Community Research and Development Information Service - CORDIS

FP7

HIT HIDDEN HIV Report Summary

Project ID: 305762
Funded under: FP7-HEALTH
Country: France

Final Report Summary - HIT HIDDEN HIV (Paving the way towards HIV eradication/control)

Executive Summary:
Major advances in HIV/AIDS treatment regimens have fundamentally altered the natural history of the disease and sharply reduced HIV-related morbidity and mortality in countries where such treatments are accessible. The most notable advance is the use of combination antiretroviral therapy or ART. However, ART is unable to achieve virus eradication or “sterilizing cure”. Indeed, in most cases viral rebound is observed after ART interruption. Thus, life-long treatment is currently needed to control HIV. Drug resistance, cumulative side-effects and high cost, represent major drawbacks of such treatments. The persistence of HIV in treated patients results from the establishment of a viral reservoir insensitive to ART and poorly visible to the immune system. Thus, understanding HIV persistence and developing drugs able to flush out HIV, in order to achieve viral eradication or “sterilizing cure” remain outstanding challenges. The partners in this collaborative research project consortium have long-term expertise in studying HIV-1/SIV replication and pathogenesis. They have complementary skills in the fields of molecular and cellular virology, immunology, and physiopathology of infectious diseases. Fruitful collaborations have been successfully established, as assessed by joint publications in first-rank journals. Teaming up with a SME specialized in the analysis and screening of natural molecules will add a translational dimension to the project. All approaches are highly innovative and currently being implemented within the laboratories of the participating institutions. They are expected to lead to pre-clinical developments within the time frame of this project.
The project was successfully conducted since most of our objectives were achieved (30 publications in high impact journal). Major discoveries were achieved. Indeed, we were successful in identifying key regulators of HIV persistence and permissiveness. Cellular factors which play role in host defense mechanisms against HIV were identified and their role in HIV induced inflammation was established. The major breakthrough is the identification of a biomarker of HIV persistent cells in vivo. This discovery will certainly open new research avenues towards the characterization, control and eradication of the latent HIV reservoir. This discovery will likely have a tremendous impact on the research conducted in the field of HIV cure both at basic and therapeutic approaches. Our discovery that CD32a+ CD4 T lymphocytes harbor the elusive HIV-1 reservoir is a crucial step towards specific targeting and elimination of this HIV-1 reservoir.

Project Context and Objectives:
HIV infection is efficiently controlled but not cured by Anti-Retroviral Therapy (ART) (Deeks et al., 2012). This is due to the establishment, early after primary infection, of a viral reservoir that is responsible for the persistence of low levels of plasma viremia in patients under suppressive ART (Chun et al., 1997; Finzi et al., 1997). Consequently, viral rebound is observed immediately after ART interruption. HIV persistence may arise from ongoing residual virus replication and/or from latently-infected cells defined as the cellular reservoir in which long-lived resting memory CD4+ T cells harbouring an integrated but transcriptionally silent provirus represent the largest pool in the blood (Chomont et al., 2011; Chomont et al., 2009; Hakre et al., 2011). Addressing the source of HIV persistence is required to achieve a cure for HIV. If ongoing replication remains in ART-treated patients, then intensification of treatment will be useful in a functional cure strategy aimed at “remission” (Barouch and Deeks, 2014; Buzon et al., 2010; Hatano et al., 2013). However, treatment intensification will have no impact on long-lived latently infected cells. Thus, targeting the sources of HIV persistence is required to achieve HIV cure in addition to ART. This can be achieved either by a so-called “Shock and Kill” strategy, which would employ virus-reactivating agents in combination with ART, or by a “Kill” approach whereby latently infected cells would be directly targeted and killed (Barouch and Deeks, 2014; Trono et al., 2010). A “Shock and kill” strategy will require deep understanding of the molecular mechanisms involved in the establishment and maintenance of transcriptionally silent proviruses. The “Kill” strategy is conditioned by the identification of specific marker(s) that differentiate latently infected cells from their non-infected counterparts.
Current approaches to purging the HIV-1 reservoir involve pharmacologic induction of HIV-1 transcription and subsequent killing of infected cells by cytolytic T lymphocytes (CTLs) or viral cytopathic assays (Barouch and Deeks, 2014; Deeks et al., 2013). Thus, many laboratories, including ours, have investigated the molecular mechanism involved in the establishment and maintenance of HIV-1 transcriptional silencing in order to better tackle the problem of virus reactivation and subsequent eradication by ART (Hakre et al., 2011; Siliciano and Greene, 2011; Trono et al., 2010). It appears from all these studies that HIV latency is a multifactorial process that involves many cellular pathways (Siliciano and Greene, 2011; Trono et al., 2010; Van Lint et al., 2013) including insufficient levels of key cellular transcription factors involved in viral promoter activation (Bosque and Planelles, 2009; Novis et al., 2013; Tyagi and Karn, 2007; Tyagi et al., 2010; Williams et al., 2006), the effect of the site of integration and the chromatin environment (Brady et al., 2009; Maldarelli et al., 2014; Sherrill-Mix et al., 2013; Singh et al., 2010), recruitment of chromatin repressors including HDACs and HMTases among others (du Chene et al., 2007; Marban et al., 2007; Tyagi et al., 2010; Van Lint et al., 1996; Wagschal et al., 2012), and transcriptional interference (Lenasi et al., 2008; Shan et al., 2011; Van Lint et al., 2013). Several Latency-Reversing Agents (LRAs) including the NF-kB activator (prostratin), HDAC inhibitors (TSA, Valproic acid VPA, SAHA), inhibitors of DNA-methylation (AzaC), IL2, IL7 and the viral transactivator Tat, have been successfully used in vitro to reactivate transcriptionally latent provirus in cellular models. However, no decrease in either the size of the viral reservoir or the reactivation of latent provirus occurred with HDACi, IL2 or IL7 when used in patients (Barouch and Deeks, 2014; Trono et al., 2010). More recently, it has been reported that none of the considered LRAs induced outgrowth of HIV-1 from the latent reservoir (PBMCs) of patients on ART (Bullen, Laird et al. 2014). These discrepancies may originate from the differences between the T cell biology in vivo and in in vitro models used to decipher the molecular mechanisms of viral latency. Indeed, primary T cell-based models require an activating signal to allow infection and subsequent removal of the activating signal to re-establish a quiescent state (Spina et al., 2013). Heterogeneous responses to LRAs in different in vitro established cellular models have been reported (Spina et al., 2013). Because resting CD4 T cells are non-permissive to HIV, there are no studies exploring the mechanism of viral latency in these cells. Additionally, most of the data on the latent reservoirs of HIV comes from studies using blood-derived cells, which can be misleading since HIV reservoir also resides in lymphocyte-rich tissues such as the peripheral lymph nodes, ileum and spleen (Barouch and Deeks, 2014; Deeks et al., 2013). Reducing the viral reservoir to an undetectable level for many years, as could be achieved in the two Boston patients and the Baby from Mississippi, was not enough to prevent viral rebound after ART interruption (Barouch and Deeks, 2014). Thus, to be effective, a “Shock and Kill” strategy will require the eradication of all replication-competent virus that persists in ART-treated patients (Barouch and Deeks, 2014; Hill et al., 2014). This is unlikely, given the recent data showing that only weak induction of viral RNA is observed when PBMCs from ART-treated patients were treated with LRAs (Bullen et al 2014; Ho YC et al, 2013). When used in vivo, HDACi shows a marginal induction of viral RNA, no induction of viral protein and no effect on the size of HIV reservoir (Archin et al., 2012; Barouch and Deeks, 2014). Thus, there is a need for alternative and/or complementary approaches.
The “Kill” strategy can be achieved either by a therapeutic vaccine or through the identification of specific biomarkers for HIV-1 persistence that can be used to specifically target latently infected cells. The latter represents one of the highest current priorities of the field. These biomarkers should allow detection, quantification and targeting of the total body burden of replication-competent HIV-1 during ART. Whether latently infected cells express markers that distinguish them from their non-infected counterparts, due to a cellular response to viral infection or to virion-associated viral proteins released into the cells upon viral entry, is unknown. Interestingly, among virion-associated proteins, accessory proteins Vif and Vpr are capable of modifying host gene expression through their ability to bind chromatin or their ability to interact and regulate cellular transcription factors and chromatin modifiers. Such an effect is particularly plausible for Vpr, which is heavily packaged into virions (700 molecules per virion). However, a reliable model to explore this hypothesis is currently lacking.
The discrepancies in viral reactivation observed between in vitro derived models and cells isolated from ART-treated patients may originate from the differences between the T cell biology in vivo and in the in vitro models used to decipher the molecular mechanisms of viral latency. Indeed, primary T cell-based models require an activating signal to allow infection and subsequent removal of the activating signal to re-establish a quiescent state. The ideal in vitro model will consist of latently infecting resting CD4 T cells in the absence of an activating signal that can be used to decipher the molecular basis of latency. The fact that resting CD4 T cells are non-permissive to HIV has hindered the development of such model. Another important limitation is that most of the data on latent reservoirs comes from studies using blood-derived cells, which can be misleading since the HIV reservoir also resides in lymphocyte-rich tissues such as the peripheral lymph nodes, ileum and spleen. Thus, reliable in vitro and in vivo models are required to deepen our understanding of the complex interaction between HIV, its host and therapeutic tools to achieve a cure for HIV.
Two main lines of research were proposed. The first starts from the recent discovery by partners of this consortium of the immune-modulator Samhd1 as the cellular factor restricting HIV-1 infection in myeloid cells. The partners explore a role for Samhd1 in immune activation and inflammation, and its impact on the balance between viral replication and persistence. To understand further HIV persistence, the partners study the molecular mechanisms underlying post-integrative latency. The second aims at identifying and optimizing novel naturally occurring inducers of HIV reactivation. Additionally, a novel non-human primate model is developed, allowing the study of viral persistence in vivo by tracking latently-infected cells.
The project aims at: (i) increasing knowledge on the contribution of immune activation and inflammation to HIV persistence (ii) deciphering the cellular and molecular mechanisms of viral persistence (iii) translating this basic knowledge into novel targets to achieve a cure for HIV/AIDS.

Project Results:
WP1: Development of a Non-human primate model for HIV latency
Efforts toward the development of therapies aimed at targeting the HIV reservoir are complicated by the evidence that HIV persistence is the result of persistent productive and non-productive infection in a number of cell types and through a variety of mechanisms (Trono et al., 2010; Van Lint et al., 2013). Thus, it’s unlikely that latently infected primary T cells represent the only source of viral rebound after ART interruption. To facilitate the advancement of our knowledge in this new area of research, in vitro models of HIV persistence in different cellular reservoirs have been developed, particularly in CD4+ T-cells that represent the largest pool of persistently infected cells in blood. Whereas each model presents clear advantages, they all share one common limitation: they are systems attempting to recapitulate extremely complex virus-cell interactions occurring in vivo, which we know very little about. Potentially conflicting results arising from different models may be difficult to interpret without validation using clinical samples. Addressing these issues merits careful consideration for the identification of valid targets and the design of effective strategies for therapy, which will increase the success of efforts toward HIV eradication. In this perspective, the development of an animal model to study viral persistence in which tracking, localizing, sorting and validating identified targets is needed. Such an animal model will bring answers to key questions in the field of viral persistence such as:
a-What is the source of viral rebound after ART interruption?
b-Where do HIV-infected persistent cells localize in vivo?
c-What are the cellular factors and the mechanisms involved in the establishment and maintenance of transcriptionally latent provirus in vivo.
d- Do latently infected cells express a specific marker differentiating them from their non-infected counterparts?
To establish the CRE-based SIV/macaque latency model.
Establishing the NHP-model dedicated to decipher the molecular and the cellular mechanisms of HIV persistence has encountered some difficulties. First, the SIV molecular clone expressing the CRE recombinase as nef-IRES-cre turned to be unstable. The IRES-Cre transgene was removed by recombination after few rounds of infection. Sequencing the SIVCre mutant allowed us to precisely determine the sequences responsible for the recombination. The identified sequence was mutated and a new construct was generated. In addition we have also made a new construct in which the Cre recombinase was expressed as an independent transcription unit under the control of the CMV promoter. Both SIVnef-IRES-Cre and SIV-CMVCre molecular clones were tested for replication and Cre expression first in vitro and in animals. Cre expression from both viruses was found to be stable in vitro and in vivo. Interestingly, Cre expression was higher in SIV-CMV Cre. This results in increased recombination efficiency when infection the dual color transduced cells. Second, we optimized the transduction of CD34 cells with the dual color vector expressing GFP and RFP. Using Hu-mice we were able to show that transduction of CD34 cells with the dual color vector does not affect immune reconstitution. Expression of GFP was stable up to 4 months post engraftment. Based on the above encouraging results, a cohort of 4 NHP reconstituted with transduced CD34 cells has been generated. Reconstituted, were infected with SIV Cre. Analyses were performed as depicted in table 1.
Table 1. Summary of the experimental procedure to assess infectivity and stability of SOCG and SORCG in vivo.

Unfortunately, we were unable to detect any GFPRFP+ cells in blood or in LN of infected animals. Viral sequence analyses revealed the absence of the Cre coding sequence due to recombination in vivo.
We next infected animals with SIVmac239-Nefopt-CMV-GFP (SOCG) and SIVmac239-Nefopt-RFP-CMV-GFP SORCG (Figure 1). The in vivo replication kinetic for both viruses displayed standard characteristics for SIVmac239 infection in macaque. However, GFP or GFP/RFP positive cells were not detected in PBMC and LN by FACS analysis.
Figure 1. In vivo replication of SIVmac239-Nefopt-CMV-GFP (SOCG) and SIVmac239-Nefopt-RFP-CMV-GFP SORCG. The in vivo replication kinetic for both viruses displayed standard characteristics for SIVmac239 infection in macaque. However, GFP or GFP/RFP positive cells were not detected in PBMC and LN by FACS analysis.

Taken together, while the dual color constructs were stable including in animals, the SIV recombinant viruses producing the CRE recombinase or expressing GFP were unstable reflecting the high recombination properties for the SIV.

As mentioned in RP2 report, we have generated HIV based constructs to achieve a similar objectives in Humanized mouse model (Figure 2).

Figure 2: Vectors generated to establish a CRE-based HIV/Hu-mice latency model. (A) Schematic representation of the lentiviral vector expressing GFP and RFP (HR-4lox). Shown are the Cre induced recombination events required for the expression of the RFP. (B) Schematic representation of HIV-1 molecular clones expressing Cre recombinase. Cre is expressed together with Nef by the R5-HIV1-Nef-IRES-Cre construct via an internal ribosome entry site (IRES) or by R5-HIV1-CMV-CRE as an independent transcriptional unit under the control of CMV promoter.

The Humice model was developed in collaboration with the laboratory of Roberto Speck (University Hospital of Zurich, Switzerland). To demonstrate the in vivo proof of concept of our animal model, we compared the reconstitution and the infectivity of 2 Hu-mice reconstituted with mock-transduced CD34 cells to 2 Hu-mice reconstituted with CD34 cells transduced with HR-4lox reporter construct. Immune reconstitution was optimal and similar in all animals. GFP expressing cells in the bone marrow and blood of CD34GFP+ reconstituted animals were detected at high frequency and stable for 5 months post reconstitution (data not shown). All the animals showed efficient and equivalent HIVCRE infection kinetics. RFP+GFP+ CD4 T cells were detected in the spleen of CD34GFP+ reconstituted animals as compared to non-infected animal (Figure 3). No RFP+ CD4 T cells were detected in GFP negative CD4 T cells. These results show an in vivo proof of concept of our strategy.

Figure 3: Facs analyses showing the presence of human CD45+, CD3+, CD4+ expressing GFP and RFP in the spleen of representative hu-mice immune reconstituted with CD34GFP+ cells and infected with HIV1CMVCRE (animal#100) or non-infected (animal#92). Animals were sacrificed 5 weeks after infection and viral RNA in plasma was 4.6x105copies/ml for the infected animal.
In conclusion, while we failed to achieve the NHP model, we were able to successfully develop the Humice model to achieve the same objectives.

Identification of specific biomarkers of latently infected cells.
Resting CD4 T cells are non-permissive to HIV. This has made difficult the establishment of an in vitro model to study viral latency in resting primary CD4 T cells that have received no prior activation signal to allow viral infection. Our discovery that SAMHD1 is responsible for viral restriction in non-dividing cells including resting CD4 T cell offers an opportunity to overcome this limitation. Indeed, treatment of resting CD4 T cells with viral like particles containing the HIV-2 accessory protein Vpx (VLP-Vpx) overcomes the restriction imposed by SAMHD1 allowing the virus to complete early steps of its life cycle up to integration (Figure 1). Interestingly, integrated provirus remains silent in the absence of an activating signal. VLP-Vpx-treated cells will be infected with HIV expressing GFP as an independent transcriptional unit under the control of the CMV promoter, which is active in resting T cells, at the end of nef gene (HIV-1CMV-GFP) to establish an in vitro resting CD4 T-cell model to study the molecular mechanisms of viral latency and to determine whether latently infected cells express specific genes that can constitute a signature distinguishing them from their non-infected counterparts.
Figure 1: Depleting SAMHD1 in quiescent CD4 T cells allows HIV-1 reverse transcription and integration but not viral transcription. PBMCs from healthy donors were treated overnight by empty or Vpx containing Viral Like Particles (VLP-Vpx) and subsequently infected with (a) HIV-1 nef-IRES GFP in which GFP expression is driven by the viral LTR or (b) an HIV expressing GFP as an independent transcriptional unit at the end of nef gene (HIV-1CMV-GFP). Of note, unlike the HIV LTR, CMV promoter is active in quiescent CD4 T cells. The number of GFP positive quiescent CD4 T cells was assessed by FACS analysis at day 4 post infection. Bar graphs show the fold increase of total HIV-DNA measured by quantitative PCR in quiescent CD4 T cells at day 3 post infection.

Using an in vitro model of non-productively HIV-infected quiescent CD4 T cells, we reveal a gene expression signature of 103 upregulated genes specific for HIV-latently infected resting CD4 T cells, 16 of which are transmembrane proteins. In vitro screening for their surface expression in HIV-infected quiescent CD4 T cells, revealed Marker 1 to be by far the most highly induced, with no detectable expression in bystander cells. Strikingly, productive HIV-1 infection of TCR-stimulated CD4 T cells is not associated with Marker1 induction arguing for quiescence-dependent mechanism. Remarkably, using blood from HIV-infected participants under suppressive antiretroviral therapy (ART), we identified a subpopulation of 0.012% of total CD4 T cells expressing Marker1 and hosting up to 3 HIV-DNA copies per cell (median of 0.56). This reservoir contains an inducible replication-competent provirus and can be predominant in some participants since its depletion results in dramatic delay in virus production. Our discovery that Marker1+ lymphocytes harbor the elusive HIV-1 reservoir is a crucial step towards specific targeting and elimination of this HIV-1 reservoir to achieve a cure for AIDS (Descours B. Patitjean G et al. Nature in revision).

WP2: Fundamental mechanisms of latency
To our current knowledge long lived resting memory CD4+ T cells harboring an integrated but transcriptionally silent provirus represent the largest pool of the latent viral reservoir in the body (Siliciano and Siliciano, 2004; Trono et al., 2010). Identification and elimination of the source of HIV persistence is required to achieve HIV cure. Accumulating evidence suggests that intensification of treatment is insufficient to eradicate the virus because it has no impact on long lived latently infected cells (Maldarelli, 2011). Thus, HIV eradication will require therapy targeting latently infected cells in addition to ART. This can be achieved either by virus reactivating agents in combination with ART or by directly targeting and killing latently infected cells (Fonseca et al., 2011; Trono et al., 2010). The aim of this work package is to deeper our understanding of the molecular mechanisms involved in the establishment and the maintenance of transcriptionally silent proviruses and to generates novel expression modules associated with permissive expression.
Objective 1: Understanding how NELF establishes and maintain HIV-1 transcriptional latency
Within this objective, transcription elongation block imposed by the negative transcription elongation factor NELF was identified as the rate limiting step controlling transcription from the HIV LTR. To gain insight into NELF-mediated transcriptional repression of HIV-1 promoter, we purified the NELF complex and identified its associated partners using tandem affinity chromatography coupled to Mass spectrometry (Figure 1). We found that NELF forms at least three distinct complexes: NELF core complex, NELF-Spt5 containing Integrator complex subunit 3 (INTS3) and NELF-Spt5 containing all Integrator complex subunits (INTScom) and RNAPII. Using Chromatin immunoprecipitation analyses, we were able to show that transcriptionally silent HIV promoter is bound by NELF-Spt5-Integrator complex (Figure 2). Activation of the LTR by the viral Tat protein resulted in removal of this complex from the LTR and recruitment of the positive transcription elongation complex PTEFb. Functional analyses using quantitative RT PCR and nuclear run on assays showed that NELF-Spt5-Integrator complex controls RNAPII pausing at the viral LTR (Figure 3). In addition to controlling RNAPII pause release INTS11 catalytic subunit of the INTScom is required for the synthesis of full length HIV 1 mRNA.

Figure 1. Schematic integration of IP/ReIP and glycerol gradient sedimentation analysis: NELF forms a small (SC) and a large (LC) complex together with subunits of the Integrator Complex. SC: NELF-A/B/(C/D)/E, SPT5 and INTS3. LC: NELF-A/B/C/D/E, SPT5, RNAPII, INTS1/3/13 and the catalytic Integrator subunit INTS11.

Objective 2: Identification of NELF inhibitors
To identify which subunit of the NELF-INTS complex is required for RNAPII pausing, we used specific siRNA targeting subunits of NELF and INTS. We have used both HIV-1 promoter and genome wide analyses, to assess for specificity. We found that knock down of the INTS catalytic subunit INTS11 and its regulatory subunit INTS9 was the most efficient in HIV LTR transcriptional derepression. Genome wide analyses demonstrated that knockdown of INTS11 and INTS9 also resulted in transcriptional activation of subset of cellular genes. We demonstrate that Integrator subunits specifically control NELF-mediated RNAPII pause/release at the HIV promoter and cellular coding genes. The strength of RNAPII pausing is determined by the nature of the NELF-associated complex. Interestingly, in addition to controlling RNAPII pause release INTS11 catalytic subunit of the INTScom is required for the synthesis of full length mRNA. Finally, we found that NELF-target genes regulated by INTScom are enriched in a 3’box sequence required for snoRNA biogenesis. Revealing these unexpected functions of INTScom in regulating RNPII pausing/release and completion of mRNA synthesis of NELF-target genes will contributes our understanding of gene expression cycle.

Figure 2. Integrator complex regulates transcription elongation at the HIV-1 LTR and at subset of cellular genes. Schematic representation of the LTR-Luciferase locus in HeLa-LTR-Luciferase cells depicting positions of primers used in ChIP (Chromatin Immuno-Precipitation) and NRO (Nuclear Run-On) assays. NROs were performed using nuclei prepared from HeLa-LTR-Luc cells transfected with the indicated siRNAs. Results are presented as fold change over control condition SCR, the average profiles of the 4 normalizations are shown. Results are presented as fold change over control condition SCR. *= p-value < 0.05; **= p-value < 0.005; ***= p-value < 0.0005, no*=no significant p-value as measured by student’s t-test. Error bars represent standard deviations (n=3). Integrator complex regulates NELF-mediated RNAPII pausing at coding genes. Venn diagram showing the intersection among differentially expressed (DE) genes identified by microarray analysis (n=3) upon depletion of -INTS11, -INTS3- or –NELF-E (p < 0.001).
Objective 3: Generation of novel expression modules associated with permissive expression
We have used a primary cell model of HIV latency for the in-depth characterization of the transcriptome of cells entering, maintaining or exiting latency at the population level. We have now completed this analysis at single cell level of latently infected cells under three conditions, either non-stimulated or stimulated by SAHA or activated through TCR stimulation. Data are still under analysis aiming at linking a specific transcriptomic signature with the success of HIV expression reactivation.
Based on the development of our previous pipeline analysis for the dynamic characterization of the joint transcriptome for the virus and the host upon HIV infection (Mohammadi et al, PLoS Pathogens 2013; web resource: http://peachi.labtelenti.org), we went a step further to analyze as well the proteome and phosphoproteome in a similar experimental setting. The joint analysis of transcriptome, proteome and phosphoproteome in a high temporal resolution identified multiple candidates that are potentially involved in promoting HIV infection (and characterized by upregulation of the candidates at both transcript and protein expression levels) or restricting HIV infection (characterized by protein degradation). These candidate proteins are currently under investigation. A manuscript as well as an updated website (Peachi2.0) are under preparation. We have used a single cell approach to identify biomarkers of specific phenotypes. As a proof-of-concept we used HIV permissiveness to validate the approach. We identified multiple markers that were enriched in HIV permissive cells and that could be used to capture susceptible cells, validating the approach (Rato et al., in preparation). We are now testing the expression of these biomarkers in a model of latently infected cells.

WP3: Characterization of natural activators of latency

Objective 1: To purify natural activators of latent HIV from human hemofiltrate.
In WP3, the working group of Prof. Dr. Frank Kirchhoff (UULM) and the Pharis Biotec GmbH (PHARIS) aimed to purify and characterize endogenous activators of latent HIV from human hemofiltrate.

To achieve this, PHARIS generated complex peptide/protein libraries from hemofiltrate obtained from dialysis patients. These libraries contain essentially all peptides and proteins smaller than 30 kDa that are circulating in the blood. About 10.000 l hemofiltrate was obtained from dialysis of patients with chronic renal failure, pooled and acidified prior to separation into eight crude extracts according to their size and charge using ion exchange chromatography. Each of these eight peptide pools was further separated into 48 fractions according to the hydrophobicity of the peptides applying reverse phase chromatography. The resulting libraries contain more than one million different natural human peptides in 384 fractions and provided the basis for screens for natural activators of latent HIV.

In the next step, the peptide libraries were utilized by UULM in comprehensive screens for activators of latent HIV in the J-LAT cell model for latency. J-LAT cell lines were derived from Jurkat cells and contain envelope-defective HIV-1 reporter constructs carrying the green fluorescent protein (GFP). Thus, reactivation of these proviral constructs leads to GFP expression and can easily be monitored by flow cytometry.

Initial screens of three independent peptide libraries identified several fractions that activate latent HIV-1 and were further investigated. For 25 of these fractions, dose-dependent activation of latent HIV in J-LAT cells without induction of cytotoxic effects was confirmed. The peptidic nature of activating agents was verified by proteinase K digest, which eliminated the activating capacity of the fractions. Purification and identification of the activating compounds in the complex peptide mixtures turned out to be a challenging task. In many cases, activities were lost during purification, at least in part due to lack of recovery during the separation steps or problem with denaturation. To some extent these problems were overcome by optimization of the purification protocols. Adjacent active hemofiltrate fractions were pooled and further purified using size-exclusion chromatography with different concentrations of urea, guanidine hydrochloride, or acetic acid were applied as eluates, instead of reversed-phase chromatography. HIV-activating fractions in J-LAT assays were further analyzed by MALDI-MS and LC-MS. These optimized approaches allowed the identification of Retinol Binding Protein 4 (RBP4) as activator of latent HIV in hemofiltrate library fractions P4-F27-38 (i.e. PH-pool 4, Fractions 27 to 38). RBP4 is mainly known as vehicle for the transport of retinol from the liver to other peripheral organs. The plasma levels of RBP4 in HIV-infected patients are similar to those found in uninfected individuals but upregulated by 7.1-fold in patients receiving HAART for 12 months(Schindler et al., 2006). RBP4 is known to induce inflammation in human endothelial cells by an NF-κB-dependent mechanism (Farjo et al., 2012) and contributes to systemic inflammation (Barazzoni et al., 2011). Furthermore, some activating fractions contained cytokines already known to stimulate T cells and hence latent HIV-1 providing further proof for the suitability of our approach.

Objective 2: To determine their potency and mechanisms in in vitro models for HIV latency.
To determine RBP4´s potency and the mechanisms underlying latent HIV activation, RBP4 was expressed in mammalian cell lines using genetic engineering technologies. PHARIS generated expression plasmids for transient and stable expression of recombinant human RBP4 with and without His-Tag. Six to ten days after transfection of HEK293T cells and culture in serum-free medium containing valoic acid for enhanced protein production, the supernatant was harvested and purified using affinity chromatography. The identity of full-length recombinant human RBP4 was confirmed by western blot analysis showing a clear band of 21kDa size after staining with an anti-RBP4 antibody. Several batches of recombinant human RBP4 proteins were produced. To determine the potency of the recombinantly produced human RBP4 (crude extracts and purified protein, with and without His-Tag), as well as commercially available recombinant human RBP4 (without Tag) from Creative Biomart (Catalog Number: RBP4-31234TH) in activating latent HIV-1, UULM incubated J-LAT 11.1 cells with different doses of RBP4. Crude extracts did not show any activity while the results with untagged RBP4 from Creative Biomart as well as purified RBP4 produced by PHARIS showed that RBP4 significantly stimulated HIV-1 gene expression at concentrations ≥12.5 µg/ml. The enhancement of latent HIV activation by RBP4 from Creative Biomart was up to 10-fold if 100µg/ml RBP4 were applied while the different RBP4 batches varied in their potency. The most potent batch produced by PHARIS enhanced the latent HIV activation about 21-fold using 100µg/ml RBP4. In contrast, His-tagged RBP4 was inactive maybe caused by steric hindrance. Thus, the results confirmed that RBP4 is an endogenous activator of latent HIV-1 proviruses in J-LAT cells.

To obtain first insights into the underlying mechanism UULM also examined the effect in two additional J-LAT cell lines differing in CpG methylation. Interestingly, RBP4 activated HIV-1 in the 11.1 and 6.3 but not in the 8.4 J-LAT cell line suggesting a possible CpG methylation-dependent mechanism. Since RBP4 is known to activate the transcription factor NF-κB that plays a key role in efficient HIV-1 gene expression, we also examined whether RBP4 might activate latent HIV through stimulation of NF-κB. Therefore, the NF-κB reporter cell lines Sup-D1 and Jurkat J4 were utilized, which carry a firefly luciferase gene driven by NF-κB response elements. However, treatment with RBP4 did not induce NF-κB activity in Sup-D1 and Jurkat J4 cells suggesting a different mechanism of HIV-1 activation. Furthermore, the influence of the RBP4 receptor STRA6 on the latent HIV activating capacity of RBP4 was examined. Experiments at UULM showed that J-LAT cells express STRA6. Hence, blocking experiments with an anti-STRA6 antibody were performed. The preincubation of J-LAT 11.1 cells with anti-STRA6 prior RBP4 addition did not lead to a significant reduction in the activation of latent HIV in J-LAT 11.1 cells suggesting that the STRA6 receptor is not involved in the potency of RBP4 to activate latent HIV.

To clarify whether RBP4 also activates latent HIV-1 in primary CD4+ T cells, UULM generated derivatives of the HIV-1 NL4-3 molecular clone expressing the Gaussia reporter gene and established methodologies for quantification of HIV-1 reactivation in its primary target cells. However, RBP4 failed to induce latent HIV-1 activation in this experimental system. The CXCR4-tropic NL4-3 molecular clone is adapted to T cell lines and may not faithfully reflect latent HIV-1 infection in vivo. To overcome this limitation, UULM generated Gaussia luciferase reporter constructs of a CCR5-tropic NL4-3 derivative as well as of several HIV-1 transmitted/founder constructs. These constructs provide an effective means to quantify latent HIV-1 activation and might reveal the possible relevance of RBP4 in primary CD4+ T cells in future studies.

RBP4 is a specific carrier for retinol and delivers it from the liver to peripheral tissues. Thus, it was examined whether retinol binding plays a role in the stimulatory effect on HIV-1. Experiments by PHARIS showed that recombinant humanRBP4 with and without His-Tag efficiently bound retinol in a dose-dependent manner with saturation at a concentration of 10µg/ml. However, RBP4 (without His-Tag) was equally active in stimulating HIV-1 in the J-LAT cell assay in the presence and absence of retinol demonstrating that the latent HIV activating effect is independent of retinol.

Objective 3: To optimize the potency of activators of latent HIV and define the underlying mechanism(s).
In order to determine whether the enhancing activity of RBP4 maps to a specific region of the protein, nine about 25 amino acid long RBP4 fragments overlapping by four to ten amino acids at both termini (depending on the amino acid sequence) were synthesized. Altogether, these overlapping peptides spanned the entire RBP4 sequence. Functional testing in the J-LAT assay showed that none of the peptide has HIV-1 enhancing activity. Thus, further structure-activity-relationship (SAR) studies are required to identify critical domains in RBP4 and to define the minimal size required for HIV-1 stimulation and ultimately to generate RBP4 derivatives with increased and broader HIV-enhancing activity.

Objective 4: To determine the levels of newly identified compounds in sera of HIV-infected individuals.
Immunoassays to determine RBP4 plasma levels are already commercially available. Using the Human RBP4 Quantikine ELISA Kit (R&D, Catalog Number: DRB400) UULM examined plasma levels of eight healthy individuals. The detected average RBP4 level was 30.0µg/ml ranging from 20.2µg/ml to 37.9µg/ml. This is in accordance with the literature stating 10-50µg/ml RBP4 plasma levels of healthy individuals (Graham et al., 2007). Notably, it has been reported that the levels of RBP4 are increased to 17-150µg/ml in patients with obesity, insulin resistance, type 2 diabetes or vascular disease (Farjo et al., 2012). Studies analyzing RBP4 plasma levels in HIV-infected individuals are rare and so far controversial. Two studies reported lower (1.1-fold to 1.2-fold) RBP4 levels in HIV-1 patients compared to healthy individuals (Kotzé et al., 2015; Beaton et al., 2004). In contrast, Schindler et al., 2006 did not detect any significant difference between these groups and an increase (7.1-fold) of RBP4 levels in patients under ART treatment. Since the relevance of RBP4 in primary cells remains elusive, the analysis of HIV-positive versus HIV-negative subjects was postponed until the relevance of this protein is clarified.

Objective 5: To assess the effect of the optimized activators on viral reservoirs in the NHP model.
As outlined in WP1, the NHP model is not yet available. Furthermore, it will be important to further clarify the role of RBP4 in primary HIV-1 target cells before testing the compound in animal models.

References
Barazzoni R, Zanetti M, Semolic A, Pirulli A, Cattin MR, Biolo G, Bosutti A, Panzetta G, Bernardi A, Guarnieri G. 2011. High plasma retinol binding protein 4 (RBP4) is associated with systemic inflammation independently of low RBP4 adipose expression and is normalized by transplantation in nonobese, nondiabetic patients with chronic kidney disease. Clin Endocrinol (Oxf).
Baeten JM, Richardson BA, Bankson DD, Wener MH, Kreiss JK, Lavreys L, Mandaliya K, Bwayo JJ, McClelland RS. 2004. Use of serum retinol-binding protein for prediction of vitamin A deficiency: effects of HIV-1 infection, protein malnutrition, and the acute phase response. Am J Clin Nutr.
Farjo KM, Farjo RA, Halsey S, Moiseyev G, Ma JX. 2012. Retinol-binding protein 4 induces inflammation in human endothelial cells by an NADPH oxidase- and nuclear factor kappa B-dependent and retinol-independent mechanism. Mol Cell Biol.
Graham TE, Wason CJ, Blüher M, Kahn BB. 2007. Shortcomings in methodology complicate measurements of serum retinol binding protein (RBP4) in insulin-resistant human subjects. Diabetologia.
Kotzé SR, Zinyama-Gutsire R, Kallestrup P, Benn CS, Gomo E, Gerstoft J, van Dam G, Mortensen OH, Ullum H, Erikstrup C. 2015. HIV and schistosomiasis in rural Zimbabwe: the association of retinol-binding protein with disease progression, inflammation and mortality. Int J Infect Dis.
Schindler K, Haider D, Wolzt M, Rieger A, Gmeinhart B, Luger A, Nowotny P, Ludvik B. 2006. Impact of antiretroviral therapy on visfatin and retinol-binding protein 4 in HIV-infected subjects. Eur J Clin Invest.

WP4: Inflammation, immune activation and HIV persistence

Chronic systemic immune activation and inflammation are hallmarks of progressive HIV infection. Immune activation not only generates activated T cell targets for the virus, further driving viral replication, but also impacts the establishment of viral persistence. For instance, some chemokines induce changes in the actin cytoskeleton of resting CD4+ T cells, allowing viral integration in the absence of lymphocyte activation and viral production. Additionally, chemokines and pro-inflammatory cytokines may directly act on integrated proviruses, by facilitating activation of the HIV LTR promoter.
The overall objective of WP4 was to provide a comprehensive analysis of the contribution of inflammation and immune activation on viral persistence.
We believe that we have reached our objectives, with about 25 articles published between 2013 and 2016 on the molecular, immunological and virological interactions between HIV and its host (see list of publications).
In summary, we have explored the behavior of dendritic cells (DCs) when they are exposed to cell-free HIV-1 and HIV-infected cells. We have demonstrated that the antiviral activity of SAMHD1 impacts antigen presentation by DC, highlighting the link that exists between restriction factors and adaptive immune responses. We have also explored the role of Vpr and observed that in T cells, this viral protein facilitates the secretion of TNF by infected cells, thus probably impacting inflammation and viral replication.
We have examined the function of other restriction factors. We have discovered that Vpr targets the SLX4 complex, which is involved in DNA repair, to induce G2M arrest and to modulate type I IFN production in infected cells. Moreover, we have shown that Vpr degrades the HLTF DNA translocase in macrophages, an enzyme also involved in DNA repair, but the consequences of this degradation remains to be further understood. We have demonstrated that the family of Interferon Induced Transmembrane proteins (IFITM) inhibits HIV-1 by an original mechanism, infiltrating the budding viral particles and preventing viral fusion. We have described the underlying mechanisms, and performed an analysis of the function and evolution of IFITM in human and non-human primates.
We have also studied the role of SAMHD1 during HIV-2 infection. We reported that HIV-2 does not efficiently infect DCs because fusion of incoming virions is inefficient. The situation is different in non-activated CD4+T cells, which can be infected by HIV-2 because of Vpx-induced degradation of SAMHD1 in these cells.
Finally, we have analyzed how the viral reservoir present in HIV-infected individuals may be targeted by broadly neutralizing antibodies (bNAbs) and killed by ADCC.
Altogether, our experiments helped understanding the role of immune activation and inflammation in HIV persistence, and allowed to establish novel tools to target the viral reservoir.
The WP4 is divided into 4 objectives that are listed below, and further detailed in the next paragraphs.
Objectives
1: To analyze the interactions of HIV-1 with Dendritic cells (DCs) and macrophages
2: To determine the role of Samhd1 in the modulation of inflammation and immune activation
3: To analyze the restriction of HIV-1 infection in quiescent CD4+ T cells
4: To study the contribution of inflammation and immune activation to HIV persistence

WP Objective 1: To analyze the interactions of HIV-1 with Dendritic cells (DCs) and macrophages
1. Demonstration of the role of SAMHD1 in controlling HIV-1 sensing and antigen presentation in Dendritic cells.
Monocyte-derived dendritic cells (MDDC) stimulate CD8 cytotoxic T lymphocytes (CTL) by presenting endogenous and exogenous viral peptides via major histocompatibility complex class I (MHC-I) molecules. MDDC are poorly susceptible to HIV-1, in part due to the presence of SAMHD1, a cellular enzyme that depletes intracellular deoxynucleoside triphosphates (dNTPs) and degrades viral RNA. Vpx, an HIV-2/SIVsm protein absent from HIV-1, antagonizes SAMHD1 by inducing its degradation. The impact of SAMHD1 on the adaptive cellular immune response remains poorly characterized. Here, we asked whether SAMHD1 modulates MHC-I-restricted HIV-1 antigen presentation. Untreated MDDC or MDDC pretreated with Vpx were exposed to HIV-1, and antigen presentation was examined by monitoring the activation of an HIV-1 Gag-specific CTL clone. SAMHD1 depletion strongly enhanced productive infection of MDDC as well as endogenous HIV-1 antigen presentation. Time-lapse microscopy analysis demonstrated that in the absence of SAMHD1, the CTL rapidly killed infected MDDC. We also report that various transmitted/founder (T/F) HIV-1 strains poorly infected MDDC and, as a consequence, did not stimulate CTL. Vesicular stomatitis virus glycoprotein (VSV-G) pseudotyping of T/F alleviated a block in viral entry and induced antigen presentation only in the absence of SAMHD1. Furthermore, by using another CTL clone that mostly recognizes incoming HIV-1 antigens, we demonstrate that SAMHD1 does not influence exogenous viral antigen presentation. Altogether, our results demonstrate that the antiviral activity of SAMHD1 impacts antigen presentation by DC, highlighting the link that exists between restriction factors and adaptive immune responses.
We also showed that in the cocultures, Samhd1 significantly inhibits productive cell-to-cell transmission to target MDDC and prevents type-I IFN response and expression of the interferon-stimulated gene MxA. Samhd1, by controlling the sensitivity of MDDC to HIV-1 infection during intercellular contacts, impacts their ability to sense the virus and to trigger an innate immune response.
These results have been published in the following articles:
Puigdomenech, I., N. Casartelli, F. Porrot, and O. Schwartz. 2013. SAMHD1 Restricts HIV-1 Cell-to-Cell Transmission And Limits Immune Detection In Monocyte-Derived Dendritic Cells. J Virol. Mar;87(5):2846-56.
Ayinde D, Bruel T, Cardinaud S, Porrot F, Prado J, Moris A, Schwartz O. SAMHD1 limits HIV-1 recognition and killing of infected dendritic cells by cytotoxic T lymphocytes. J Virol. 2015 Apr 29. pii: JVI.00069-15.

2. Identification of two cellular proteins targeted by Vpr during HIV-1 infection.
Viruses often interfere with the DNA damage response to better replicate in their hosts. The HIV auxiliary protein Vpr potently blocks the cell cycle at the G2/M transition. We observed that G2/M arrest results from untimely activation of the structure-specific endonuclease (SSE) regulator SLX4 complex (SLX4com) by Vpr, a process that requires VPRBP-DDB1-CUL4 E3-ligase complex. Direct interaction of Vpr with SLX4 induced the recruitment of VPRBP and kinase-active PLK1, enhancing the cleavage of DNA by SLX4-associated MUS81-EME1 endonucleases. G2/M arrest-deficient Vpr alleles failed to interact with SLX4 or to induce recruitment of MUS81 and PLK1. Furthermore, knockdown of SLX4, MUS81, or EME1 inhibited Vpr-induced G2/M arrest. In addition, we show that the SLX4com is involved in suppressing spontaneous and HIV-1-mediated induction of type 1 interferon and establishment of antiviral responses. Thus, our work not only reveals the identity of the cellular factors required for Vpr-mediated G2/M arrest but also identifies the SLX4com as a regulator of innate immunity.
Additionally, using an unbiased quantitative proteomic screen, we report that Vpr down-regulates helicase-like transcription factor (HLTF), a DNA translocase involved in the repair of damaged replication forks. Vpr subverts the DDB1-cullin4-associated-factor 1 (DCAF1) adaptor of the Cul4A ubiquitin ligase to trigger proteasomal degradation of HLTF. This event takes place rapidly after Vpr delivery to cells, before and independently of Vpr-mediated G2 arrest. HLTF is degraded in lymphocytic cells and macrophages infected with Vpr-expressing HIV-1. Our results reveal a previously unidentified strategy for HIV-1 to antagonize DNA repair in host cells.
These results have been published in the following articles:
Nadine Laguette; Christelle Brégnard; Pauline Hue; Jihane Basbous; Ahmad Yatim; Marion Larroque; Frank Kirchhoff; Angelos Constantinou; Bijan Sobhian; Monsef Benkirane Premature activation of the SLX4 complex by Vpr promotes G2/M arrest and escape from innate immune sensing. Cell. 2014, DOI: 10.1016/j.cell.2013.12.011, PMID: 24412650
Lahouassa H, Blondot ML, Chauveau L, Chougui G, Morel M, Leduc M, Guillonneau F, Ramirez BC, Schwartz O, Margottin-Goguet F. HIV-1 Vpr degrades the HLTF DNA translocase in T cells and macrophages. Proc Natl Acad Sci U S A. 2016 May 10;113(19):5311-6. doi: 10.1073/pnas.1600485113. Epub 2016 Apr 25.

WP Objective 2: To determine the role of Samhd1 in the modulation of inflammation and immune activation (see also other WP Objective 1)
1. Analysis of the permissivity of subsets of macrophages to HIV-1 infection and characterization of the underlying mechanisms.
In order to develop strategies to prevent HIV-1 (human immunodeficiency virus type 1) transmission, it is crucial to better characterize HIV-1 target cells in the female reproductive tract (FRT) mucosae and to identify effective innate responses. Control of HIV-1 infection in the decidua (the uterine mucosa during pregnancy) can serve as a model to study natural mucosal protection. Macrophages are the main HIV-1 target cells in the decidua. Here we report that in vitro, macrophages and T cells are the main HIV-1 targets in the endometrium in nonpregnant women. As reported for decidual macrophages (dM), endometrial macrophages (eM) were found to have an M2-like phenotype (CD68+ CD163+ CD206+ IL-10high). However, eM and dM may belong to different subpopulations, as they differently express certain markers and secrete different amounts of proinflammatory and anti-inflammatory cytokines. We observed strong expression of the SAMHD1 restriction factor and weak expression of its inactive form (pSAMHD1, phosphorylated at residue Thr592) in both eM and dM. Infection of macrophages from both tissues was enhanced in the presence of the viral protein Vpx, suggesting a role for SAMHD1 in the restriction of HIV-1 infection. This study and further comparisons of the decidua with FRT mucosae in nonpregnant women should help to identify mechanisms of mucosal protection against HIV-1 infection.
These results have been published in the following article:
Quillay H, El Costa H, Marlin R, Duriez M, Cannou C, Chretien F, Fernandez H, Lebreton A, Ighil J, Schwartz O, Barre-Sinoussi F, Nugeyre MT, Menu E. 2015. Distinct Characteristics of Endometrial and Decidual Macrophages and Regulation of Their Permissivity to HIV-1 Infection by SAMHD1. J Virol 89:1329-1339.

WP Objective 3: To analyze the restriction of HIV-1 infection in CD4+ T cells
1. Analysis of the restriction of HIV-1 infection induced by the Interferon-induced transmembrane proteins (IFITM1-3).
The interferon-induced transmembrane (IFITM) proteins protect cells from diverse virus infections by inhibiting virus-cell fusion. IFITM proteins also inhibit HIV-1 replication through mechanisms only partially understood. We show that when expressed in uninfected lymphocytes, IFITM proteins exert protective effects during cell-free virus infection, but this restriction can be overcome upon HIV-1 cell-to-cell spread. However, when present in virus-producing lymphocytes, IFITM proteins colocalize with viral Env and Gag proteins and incorporate into nascent HIV-1 virions to limit entry into new target cells. IFITM in viral membranes is associated with impaired virion fusion, offering additional and more potent defense against virus spread. Thus, IFITM proteins act additively in both productively infected cells and uninfected target cells to inhibit HIV-1 spread, potentially conferring these proteins with greater breadth and potency against enveloped viruses.
We have also analyzed the role of amino-terminal mutants of IFITM3 that prevent ubiquitination and endocytosis. We observed that the mutanst are more abundantly incorporated into virions and exhibit enhanced inhibition of HIV-1 fusion. An analysis of primate genomes revealed that IFITM3 is the most ancient antiviral family member of the IFITM locus and has undergone a repeated duplication in independent host lineages. Some IFITM3 genes in nonhuman primates, including those that arose following gene duplication, carry amino-terminal mutations that modify protein localization and function. This suggests that "runaway" IFITM3 variants could be selected for altered antiviral activity. Furthermore, we show that adaptations in IFITM3 result in a trade-off in antiviral specificity, as variants exhibiting enhanced activity against HIV-1 poorly restrict influenza A virus. Overall, we provide the first experimental evidence that diversification of IFITM3 genes may boost the antiviral coverage of host cells and provide selective functional advantages.
These results have been published in the two following articles:
Compton A, Bruel T, Porrot F, Mallet A, Sachse M, Euvrard M, Liang C, Casartelli N, and Schwartz O Interferon-induced transmembrane proteins incorporate into HIV-1 virions and impair viral cell-to-cell spread. 2014 Cell Host & Microbe Dec 10;16(6):736-47
Compton A, Roy N, Porrot F, Billet A, Liang C, Yount J, Casartelli N, and Schwartz O. Naturally occurring mutation in primate IFITM3 allows escape from post-translational regulation and enhances anti-HIV activity. EMBO Rep. 2016 Sep 6. pii: e201642771

2. Analysis of the restriction of HIV-1 infection induced by SAMHD1 in CD4+ T cell subsets.
HIV-1 replication depends on the state of cell activation and division. It is established that SAMHD1 restricts HIV-1 infection of resting CD4 T cells. The modulation of SAMHD1 expression during T-cell activation and proliferation, however, remains unclear, as well as a role for SAMHD1 during HIV-1 pathogenesis. SAMHD1 expression was assessed in CD4 T cells after their activation and in-vitro HIV-1 infection. We performed phenotype analyzes using flow cytometry on CD4 T cells from peripheral blood and lymph nodes from cohorts of HIV-1-infected individuals under antiretroviral treatment or not, and controls. We show that SAMHD1 expression decreased during CD4 T-cell proliferation in association with an increased susceptibility to in-vitro HIV-1 infection. Additionally, circulating memory CD4 T cells are enriched in cells with low levels of SAMHD1. These SAMHD1 cells are highly differentiated, exhibit a large proportion of Ki67 cycling cells and are enriched in T-helper 17 cells. Importantly, memory SAMHD1 cells were depleted from peripheral blood of HIV-infected individuals. We also found that follicular helper T cells present in secondary lymphoid organs lacked the expression of SAMHD1, which was accompanied by a higher susceptibility to HIV-1 infection in vitro. We demonstrate that SAMHD1 expression is decreased during CD4 T-cell activation and proliferation. Also, CD4 T-cell subsets known to be more susceptible to HIV-1 infection, for example, T-helper 17 and follicular helper T cells, display lower levels of SAMHD1. These results pin point a role for SAMHD1 expression in HIV-1 infection and the concomitant depletion of CD4 T cells.
These results have been published in the following article:
Ruffin N, Brezar V, Ayinde D, Lefebvre C, Schulze Zur Wiesch J, van Lunzen J, Bockhorn M, Schwartz O, Hocini H, Lelievre JD, Banchereau J, Levy Y, Seddiki N Resident and circulating effector/memory CD4+ T-cells lacking SAMHD1 are highly sensitive to HIV-1 infection. AIDS 13;29(5):519-30

3. Analysis of the restriction of HIV-1 infection induced by SUN2.
In a previous screen of putative interferon-stimulated genes, SUN2 was shown to inhibit HIV-1 infection in an uncharacterized manner. SUN2 is an inner nuclear membrane protein belonging to the linker of nucleoskeleton and cytoskeleton complex. We have analyzed the role of SUN2 in HIV infection. We report that in contrast to what was initially thought, SUN2 is not induced by type I interferon, and that SUN2 silencing does not modulate HIV infection. However, SUN2 overexpression in cell lines and in primary monocyte-derived dendritic cells inhibits the replication of HIV but not murine leukemia virus or chikungunya virus. We identified HIV-1 and HIV-2 strains that are unaffected by SUN2, suggesting that the effect is specific to particular viral components or cofactors. Intriguingly, SUN2 overexpression induces a multilobular flower-like nuclear shape that does not impact cell viability and is similar to that of cells isolated from patients with HTLV-I-associated adult T-cell leukemia or with progeria. Nuclear shape changes and HIV inhibition both mapped to the nucleoplasmic domain of SUN2 that interacts with the nuclear lamina. This block to HIV replication occurs between reverse transcription and nuclear entry, and passaging experiments selected for a single-amino-acid change in capsid (CA) that leads to resistance to overexpressed SUN2. Furthermore, using chemical inhibition or silencing of cyclophilin A (CypA), as well as CA mutant viruses, we implicated CypA in the SUN2-imposed block to HIV infection. Our results demonstrate that SUN2 overexpression perturbs both nuclear shape and early events of HIV infection.
These results have been published in the following article:
Donahue DA, Amraoui S, di Nunzio F, Kieffer C, Porrot F, Opp S, Diaz-Griffero F, Casartelli N, Schwartz O. SUN2 overexpression deforms nuclear shape and inhibits HIV. J Virol. 2016 Feb 10. pii: JVI.03202-15.

WP Objective 4: To study the contribution of inflammation and immune activation to HIV persistence
1. Analysis of the infection of resting CD4+ T cells and dendritic cells by HIV-1 and HIV-2.
Human Immunodeficiency Virus-type 2 (HIV-2) encodes Vpx that degrades SAMHD1, a cellular restriction factor active in non-dividing cells. HIV-2 replicates in lymphocytes but the susceptibility of monocyte-derived dendritic cells (MDDCs) to in vitro infection remains partly characterized. Here, we investigated HIV-2 replication in primary CD4+ T lymphocytes, both activated and non-activated, as well as in MDDCs. We focused on the requirement of Vpx for productive HIV-2 infection, using the reference HIV-2 ROD strain, the proviral clone GL-AN, as well as two primary HIV-2 isolates. All HIV-2 strains tested replicated in activated CD4+ T cells. Unstimulated CD4+ T cells were not productively infected by HIV-2, but viral replication was triggered upon lymphocyte activation in a Vpx-dependent manner. In contrast, MDDCs were poorly infected when exposed to HIV-2. HIV-2 particles did not potently fuse with MDDCs and did not lead to efficient viral DNA synthesis, even in the presence of Vpx. Moreover, the HIV-2 strains tested were not efficiently sensed by MDDCs, as evidenced by a lack of MxA induction upon viral exposure. Virion pseudotyping with VSV-G rescued fusion, productive infection and HIV-2 sensing by MDDCs. Vpx allows the non-productive infection of resting CD4+ T cells, but does not confer HIV-2 with the ability to efficiently infect MDDCs. In these cells, an entry defect prevents viral fusion and reverse transcription independently of SAMHD1. We propose that HIV-2, like HIV-1, does not productively infect MDDCs, possibly to avoid triggering an immune response mediated by these cells.
These results have been published in the following article:
Chauveau L, Puigdomenech I, Ayinde D, Roesch F, Porrot F, Bruni D, Visseaux B, Descamps D, Schwartz O. 2015. HIV-2 infects resting CD4+ T cells but not monocyte-derived dendritic cells. Retrovirology 12:2.

2. Analysis of TNF alpha production by HIV-1 infected T cells and role of Vpr.
The HIV-1 accessory protein Vpr displays different activities potentially impacting viral replication, including the arrest of the cell cycle in the G2 phase and the stimulation of apoptosis and DNA damage response pathways. Vpr also modulates cytokine production by infected cells, but this property remains partly characterized. Here, we investigated the effect of Vpr on the production of the proinflammatory cytokine tumor necrosis factor (TNF). We report that Vpr significantly increases TNF secretion by infected lymphocytes. De novo production of Vpr is required for this effect. Vpr mutants known to be defective for G2 cell cycle arrest induce lower levels of TNF secretion, suggesting a link between these two functions. Silencing experiments and the use of chemical inhibitors further implicated the cellular proteins DDB1 and TAK1 in this activity of Vpr. TNF secreted by HIV-1-infected cells triggers NF-κB activity in bystander cells and allows viral reactivation in a model of latently infected cells. Thus, the stimulation of the proinflammatory pathway by Vpr may impact HIV-1 replication in vivo.
These results have been published in the following article:
Roesch F, Richard L, Rua R, Porrot F, Casartelli N, Schwartz O. Vpr enhances TNF production by HIV-1 infected T cells. J Virol. 2015 Sep 23. pii: JVI.02098-15.

3. Elimination of HIV-1 infected T cells by broadly neutralizing antibodies
The Fc region of HIV-1 Env-specific broadly neutralizing antibodies (bNAbs) is required for suppressing viraemia, through mechanisms which remain poorly understood. Here, we identify bNAbs that exert antibody-dependent cellular cytotoxicity (ADCC) in cell culture and kill HIV-1-infected lymphocytes through natural killer (NK) engagement. These antibodies target the CD4-binding site, the glycans/V3 and V1/V2 loops on gp120, or the gp41 moiety. The landscape of Env epitope exposure at the surface and the sensitivity of infected cells to ADCC vary considerably between viral strains. Efficient ADCC requires sustained cell surface binding of bNAbs to Env, and combining bNAbs allows a potent killing activity. Furthermore, reactivated infected cells from HIV-positive individuals expose heterogeneous Env epitope patterns, with levels that are often but not always sufficient to trigger killing by bNAbs. Our study delineates the parameters controlling ADCC activity of bNAbs, and supports the use of the most potent antibodies to clear the viral reservoir.
These results have been published in the following article:
Bruel T, Guivel-Benhassine F, Amraoui S, Malbec M, Richard L, Bourdic K, Donahue DA, Lorin V, Casartelli N, Noël N, Lambotte O, Mouquet H, Schwartz O. Elimination of HIV-1 infected cells by broadly neutralizing antibodies. Nature Communications, 2016 Mar 3;7:10844. doi: 10.1038/ncomms10844

WP5: Clinical translation and disease cohorts (Partner 4, CHUV)
The main objective of WP5 is to establish the conditions for collaborative cohorts to be used for validation and clinical translation of the results obtained within the previous WPs. A surrogate objective is to use human sample material to investigate the feature of productive or latent HIV infection, as well as uncovering the mechanisms linked to HIV latency reactivation.
This clinical WP is thus tightly linked to the identification of molecules and gene candidates involved in HIV from other WP. Obviously, the nature of the emerging candidates to assess determined the design of the study that uses clinical data, bioresource materials (serum, plasma, viable cells), or the clinical structure to recruit individuals for fresh material/large sample donation.

Objective 1: To establish the conditions for work in collaboration with the Swiss HIV Cohort Study.
The Swiss HIV Cohort Study (SHCS) was established in 1988. It is an ongoing, nationwide, multicenter, clinic-based observational study with continuous enrolment and semi-annual study visits. Socio-demographic, clinical and laboratory data are recorded in detail. The SHCS is estimated to cover about 70% of people with HIV living in Switzerland. Until November 2016, 19’523 patients have been enrolled, 4,906 have died during active follow-up and 5,225 were declared as loss to follow-up. From the 9’392 patients currently followed, 28% are women. Biological repository for 19’523 patients contains over 1 million of plasma/serum samples and aliquots of viable cells.

The conditions for clinical translation in the context of the SHCS were successfully established. The Swiss HIV Cohort Study (SHCS) approved the project. The database of the SHCS cohort was successfully used for assessing the role of gene variation and expression on HIV replication in HIV+ individuals as illustrated in objective 2.

Objective 2: To conduct analyses and validation studies using the cohort clinical database and biological repository.
The database of the SHCS cohort has been successfully used for patient selection and biological material selection from the biological repository.

In particular, it has been used to analyze the role of an endogenous fragment of human albumin in antagonizing CXCR4 and in displaying anti-inflammatory properties, in collaboration with Partners 3 and 5 (1). This study aimed at screening a blood-derived peptide library for inhibitors of CXCR4-tropic HIV-1 strains and identified a 16-amino acid fragment of serum albumin (named EPI-X4) as being an effective and highly specific CXCR4 antagonist. Detection of EPI-X4 was performed in the plasma and serum from 102 HIV-infected individuals from the SHCS representing the full spectrum of viral load and disease progression. Samples were collected while the individuals were treatment naive. Although available, more samples were not necessary for further validating the hypothesis.
The identification of active peptides from human hemofiltrates was carried in WP3 by Partners 3 and 5, i.e. groups of Prof. Dr. Frank Kirchhoff (UULM) and Prof. Wolf-Georg Forssmann (Pharis Biotech). They identified a potential candidate, GBP4, able to reactivate latent HIV in a Jurkat cell line model (J-LAT). However, GBP4 activity was not confirmed in primary cells, and requires further investigation and characterization before assessment in the context of SHCS samples.
We develop multiple bioassays and molecular approaches in order to (i) identify novel candidates potentially involved in HIV latency and persistence as well as in HIV productive infection, (ii) validate functionally the role of the gene candidate on HIV replication, and (iii) characterize host cell gene-specific features (genomic variants, conservation across mammals, expression level) in the context of primary samples from healthy donors and from the SHCS repository and correlate them with viral features (viral setpoint, viral load, HIV replication). For this, we implemented genome and whole-transcriptome analyses using state-of-the-art techniques such as genome-wide SNP analysis, exome sequencing and RNA-Seq deep sequencing of diverse cellular subtypes, at population or single-cell level (fluidgm technology), and under different treatment conditions (non-stimulated, stimulated with latency reversing agents, or activated by T-cell receptor stimulation).
We have performed multiple studies to investigate human genetic variation and truncations in the human genome and in immune genes in particular, using biological material from healthy individuals as well as HIV+ individuals selected from the SHCS (2, 3). These genome-wide data were then correlated with viral parameters, providing useful information on the impact of host genetics on HIV acquisition and HIV disease progression. We have generated a public resource named GuavaH (Genomic Utility for Association and Viral Analyses in HIV, http://www.GuavaH.org), to support multipurpose analysis of genome-wide genetic variation and gene expression profile across multiple phenotypes relevant to HIV biology, and that should be a valuable tool for the scientific community.
We brought these data one step further to identify novel restriction factors (4). For this, we hypothesized that HIV restriction factors would respond to the following criteria: be under positive selection pressure, be induced during HIV-1 infection, be up-regulated by interferons, and/or interact with viral proteins. This analysis identified 30 putative restriction factors. To date, the restriction mechanism of GBP5 and APOL6 were further investigated (5)(Pyndiah et al, in preparation). The other restriction factor candidates will be analyzed as well in the future.
We developed an approach aimed at the dynamic in-depth characterization of the joint transcriptome of the virus and the host upon HIV infection (6). Briefly, cells were exposed to a high number of HIV particles to allow universal infection. Cells were then collected every two hours, over a period of 24 hours, to determine viral replication intermediates (reverse transcription, integration, transcription, translation, and release) and their impact on the cellular transcriptional response (analyzed by SAGE-Seq). This unprecedented study provided a coherent time frame of viral progression, needed for modeling cellular transcriptome modulation concomitant to viral progression. It first revealed the profound reprogramming of the host cell upon HIV infection with a massive early shutdown of genes, including restriction factors. A second phase was characterized by the progressive upregulation of genes likely to be required to promote HIV life cycle. These latter genes might be further investigated to analyze their exact role on HIV replication. The data generated by this study were used for mathematically modeling and were rendered publically available on internet at http://www.peachi.labtelenti.org for customized querying by the scientific community. This website has proven to be useful as many scientists started their investigation by first assessing the expression of specific genes of interest in this system, thereby providing a first reference for HIV analysis. Coherent time frame of viral progression, needed for modeling cellular transcriptome modulation concomitant to viral progression
In addition, the cellular RNA collected in this work was further used in order to investigate the non-coding transcriptome, and in particular the lncRNAome, in collaboration with the group of Linos Vandekerckhove (7). This analysis highlighted a different transcriptinal regulation profile as compared to protein coding mRNAs and demonstrated that lncRNAs add a new dimension to the HIV-host interplay. This study calls for future investigation of lncRNA and lncRNA-mRNA pairs to understand their exact contribution in modulating HIV replication.
We further developed this HIV-host dynamic approach by repeating the infection experiment, and collecting this time additional samples to characterize the proteome and phosphoproteome (Golumbeanu et al, in preparation). This proteo-transcriptomic analysis of the cell upon HIV infection provided very interesting results that we are still currently analyzing and validating. In particular, we were able to detect 12829 transcripts, 3623 proteins and 572 phosphorylated proteins. Many analyses and experiments will be derived from this study in the near future to identify novel gene products involved in HIV replication, either promoting it or blocking it. For now, we have started investigating a few candidate genes that were characterized by a discrepancy between the RNA and protein levels. Indeed, we hypothesized that genes that were upregulated or not modulated at the RNA level but downregulated at the protein level might be restriction factors that HIV specifically degrades. We identified 26 candidate genes that fulfill these criteria and that we decided to test in vitro. We are currently cloning these genes. In parallel to this work, we will update the peachi webresource and implement a new version, peachi 2.0, that will accommodate protein data in addition to transcriptomic data and that will be publically available and queryable by the scientific community.
In order to investigate HIV latency, we first investigated CD4+ T cells isolated from HIV+ individuals selected from the SHCS. When these cells were treated with control, SAHA (histone deacetylase inhibitor) or activated by T-cell receptor (TCR) stimulation, viruses were produced mostly by TCR-stimulated cells, but not with mock treatment or SAHA. This study indicated that SAHA might not work as initially suggested in reactivating viral particle production from latently infected cells. To further investigate this question, and taking advantage of our established expertise in genome-wide analysis, we used a primary CD4+ T cell model of HIV latency for the in-depth characterization of the transcriptome of cells entering, maintaining or exiting latency at the population level (8). This study indicated that HIV latency can be due to transcriptional silencing but that additional mechanisms may contribute as well, including post-transcriptional blocks and the cellular state. It also allowed providing additional accuracy when evaluating latent cell reactivation, in particular by discriminating between increase of cell-associated viral RNA from cell-free viral RNA (and thus particle production). We have generated here again an additional web resource named Litchi (http://www.litchi.labtelenti.org) where the expression of individual genes can be queried in CD4+ T cell across different stages of latency or under different treatments. We are pursuing this study by investigating (i) another primary model of HIV latency, and (ii) the transcriptome of latent cells under different stimulations at the single-cell level, isolated from this primary cell model of HIV latency or from cells from HIV+ individuals selected from the SHCS. Data are still under analysis aiming at linking a specific transcriptomic signature with the success of HIV expression reactivation.

We also implemented and developed a single cell approach to identify specific biomarkers of HIV latency. As a proof-of-concept, we used susceptibility to HIV as a model to identify biomarkers of HIV permissiveness (9, 10)(Rato et al, in preparation). Using state-of-the-art technology for single cell RNA-Seq analysis, we used fluidigm technology to characterize the transcriptome of 85 individual cells isolated from a hyperpermissive donor and 80 individual cells isolated from a hypopermissive donor. We then focused and completed this analysis by characterizing the cell surface expression of 332 proteins. We uncovered a few gene candidates with differential expression between the two donors that correlated with HIV permissiveness. To functionally validate these candidates, cells from multiple donors were first sorted by FACs according to their level of candidate gene expression before HIV infection. Most candidates were successfully validated functionally, identifying multiple proteins marking the cell permissive to HIV. This approach may be applied to any specific phenotype of interest and we are now applying it to identify specific biomarkers of HIV latency.

Objective 3: To establish the cohort as a recruitment platform of specific populations/dedicated sampling.
We have developed all the assays and pipelines necessary to characterize candidate biomarkers of HIV latency and candidate molecules as HIV latency reactivation agents. We depend on the results from other work-packages to continue our work and to test them in the context of the Swiss HIV Cohort Study. To date, we have used biological samples from archived biological repository or collected fresh blood from SHCS participants to directly investigate the amount of latent cells and their ability to be reactivated ex vivo (HIV latency investigation described in objective 2). We estimate that we have fulfilled our objectives, although we did not identify yet a candidate biomarker of HIV latency to be further tested in the context of a specific clinical trial of the Swiss HIV Cohort Study.

References
1. Zirafi O, Kim KA, Standker L, Mohr KB, Sauter D, Heigele A, et al. Discovery and characterization of an endogenous CXCR4 antagonist. Cell reports. 2015;11(5):737-47.
2. Bartha I, Rausell A, McLaren PJ, Mohammadi P, Tardaguila M, Chaturvedi N, et al. The Characteristics of Heterozygous Protein Truncating Variants in the Human Genome. PLoS computational biology. 2015;11(12):e1004647.
3. Bartha I, McLaren PJ, Ciuffi A, Fellay J, Telenti A. GuavaH: a compendium of host genomic data in HIV biology and disease. Retrovirology. 2014;11:6.
4. McLaren PJ, Gawanbacht A, Pyndiah N, Krapp C, Hotter D, Kluge SF, et al. Identification of potential HIV restriction factors by combining evolutionary genomic signatures with functional analyses. Retrovirology. 2015;12:41.
5. Krapp C, Hotter D, Gawanbacht A, McLaren PJ, Kluge SF, Sturzel CM, et al. Guanylate Binding Protein (GBP) 5 Is an Interferon-Inducible Inhibitor of HIV-1 Infectivity. Cell host & microbe. 2016;19(4):504-14.
6. Mohammadi P, Desfarges S, Bartha I, Joos B, Zangger N, Munoz M, et al. 24 hours in the life of HIV-1 in a T cell line. PLoS pathogens. 2013;9(1):e1003161.
7. Trypsteen W, Mohammadi P, Van Hecke C, Mestdagh P, Lefever S, Saeys Y, et al. Differential expression of lncRNAs during the HIV replication cycle: an underestimated layer in the HIV-host interplay. Scientific reports. 2016;6:36111.
8. Mohammadi P, di Iulio J, Munoz M, Martinez R, Bartha I, Cavassini M, et al. Dynamics of HIV latency and reactivation in a primary CD4+ T cell model. PLoS pathogens. 2014;10(5):e1004156.
9. Julia M, Telenti A, Rausell A. Sincell: an R/Bioconductor package for statistical assessment of cell-state hierarchies from single-cell RNA-seq. Bioinformatics. 2015;31(20):3380-2.
10. Ciuffi A, Quenneville S, Rausell A, Rato S, Muñoz M, Telenti A, editors. Single-cell analysis identifies biomarkers for HIV permissiveness Abstract OP2.1). Seventh International Workshop on HIV Persistence during Therapy; 2015 December 2015; Miami (FL), USA: Journal of Virus Eradication.

Potential Impact:
Hit Hidden HIV is a project mainly devoted to decipher different aspects of HIV persistence, which is one of the priorities and one of the most challenging aspect of HIV/AIDS research. The ultimate goal is translate this knowledge into efficient therapeutic strategy toward HIV cure. Major breakthrough were achieved within the frame work of this project.
We estimate that our work has already significantly impacted the scientific community regarding, among others, HIV replication, inflammation, HIV restriction, gene variation, gene expression, HIV latency and the identification of a specific cell surface biomarker expressed by HIV persistent CD4 T cell in patients under suppressive ART. In the future, we should focus on “proof of concept” to use the knowledge generated during 3 years to achieve HIV-1 cure in patients undergoing highly active antiretroviral therapy. Indeed, the identification of a biomarker for HIV persistent cells together with the increased knowledge on use of broadly neutralizing antibodies open the possibility to clinical trials aiming at eliminating viral reservoir. Importantly, this European support also allowed (i) exchanging ideas with top scientists, within the collaborative consortium, as well as with other scientists, (ii) starting novel collaborations with top European researchers, and (iii) helped the careers of all investigators that participated in this project.
The potential societal impacts include future novel strategies to target the viral reservoir. Classical Antiretroviral treatment (ART) is able to control the infection but is unable to permanently eradicate HIV from infected people leaving reservoir of virus within the population. We can envisage two related impacts on the European Union. An impact on health, because the persistence of latent HIV reservoirs results in increase of occurrence of other pathologies in HIV-1 infected individuals under successful ART. A financial impact because ART is a life-long treatment with a heavy burden on public health care. The problem is even more pronounced in countries under development, where ART is not financially and logistically sustainable, although major progress have been obtained very recently in the access to treatment in such countries. Taken together, these considerations highlight that the “Hit Hidden HIV” consortium meets both the needs of EU citizens and of Europe’s agenda for life sciences, which are based on the needs of its citizens. Furthermore, the consortium has a strong international connotation. Hence, the consortium responds to the urge of the Health FP7 work program to tackle global health problems, even if the impact on the EU is much less as compared to other countries outside the EU.
Dissemination activities
For dissemination, the results were made accessible to the scientific community, through multiple presentations at international meetings, publications and publically available web resources allowing customized queries. Many investigations are still ongoing and acknowledgements to the European Union’s Seventh Framework Program FP7/2007-2013/under grant agreement n°305762/Hit Hidden HIV grant will thus be specified in future publications and international meeting presentations. Several patents were published. Newly developed reagents, proviral HIV-1 reporter constructs and protocols will be made available to the scientific community. Some of the partners were also very active at presenting their work to public audience both in their national media and through open public seminars.
Major achievements
The project aims at (i) identifying key cellular and molecular mechanisms of viral persistence (ii) increasing knowledge on the contribution of immune activation and inflammation to HIV persistence (iii) translating this basic knowledge into novel targets and strategies to achieve a cure for HIV/AIDS.
*The identification of CD32a as biomarker of HIV persistent CD4 T cell in HIV infected individuals under suppressive ART is certainly one of the major breakthrough achieved within the frame of the Hit hidden HIV project. There is no doubt that the identification of this marker will open lots of new research avenues towards the characterization, control and eradication of the latent HIV reservoir. This discovery will likely have a tremendous impact on the research conducted in the field of HIV cure. Indeed, this discovery establish the basis for a direct “Kill” strategy towards elimination of the reservoir, which is required to achieve a cure for HIV.
*Our results obtained through the HIT HIDDEN HIV consortium opened interesting questions about how HIV-1 interacts with CD4+ lymphocytes and dendritic cells. We have also analyzed how to target the viral reservoir present in HIV-1 infected individuals under successful Anti-Retroviral Treatment (ART). Our next aim will be to characterize further the effect of broadly neutralizing antibodies (bNAbs) that may bind to HIV-1 Env glycoproteins at the surface of reactivated cells. We are exploring what could be the best reactivator of viral latency, combined to various bNAbs to eliminate or significantly decrease the viral reservoir.
*Our analysis of the effect of the anti-HIV-1 broadly neutralizing antibodies (bNAbs) is continuing. We demonstrated that some of the most potent antibodies target the viral reservoir in samples from HIV-1 infected individuals. We intend to study the underlying cellular and molecular mechanisms, to define the optimal combinations of bNAbs that could be used in future clinical trials. Partner 1 identified a marker of latently infected cells and we will determine with his group if antibodies directed against this marker may eliminate the viral reservoir. We will perform killing assays in cell culture systems, combining antibodies targeting the marker of the reservoir and bNAbs.
*The identification of RBP4 provides proof-of-concept evidence that examination of blood-derived peptide libraries allows to discover newly endogenous agents activating latent HIV-1 proviruses. Some enhancing fractions contained known stimulatory cytokines but the results suggest that several as-yet-unknown endogenous enhancers of latent HIV-1 proviruses remain to be discovered. Improved purification methods, together with newly developed screens in primary HIV-1 latency models, should allow the identification of these factors. It will also be of interest to use peptide/protein libraries derived from e.g. immune-activated and/or HIV-infected individuals or lymphatic organs that might represent the major latent reservoirs of HIV-1. Since the identified peptides are naturally present in the human body they might play relevant physiological roles in the establishment and maintenance of HIV latency. Furthermore, endogenous peptides or improved derivatives thereof are promising drug candidates since they are less likely to induce undesired side-effects than small-molecule agents.

Describing gene expression signature for HIV permissiveness is a great discovery. The data were made quickly available to the scientific community to share knowledge and understanding related to HIV issues. For this, we generated multiple web resources that are publically available and that is easy to customize and to use. In particular, any gene of interest can be queried and multiple information can be retrieved, including gene variants, splice variants, expression in resting CD4+ T cells, expression in TCR-activated cells, time of expression, and so on. We are convinced that these resources are helpful and valuable.

List of Websites:
Project public website address: http://www.hithiddenhiv.org/

Contact:
Dr. Monsef BENKIRANE
Molecular Virology Lab, Institute of Human Genetics - CNRS
141, rue de la Cardonille, 34396 Montpellier - Cedex 5, France
Phone +33-(0)4 34 35 99 96, FAX +33-(0)4 34 35 99 01
E-mail: monsef.benkirane@igh.cnrs.fr

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