of proviruses from the nuclear lamin at 48 h (black bars) and 13 d postinfection (red bars) (P = 5.76e-08, KS test). (B) Data are represented in bar graphs as the percentage of γ-H2AX foci in the three concentric zones of equal surface area. The peripheral zone is in black, the middle zone in light gray, and the inner zone in gray.

We next applied SCIP to cells where HIV-1 integration is retargeted toward heterochromatin and intergenic regions, thus altering the physiological lentiviral integration preference toward gene-rich units (36). This experimental system was obtained by expressing in target cells a chimera LEDGF/p75 engineered to contain an alternative chromatin-binding domain, CBX1, at its N terminus (CBX-LEDGF325–530). The topological analysis of viral associated γ-H2AX foci in CBX1 hybrid cell lines revealed that proviral DNA is randomly distributed in the nuclei, showing no preferential localization toward the nuclear periphery (Fig. 6 A and B; see Fig. S8 C and D for background foci distribution).

Fig. 6.

Fig. 6.

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Analysis of the proviral DNA retargeting. (A) Distribution of the distances of HIV-1 γ-H2AX foci from the nuclear lamin in HeLa control cells (black bars) and in cells expressing CBX-LEDGF325–530 (blue bars) (P = 2.147e-06, KS test). (B) Data are represented in bar graphs as the percentage of γ-H2AX foci in the three concentric zones of equal surface area as in Fig. 5B.

Therefore, SCIP analysis performed in infected cells showed that HIV-1 preferentially integrate at the nuclear border in normal conditions, and random distribution is observed in cells engineered to retarget integration toward noncanonical spots of the chromatin. Finally, proviral DNA localization is dynamic because repositioning could be observed at later time points from infection.

Discussion

Here we report SCIP, an imaging technique to efficiently visualize HIV-1 DNA integrated into the host genome of individual cells. The possibility to analyze viral integrated DNA within structurally intact nuclei of individual cells allowed studying the topology of HIV-1 in the nuclear compartment. This analysis revealed that at 48 h postinfection, HIV-1 is preferentially localized in the nuclear periphery. The observed spatial distribution is consistent with the one obtained by analyzing fluorescently labeled HIV-1 protein complexes at the nuclear level (14). It is interesting to note that the same analysis performed at day 13 from infection revealed a relocalization of proviral DNA toward the center of the nucleus with a random distribution in the nuclear compartment. The localization of PICs and provirus in the periphery of the nucleus at early time points strongly suggests that the virus integrates soon after its transition through the nuclear envelope. Nevertheless, once the HIV-1 genome is stably integrated into the host peripheral chromatin, its radial distribution substantially changes over time probably owing to a rearrangement of the chromosome territory where the integration occurred. We observed that in cells engineered to retarget integration toward nonphysiological integration spots (CBX-LEDGF325–530), proviral DNA does not show any preferential nuclear distribution. These observations clearly indicate that in addition to a linear preference of HIV-1 toward gene-rich regions of the chromatin (36), the virus also acquires a specific spatial orientation within the nuclear compartment.

It is remarkable that SCIP does not detect linear or circular nonintegrated viral DNA. This could be due to the conformation of the viral cDNA within the structure of the preintegration complex, where the I-SceI target site would not be accessible to endonucleases because masked by viral and cellular factors (37). An alternative explanation could be that nonintegrated forms of viral DNA may not be adequately chromatinized to determine γ-H2AX accumulation at I-SceI sites.

Nevertheless, the presence of nonintegrated viral forms can be indirectly reported in γ-H2AX negative cells by the presence of the GFP expressed from the nonintegrated viruses. Therefore, by combining GFP and SCIP it is possible to determine three different infectivity conditions: (i) HIV-1 infection leading to completed integration revealed by cells positive for both γ-H2AX and GFP signals, (ii) viral DNA reaches the nuclei of the cells but integration is impaired revealed by cells negative for γ-H2AX and positive for GFP owing to the expression of the nonintegrated DNA, and (iii) absence of viral DNA in the nuclei as a result of replication impairments upstream from nuclear import, revealed by the lack of both GFP and γ-H2AX signals.

An additional important feature of SCIP is the possibility to analyze individual infected cells without culture clonal selection, as opposed to the bulk analysis performed with conventional approaches. This advancement of the SCIP technique revealed that cells are heterogeneously infected showing different densities of HIV-1 genomes per cell (Figs. S4 and S5). This observation is consistent with former reports showing that clones derived from the same infection display variable numbers of proviruses (38, 39).

The power of SCIP to monitor expression /integration in individual cells might be key to investigate the still obscure mechanisms leading to HIV-1 latency (40). Taken together, these data strongly demonstrated that SCIP is a robust and clear-cut readout for understanding HIV-1 replication in details such as cellular conditions or factors affecting HIV-1 nuclear entry and the topology of integration at the individual cell level. The technical advantages and the multiple endpoints detection provided by this system are expected to contribute as a powerful tool also for large-scale analysis. In fact, high content analysis of γ-H2AX foci was reported (41). Similarly, by coupling SCIP to a high-throughput fluorescence microscopy platform, the technique here reported may become a systematic qualitative and quantitative screen for HIV-1 potential cofactors and antiretroviral drugs.

Materials and Methods

For a detailed description, please refer to SI Materials and Methods. It includes detailed procedures for confocal microscopy imaging acquisition and analysis. It also includes description of constructs engineering, virus production, cell culture conditions, and immunofluorescence procedures.

Supplementary Material

Supporting Information
supp_110_14_5636__index.html (6.8KB, html)

Acknowledgments

The authors are grateful to the laboratory of W. Thys and M. Giacca for valuable discussion and to A. Calvello for technical assistance. This work was supported by grants from the European Union Seventh Framework Programme (THINC, Health-2008-201032), by the Istituto Superiore di Sanità Italian AIDS Program (Grant 40H90), by the Provincia Autonoma di Trento (COFUND Project, Team 2009 – Incoming), and by FIRB 2008 Futuro in Ricerca (RBFR08HSWG).

Footnotes

The authors declare no conflict of interest.

*This Direct Submission article had a prearranged editor.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1216254110/-/DCSupplemental.

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