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HIV reproducibly establishes a latent infection after acute infection of T cells in vitro

2003; Springer Nature; Volume: 22; Issue: 8 Linguagem: Inglês

10.1093/emboj/cdg188

1460-2075

Albert Jordan, Dwayne A. Bisgrove, Eric Verdin,

Cytomegalovirus and herpesvirus research

Article15 April 2003free access HIV reproducibly establishes a latent infection after acute infection of T cells in vitro Albert Jordan Albert Jordan Gladstone Institute of Virology and Immunology, University of California, San Francisco, CA, 94141 USA Present address: Center for Genomic Regulation, Passeig Marítim, 37–49, E-08003 Barcelona, Spain Search for more papers by this author Dwayne Bisgrove Dwayne Bisgrove Gladstone Institute of Virology and Immunology, University of California, San Francisco, CA, 94141 USA Search for more papers by this author Eric Verdin Corresponding Author Eric Verdin Gladstone Institute of Virology and Immunology, University of California, San Francisco, CA, 94141 USA Department of Medicine, University of California, San Francisco, CA, 94141 USA Search for more papers by this author Albert Jordan Albert Jordan Gladstone Institute of Virology and Immunology, University of California, San Francisco, CA, 94141 USA Present address: Center for Genomic Regulation, Passeig Marítim, 37–49, E-08003 Barcelona, Spain Search for more papers by this author Dwayne Bisgrove Dwayne Bisgrove Gladstone Institute of Virology and Immunology, University of California, San Francisco, CA, 94141 USA Search for more papers by this author Eric Verdin Corresponding Author Eric Verdin Gladstone Institute of Virology and Immunology, University of California, San Francisco, CA, 94141 USA Department of Medicine, University of California, San Francisco, CA, 94141 USA Search for more papers by this author Author Information Albert Jordan1,2, Dwayne Bisgrove1 and Eric Verdin 1,3 1Gladstone Institute of Virology and Immunology, University of California, San Francisco, CA, 94141 USA 2Present address: Center for Genomic Regulation, Passeig Marítim, 37–49, E-08003 Barcelona, Spain 3Department of Medicine, University of California, San Francisco, CA, 94141 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (2003)22:1868-1877https://doi.org/10.1093/emboj/cdg188 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The presence of latent reservoirs has prevented the eradication of human immunodeficiency virus (HIV) from infected patients successfully treated with anti-retroviral therapy. The mechanism of postintegration latency is poorly understood, partly because of the lack of an in vitro model. We have used an HIV retroviral vector or a full-length HIV genome expressing green fluorescent protein to infect a T lymphocyte cell line in vitro and highly enrich for latently infected cells. HIV latency occurred reproducibly, albeit with low frequency, during an acute infection. Clonal cell lines derived from latent populations showed no detectable basal expression, but could be transcriptionally activated after treatment with phorbol esters or tumor necrosis factor α. Direct sequencing of integration sites demonstrated that latent clones frequently contain HIV integrated in or close to alphoid repeat elements in heterochromatin. This is in contrast to a productive infection where integration in or near heterochromatin is disfavored. These observations demonstrate that HIV can reproducibly establish a latent infection as a consequence of integration in or near heterochromatin. Introduction Combination anti-retroviral therapy can control HIV-1 replication and delay disease progression. However, despite the complete suppression of detectable viremia in many patients, viremia re-emerges rapidly after interruption of treatment, consistent with the existence of a latent viral reservoir (Finzi et al., 1997; Wong et al., 1997). This reservoir is thought to consist mainly of latently infected resting memory CD4+ T cells (recently reviewed by Chun and Fauci, 1999; Butera, 2000; Pierson et al., 2000). Due to the long half-life of this reservoir (44 months), it has been estimated that its total eradication with current treatment would require over 60 years (Finzi et al., 1999). Latently infected cells contain replication-competent integrated HIV-1 genomes that are blocked at the transcriptional level, resulting in the absence of viral protein expression. HIV depends on both cellular and viral factors for efficient transcription of its genome, and the activity of the HIV promoter is tightly linked to the level of activation of its host cell. HIV transcription is characterized by two temporally distinct phases. The early phase occurs immediately after integration and relies solely on cellular transcription factors. Because of a transcriptional elongation defect in the basal HIV promoter, most transcripts cannot elongate efficiently and terminate rapidly after initiation. However, a few transcripts elongate throughout the genome, resulting in transcription of the viral transactivator Tat. The late phase of transcription occurs when enough Tat protein has accumulated. Tat binds to TAR, recruits the pTEFb complex and causes the hyperphosphorylation of RNA polymerase II, dramatically increasing its ability to elongate (Karn, 1999). To understand how postintegration latency is established, an in vitro cell system reflecting the state of HIV-1 latency is required. While several latently infected cell lines have been established after HIV infection, the proviruses integrated in these cell lines harbored mutations in their Tat–TAR transcriptional axis (Emiliani et al., 1996, 1998) or in NF-κB binding sites in the HIV promoter (Antoni et al., 1994). The presence of mutations in HIV-infected latent cell lines has raised questions about the significance of these lines to latency in vivo. However, the very presence of these mutations has also strengthened the concept that transcription inhibition is critical to the establishment and maintenance of HIV latency. We reported previously that the site of integration of the HIV-1 provirus into the cell genome affects its basal Tat-independent transcriptional activity (Jordan et al., 2001). We therefore predicted that a small fraction of integration sites might lead to such low basal promoter activity that no Tat mRNA would accumulate. Such a provirus would be locked in the early phase of transcription, resulting in functional latency. In this manuscript, we have used recombinant viruses that express green fluorescent protein (GFP) to selectively enrich for rare cells blocked at the transcriptional level during an acute infection. Using this approach, we have isolated a large number of clonal cell lines harboring HIV in a latent state. The latent provirus can be reactivated at the transcriptional level in these cell lines by a variety of agents. This study documents that HIV reproducibly establishes a latent infection with low frequency after acute infection of T cells in vitro. Results Establishment of an in vitro model of HIV-1 latency To determine whether unique integration events can lead to latency, we used an HIV-based retroviral vector containing the Tat and GFP open reading frames both under the control of the HIV promoter in the 5′ long terminal repeat (LTR). We infected a culture of the lymphocytic cell line Jurkat with viral particles containing this vector and used differential fluorescence-activated cell sorting (FACS) based on GFP expression (Figure 1A). First, we infected Jurkat cells with the LTR–Tat–IRES–GFP virus at a low m.o.i. and isolated GFP-negative cells by FACS 4 days after infection (Figure 1A). This population presumably harbored both uninfected cells and cells with transcriptionally silenced proviruses. To activate HIV expression, we treated this population with TPA or tumor necrosis factor α (TNF-α) and purified GFP-positive cells by FACS (Figure 1A). These cells, representing 100-fold enrichment) in or near alphoid repeats in latent cells in comparison to productively infected cells (Figure 3E). Similar results were obtained after infection of human peripheral blood mononuclear cells (PBMCs) with the HIV-derived vector (data not shown). Cloning and sequencing of several PCR products obtained after amplification with the alphoid specific primers confirmed that these contained HIV integration events into alphoid DNA (data not shown). Transcriptional activation of the HIV promoter in latently infected cells We have tested a number of biological and chemical agents for their ability to reactivate latent HIV expression. Several NF-κB activators, including TPA, TNF-α and PHA, independently induced HIV expression, as expected, since NF-κB strongly induces the HIV promoter activity. TPA was the strongest inducer tested (Figure 4). All treatments increased the number of GFP-positive cells and the mean fluorescence intensity reflective of GFP levels (data not shown). Incubation with anti-CD3 antibodies, which crosslink the surface T-cell receptors, an alternative pathway leading to NF-κB activation, also induced GFP expression (data not shown). These results suggest that activation of the NF-κB pathway may boost HIV transcription initiation, the production of Tat, and transition to Tat-dependent transcriptional activation. Trichostatin A (TSA), an inhibitor of histone deacetylases also activated expression but to a lesser extent than NF-κB activators and only in some cell lines (see clones A1 as an example, Figure 4). Treatment with 5-axa-2-deoxycytidine (aza-dC), an inhibitor of DNA methylation, had little effect on the fraction of cells induced to transcribe HIV alone or in combination with a histone deacetylase inhibitor (Groudine et al., 1981; Chen et al., 1997; data not shown). Figure 4.Transcriptional activation of the HIV promoter in latently infected cells. Cells from clones 82, A1, A7 and A10 were treated as described in Materials and methods with several indicated agents and LTR expression was measured by flow cytometry. Data are expressed as percentage of cells becoming GFP-positive after a 24 h treatment. Download figure Download PowerPoint Latent cell lines containing a full-length integrated HIV genome To confirm that HIV latency can be established in the context of a full-length provirus, we used a recombinant HIV molecular clone containing the GFP open reading frame in place of the Nef gene (Bieniasz and Cullen, 2000) (HIV-R7/E−/GFP; Figure 5A). To restrict our analysis to a single infection cycle, the env gene was suppressed by introduction of a frameshift mutation. This defect was complemented by coexpression of a VSV-G envelope protein to generate pseudotyped viral particles. We infected a culture of the lymphocytic cell line Jurkat with viral particles containing this HIV genome and used differential FACS based on GFP expression to isolate GFP-negative cells by FACS 4 days after infection (Figure 5B). Based on our previous experiments, we predicted that this population harbored both uninfected cells and cells with transcriptionally silenced proviruses. To activate HIV expression, we treated this population with TNF-α and observed that a small fraction of the cell population (1.9%) became positive. These activated cells were purified by FACS based on GFP expression levels (Figure 5B). These cells were both grown as a group and individually sorted for further characterization. Re-analysis after sorting showed that a small proportion of the cells had no GFP expression, indicating transcriptional silencing has occurred after withdrawal of TNF-α (Figure 5B). Flow cytometry analysis of individual clones showed low basal GFP expression (Figure 5C). After TNF-α treatment, HIV expression was increased in all clones both in terms of the fraction of cells that became GFP-positive (Figure 5C) but also in terms of mean fluorescence intensity (data not shown). Measurement of virus-specific mRNA showed that the mechanism of latency in these clones was controlled at the transcriptional level (Figure 1C, clones F11 and G10). Levels of viral mRNA after activation were similar to those measured after a productive HIV infection (compare clones F11 and G10 with NL4-3, Figure 1C). Similar results were obtained using northern blot analysis (data not shown). Analysis of HIV-specific protein expression in several clones by western blotting using an antiserum from an HIV-infected individual showed no detectable expression of HIV proteins under basal conditions (Figure 5D). Treatment of the same clones with TNF-α led to a dramatic increase in HIV protein expression, particularly the gag p55 precursor (Figure 5D). When the culture supernatants of the same clones were examined for HIV-specific p24 expression, no or low picogram amounts could be detected under basal conditions (Table I). Treatment with TNF-α led to a >1000-fold increase in p24 measurement for several representative clones (Table I). These observations demonstrate that transcriptional latency can also be established in the context of a full-length HIV infection. Figure 5.Establishment of latently infected cell lines with a full-length HIV provirus. (A) Genome organization of a molecular clone of HIV encoding GFP and containing a frameshift mutation in env. (B) Schematic representation of protocol for enrichment of latently infected cells after infection of Jurkat cells with HIV-R7/E−/GFP (see text for details). (C) Clonal cell lines isolated using the procedure described above were analyzed for GFP expression under basal and stimulated conditions (24 h treatment with TNF-α). (D) Western blot analysis of four representative Jurkat clones latently infected with HIV-R7/E−/GFP. Clones were treated for 24 h with TNF-α (10 ng/ml) and cell lysates were analyzed by western blotting using an antiserum from an HIV-infected individual (provided by the NIH AIDS Research and Reagent Reference Program). A predominant band of 55 kDa corresponds to the Gag precursor protein. The same samples were analyzed using an antiserum specific for α-tubulin to ensure equal loading. Download figure Download PowerPoint Table 1. Low to undetectable HIV protein expression in several latently infected clonal cell lines under basal conditions GFP-positive (%) GFP signal (MFI) p24 (pg/ml) TNF-α − TNF-α + TNF-α − TNF-α + TNF-α − TNF-α + Clone 15.4 <1 46 ± 6 6 ± 0.5 188 ± 34 7 ± 12 6066 ± 1960 Clone 6.3 <1 27 ± 9 5 ± 0.2 135 ± 43 0 10 100 ± 4573 Clone 8.4 <1 77 ± 6 5 ± 0.3 488 ± 74 0 32 967 ± 10 537 Clone 9.2 <1 75 ± 7 7 ± 0.5 522 ± 61 23 ± 6 41 067 ± 9100 Clone 10.6 <1 96 ± 1 5 ± 1.4 645 ± 45 14 ± 3 85 500 ± 5981 Five representative Jurkat clones latently infected with HIV-R7/E−/GFP (same clones as shown in Figure 5D) were treated for 24 h with TNF-α (10 ng/ml). HIV expression was quantified using flow cytometry for GFP. Results are shown both as the fraction of cells expressing GFP above background (control Jurkat cells) and as the mean fluorescence intensity (MFI). Cell culture supernatant fluids were also assayed for the Gag-derived p24 HIV protein using an ELISA. Average of three measurements ± SD are shown. Discussion We present evidence that HIV can reproducibly lead to a state of transcriptional silencing and true postintegration latency. We have used a FACS-based protocol to highly enrich for latently infected cells and show that HIV integration leads with low frequency to integration sites that cannot support basal transcription of the HIV promoter. The low frequency of integration events leading to latency (∼1%) is likely to be the reason why this phenomenon has eluded discovery until now. Integration into alphoid repetitive DNA, a component of centromeric heterochromatin, occurred frequently in our latent clone population. A recent comprehensive analysis of the site of integration of HIV during a productive infection showed that HIV integrates preferentially in actively transcribed genes (69% of integrations sites; Schroder et al., 2002). In the same study, α satellite integration represented <1% of all integration events (Schroder et al., 2002). In contrast, in our small sample of sequenced integration sites from latent infections, we observed 50% (four of eight) of integration sites in alphoid repeats. Analysis of a larger sample using the alphoid repeat PCR assay indicated that the true frequency of alphoid repeat integration is probably lower, between 20 and 30% in latent cells (data not shown). Heterochromatin is found in transcriptionally inactive regions of the genome and is associated with specialized chromosome structures, such as centromeres and telomeres. It is characterized by a strongly condensed nucleosomal structure that impairs access to the underlying DNA for transcription factors and the basal transcriptional machinery. The unique properties of hetero chromatin are associated with the incorporation of particular histone variants, unique post-translational modifications of the histone tails and, heterochromatin-specific proteins (Wallrath, 1998; Jenuwein, 2001; Jenuwein and Allis, 2001). The transcriptional silencing effect of heterochromatin is not restricted to the region packaged into heterochromatin itself but extends to several kilobases of adjoining DNA. Heterochromatin-mediated transcriptional silencing has been characterized extensively in Saccharomyces cerevisiae, where the integration of a retrotransposon in or near heterochromatin leads to transcriptional repression and, in Drosophila, where chromosomal rearrangements (inversions, translocation), occurring as a result of X-ray irradiation, placed euchromatic genes close to a heterochromatic breakpoint. This chromosomal rearrangement led to a position-dependent inactivation of the transposed euchromatic gene. Because the expression of the rearranged euchromatic gene or of the transposon is often characterized by a variegated phenotype, transcriptional repression by integration in or near heterochromatin has been referred to as position effect variegation (Tartof, 1994). Interestingly, transcription of the HIV promoter in latent clones also shows a variegated phenotype after activation, i.e. only a fraction of the population becomes reactivated in response to a global signal. Variegated expression of the HIV promoter was also observed during progressive repression after withdrawal of the activation signal. Different clones reverted to the silenced phenotype at different rates, possibly indicating that different chromatin environments lead to transcriptional repression with different efficiencies. We propose as a working hypothesis that a repressive chromatin environment is the common underlying mechanism for transcriptional repression in all of our clones. As discussed above, we estimate that 20–30% of integration events in latent cell