Pharmacological Targeting of Sphingosine Kinases Impedes HIV-1 Infection of CD4 T Cells through SAMHD1 Modulation.

Sphingosine-1-phosphate (S1P) is a sphingolipid modulator of a myriad of cellular processes, and therapeutic targeting of S1P signaling is utilized clinically to treat multiple sclerosis. We have previously shown that functional antagonism of S1P receptors reduces cell-free, cell-to-cell, and latent HIV-1 infection in primary CD4 T cells. In this work, we examined whether targeting sphingosine kinase 1 or 2 (SPHK1/2) to inhibit S1P production would prevent infection using multiple HIV-1 primary isolates and infectious molecular clones. SPHK inhibition reduced HIV transmission between primary CD4 T cells in both cell-to-cell transmission and pretreatment coculture models. Mechanistically, pharmacological inhibition of SPHK reduced susceptibility to infection primarily by downregulating phosphorylated SAMHD1 (pSAMHD1), enhancing the activity of this innate HIV-1 restriction factor. Furthermore, genetic disruption of either SPHK1 or SPHK2 by CRISPR/Cas9 reduced phosphorylation of SAMHD1, demonstrating the role of these kinases in modulation of SAMHD1 activity. The effect of SPHK inhibition on limiting HIV-1 infection in CD4 T cells was observed irrespective of the biological sex or age of the donor, with neither variable significantly influencing the effectiveness of SPHK inhibition. Our results demonstrate that targeting SPHK inhibits transmission of HIV-1 via modulation of SAMHD1 phosphorylation to decrease permissiveness to infection in CD4 T cells and suggests that therapeutic targeting of this pathway early in infection enables development of strategies to prevent establishment of infection and hinder cell-to-cell transmission of HIV-1. IMPORTANCE HIV-1 infection, once established, requires lifelong treatment due to the ability of the virus to maintain latent infection in its host and become reactivated during an interruption in antiretroviral treatment (ART). Although preventing transmission and acquisition of HIV is an important goal, no ART thus far have exploited harnessing a component of the host immune system to combat transmission of the virus. We have previously shown that inhibition of sphingosine-1-phosphate (S1P) receptors, a component of S1P signaling, reduces HIV-1 infection in human CD4 T cells. We therefore investigated inhibition of sphingosine kinases, another element of this signaling system, in this work. We found that inhibition of sphingosine kinases 1 and 2 (SPHK1/2) could reduce HIV-1 transmission, both among CD4 T cells and between macrophages and CD4 T cells. Our research therefore suggests that therapeutic targeting of SPHK or S1P receptors may aid in the development of strategies to prevent establishment and transmission of HIV-1 infection among immune cells.

The HIV Latency Reversal Agent HODHBt Enhances NK Cell Effector and Memory-Like Functions by Increasing Interleukin-15-Mediated STAT Activation.

Elimination of human immunodeficiency virus (HIV) reservoirs is a critical endpoint to eradicate HIV. One therapeutic intervention against latent HIV is "shock and kill." This strategy is based on the transcriptional activation of latent HIV with a latency-reversing agent (LRA) with the consequent killing of the reactivated cell by either the cytopathic effect of HIV or the immune system. We have previously found that the small molecule 3-hydroxy-1,2,3-benzotriazin-4(3H)-one (HODHBt) acts as an LRA by increasing signal transducer and activator of transcription (STAT) factor activation mediated by interleukin-15 (IL-15) in cells isolated from aviremic participants. The IL-15 superagonist N-803 is currently under clinical investigation to eliminate latent reservoirs. IL-15 and N-803 share similar mechanisms of action by promoting the activation of STATs and have shown some promise in preclinical models directed toward HIV eradication. In this work, we evaluated the ability of HODHBt to enhance IL-15 signaling in natural killer (NK) cells and the biological consequences associated with increased STAT activation in NK cell effector and memory-like functions. We showed that HODHBt increased IL-15-mediated STAT phosphorylation in NK cells, resulting in increases in the secretion of CXCL-10 and interferon gamma (IFN-γ) and the expression of cytotoxic proteins, including granzyme B, granzyme A, perforin, granulysin, FASL, and TRAIL. This increased cytotoxic profile results in increased cytotoxicity against HIV-infected cells and different tumor cell lines. HODHBt also improved the generation of cytokine-induced memory-like NK cells. Overall, our data demonstrate that enhancing the magnitude of IL-15 signaling with HODHBt favors NK cell cytotoxicity and memory-like generation, and thus, targeting this pathway could be further explored for HIV cure interventions. IMPORTANCE Several clinical trials targeting the HIV latent reservoir with LRAs have been completed. In spite of a lack of clinical benefit, they have been crucial to elucidate hurdles that "shock and kill" strategies have to overcome to promote an effective reduction of the latent reservoir to lead to a cure. These hurdles include low reactivation potential mediated by LRAs, the negative influence of some LRAs on the activity of natural killer and effector CD8 T cells, an increased resistance to apoptosis of latently infected cells, and an exhausted immune system due to chronic inflammation. To that end, finding therapeutic strategies that can overcome some of these challenges could improve the outcome of shock and kill strategies aimed at HIV eradication. Here, we show that the LRA HODHBt also improves IL-15-mediated NK cell effector and memory-like functions. As such, pharmacological enhancement of IL-15-mediated STAT activation can open new therapeutic avenues toward an HIV cure.

Antibody and T-cell responses to coronavirus disease 2019 vaccination in common variable immunodeficiency and specific antibody deficiency.

BACKGROUND: Clinical trials of the mRNA coronavirus disease 2019 (COVID-19) vaccines excluded individuals with primary antibody deficiencies. OBJECTIVE: To evaluate whether antibody and T-cell responses to mRNA COVID-19 vaccination in patients with common variable immunodeficiency (CVID) and specific antibody deficiency (SAD) were comparable to those in healthy controls. METHODS: We measured antibody responses against the spike glycoprotein and the receptor-binding domain (RBD) in addition to severe acute respiratory syndrome coronavirus 2 specific T-cell responses using peripheral blood mononuclear cells 2 to 8 weeks after the subjects completed the primary 2-dose vaccine series. RESULTS: The study comprised 12 patients with CVID, 7 patients with SAD, and 10 controls. Individuals with CVID had lower immunoglobulin (Ig) G and Ig A levels against spike glycoprotein than did both individuals with SAD (P = .27 and P = .01, respectively) and controls (P = .01 and P = .004, respectively). The CVID group developed lower IgG titers against the RBD epitope than did the control group (P = .01). Participants with CVID had lower neutralizing titers than did the control group (P = .002). All participants with SAD developed neutralizing titers. All 3 groups (SAD, CVID, and control) developed antigen-specific CD4+ and CD8+ T-cell responses after vaccination. CONCLUSION: Our results suggest that patients with CVID may have impaired antibody responses to COVID-19 vaccination but intact T-cell responses, whereas patients with SAD would be expected to have both intact antibody and T-cell responses to vaccination.

The Intact Non-Inducible Latent HIV-1 Reservoir is Established In an In Vitro Primary TCM Cell Model of Latency.

The establishment of HIV-1 latency has hindered an HIV-1 cure. "Shock and Kill" strategies to target this reservoir aim to induce the latent provirus with latency reversing agents (LRAs). However, recent studies have shown that the majority of the intact HIV-1 viral reservoir found in ART-suppressed HIV infected individuals is not inducible. We sought to understand whether this non-inducible reservoir is established, and thus able to be studied, in an in vitro primary TCM model of latency. Furthermore, we wanted to expand this model system to include R5-tropic and non-B subtype viruses. To that end, we generated our TCM model of latency with an R5 subtype B virus, AD8 and an R5 subtype C virus, MJ4. Our results demonstrate that both intact and defective proviruses are generated in this model. Less than 50% of intact proviruses are inducible regardless of viral strain in the context of maximal stimulation through the TCR or with different clinically relevant LRAs including the HDAC inhibitors SAHA and MS-275, the PKC agonist Ingenol 3,20-dibenzoate or the SMAC mimetic AZD-5582. Our findings suggest that current LRA strategies are insufficient to effectively reactivate intact latent HIV-1 proviruses in primary CD4 TCM cells and that the mechanisms involved in the generation of the non-inducible HIV-1 reservoir can be studied using this primary in vitro model.Importance: HIV-1 establishes a latent reservoir that persists under antiretroviral therapy. Antiretroviral therapy is able to stop the spread of the virus and the progression of the disease but does not target this latent reservoir. If antiretroviral therapy is stopped, the virus is able to resume replication and the disease progresses. Recently, it has been demonstrated that most of the latent reservoir capable of generating replication competent virus cannot be induced in the laboratory setting. However, the mechanisms that influence the generation of this intact and non-inducible latent reservoir are still under investigation. Here we demonstrate the generation of defective, intact and intact non-inducible latent HIV-1 in a TCM model of latency using different HIV-1 strains. Thus, the mechanisms which control inducibility can be studied using this primary cell model of latency, which may accelerate our understanding of the latent reservoir and the development of curative strategies.

Selective BCL-XL Antagonists Eliminate Infected Cells from a Primary-Cell Model of HIV Latency but Not from Ex Vivo Reservoirs.

HIV persists, despite immune responses and antiretroviral therapy, in viral reservoirs that seed rebound viremia if therapy is interrupted. Previously, we showed that the BCL-2 protein contributes to HIV persistence by conferring a survival advantage to reservoir-harboring cells. Here, we demonstrate that many of the BCL-2 family members are overexpressed in HIV-infected CD4+ T cells, indicating increased tension between proapoptotic and prosurvival family members-and suggesting that inhibition of prosurvival members may disproportionately affect the survival of HIV-infected cells. Based on these results, we chose to study BCL-XL due to its consistent overexpression and the availability of selective antagonists. Infection of primary CD4+ T cells with HIV resulted in increased BCL-XL protein expression, and treatment with two selective BCL-XL antagonists, A-1155463 and A-1551852, led to selective death of productively infected CD4+ T cells. In a primary cell model of latency, both BCL-XL antagonists drove reductions in HIV DNA and in infectious cell frequencies both alone and in combination with the latency reversing agent bryostatin-1, with little off-target cytotoxicity. However, these antagonists, with or without bryostatin-1 or in combination with the highly potent latency reversing agent combination phorbol myristate acetate (PMA) + ionomycin, failed to reduce total HIV DNA and infectious reservoirs in ex vivo CD4+ T cells from antiretroviral therapy (ART)-suppressed donors. Our results add to growing evidence that bona fide reservoir-harboring cells are resistant to multiple "kick and kill" modalities-relative to latency models. We also interpret our results as encouraging further exploration of BCL-XL antagonists for cure, where combination approaches, including with immune effectors, may unlock the ability to eliminate ex vivo reservoirs. IMPORTANCE Although antiretroviral therapy (ART) has transformed HIV infection into a manageable chronic condition, there is no safe or scalable cure. HIV persists in "reservoirs" of infected cells that reinitiate disease progression if ART is interrupted. Whereas most efforts to eliminate this reservoir have focused on exposing these cells to immune-mediated clearance by reversing viral latency, recent work shows that these cells also resist being killed. Here, we identify a "prosurvival" factor, BCL-XL, that is overexpressed in HIV-infected cells, and demonstrate selective toxicity to these cells by BCL-XL antagonists. These antagonists also reduced reservoirs in a primary-cell latency model but were insufficient to reduce "natural" reservoirs in ex vivo CD4+ T cells-adding to growing evidence that the latter are resilient in a way that is not reflected in models. We nonetheless suggest that the selective toxicity of BCL-XL antagonists to HIV-infected cells supports their prioritization for testing in combinations aimed at reducing ex vivo reservoirs.

Fingolimod inhibits multiple stages of the HIV-1 life cycle.

Antiretroviral drugs that target various stages of the Human Immunodeficiency Virus (HIV) life cycle have been effective in curbing the AIDS epidemic. However, drug resistance, off-target effects of antiretroviral therapy (ART), and varying efficacy in prevention underscore the need to develop novel and alternative therapeutics. In this study, we investigated whether targeting the signaling molecule Sphingosine-1-phosphate (S1P) would inhibit HIV-1 infection and generation of the latent reservoir in primary CD4 T cells. We show that FTY720 (Fingolimod), an FDA-approved functional antagonist of S1P receptors, blocks cell-free and cell-to-cell transmission of HIV and consequently reduces detectable latent virus. Mechanistically, FTY720 impacts the HIV-1 life cycle at two levels. Firstly, FTY720 reduces the surface density of CD4, thereby inhibiting viral binding and fusion. Secondly, FTY720 decreases the phosphorylation of the innate HIV restriction factor SAMHD1 which is associated with reduced levels of total and integrated HIV, while reducing the expression of Cyclin D3. In conclusion, targeting the S1P pathway with FTY720 could be a novel strategy to inhibit HIV replication and reduce the seeding of the latent reservoir.

Human splice factors contribute to latent HIV infection in primary cell models and blood CD4+ T cells from ART-treated individuals.

It is unclear what mechanisms govern latent HIV infection in vivo or in primary cell models. To investigate these questions, we compared the HIV and cellular transcription profile in three primary cell models and peripheral CD4+ T cells from HIV-infected ART-suppressed individuals using RT-ddPCR and RNA-seq. All primary cell models recapitulated the block to HIV multiple splicing seen in cells from ART-suppressed individuals, suggesting that this may be a key feature of HIV latency in primary CD4+ T cells. Blocks to HIV transcriptional initiation and elongation were observed more variably among models. A common set of 234 cellular genes, including members of the minor spliceosome pathway, was differentially expressed between unstimulated and activated cells from primary cell models and ART-suppressed individuals, suggesting these genes may play a role in the blocks to HIV transcription and splicing underlying latent infection. These genes may represent new targets for therapies designed to reactivate or silence latently-infected cells.

Structure-Activity Relationship Analysis of Benzotriazine Analogues as HIV-1 Latency-Reversing Agents.

"Shock and kill" therapeutic strategies toward HIV eradication are based on the transcriptional activation of latent HIV with a latency-reversing agent (LRA) and the consequent killing of the reactivated cell by either the cytopathic effect of HIV or an arm of the immune system. We have recently found several benzotriazole and benzotriazine analogues that have the ability to reactivate latent HIV by inhibiting signal transducer and activator of transcription 5 (STAT5) SUMOylation and promoting STAT5 binding to the HIV long terminal repeat and increasing its transcriptional activity. To understand the essential structural groups required for biological activity of these molecules, we performed a systematic analysis of >40 analogues. First, we characterized the essential motifs within these molecules that are required for their biological activity. Second, we identified three benzotriazine analogues with similar activity. We demonstrated that these three compounds are able to increase STAT5 phosphorylation and transcriptional activity. All active analogues reactivate latent HIV in a primary cell model of latency and enhance the ability of interleukin-15 to reactivate latent HIV in cells isolated from aviremic participants. Third, this family of compounds also promote immune effector functions in vitro in the absence of toxicity or global immune activation. Finally, initial studies in mice suggest lack of acute toxicity in vivo A better understanding of the biological activity of these compounds will help in the design of improved LRAs that work via inhibition of STAT5 SUMOylation.

Microbiota promotes systemic T-cell survival through suppression of an apoptotic factor.

Symbiotic microbes impact the severity of a variety of diseases through regulation of T-cell development. However, little is known regarding the molecular mechanisms by which this is accomplished. Here we report that a secreted factor, Erdr1, is regulated by the microbiota to control T-cell apoptosis. Erdr1 expression was identified by transcriptome analysis to be elevated in splenic T cells from germfree and antibiotic-treated mice. Suppression of Erdr1 depends on detection of circulating microbial products by Toll-like receptors on T cells, and this regulation is conserved in human T cells. Erdr1 was found to function as an autocrine factor to induce apoptosis through caspase 3. Consistent with elevated levels of Erdr1, germfree mice have increased splenic T-cell apoptosis. RNA sequencing of Erdr1-overexpressing cells identified the up-regulation of genes involved in Fas-mediated cell death, and Erdr1 fails to induce apoptosis in Fas-deficient cells. Importantly, forced changes in Erdr1 expression levels dictate the survival of auto-reactive T cells and the clinical outcome of neuro-inflammatory autoimmune disease. Cellular survival is a fundamental feature regulating appropriate immune responses. We have identified a mechanism whereby the host integrates signals from the microbiota to control T-cell apoptosis, making regulation of Erdr1 a potential therapeutic target for autoimmune disease.

Interferon-Mediated Long Non-Coding RNA Response in Macrophages in the Context of HIV.

Interferons play a critical role in the innate immune response against a variety of pathogens, such as HIV-1. Recent studies have shown that long non-coding genes are part of a reciprocal feedforward/feedback relationship with interferon expression. They presumably contribute to the cell type specificity of the interferon response, such as the phenotypic and functional transition of macrophages throughout the immune response. However, no comprehensive understanding exists today about the IFN-lncRNA interplay in macrophages, also a sanctuary for latent HIV-1. Therefore, we completed a poly-A+ RNAseq analysis on monocyte-derived macrophages (MDMs) treated with members of all three types of IFNs (IFN-α, IFN-ε, IFN-γ or IFN-λ) and on macrophages infected with HIV-1, revealing an extensive non-coding IFN and/or HIV-1 response. Moreover, co-expression correlation with mRNAs was used to identify important (long) non-coding hub genes within IFN- or HIV-1-associated gene clusters. This study identified and prioritized IFN related hub lncRNAs for further functional validation.

Sex Influences SAMHD1 Activity and Susceptibility to Human Immunodeficiency Virus-1 in Primary Human Macrophages.

BACKGROUND: Macrophages are major targets for HIV-1, contribute to viral propagation in vivo, and are instrumental in the pathogenesis of HAND. While it is known that host sex affects HIV-1 viremia and influences the severity of HIV-1-associated neurocognitive disease, a cellular or molecular basis for these findings remains elusive. METHODS: We explored whether sex affects HIV-1 infectivity of primary human macrophages and CD4+ T cells in vitro. RESULTS: Macrophages derived from female donors were less susceptible to HIV-1 infection than those derived from males. This sex-dependent difference in macrophage infectivity was independent of the requirement for CD4/CCR5-mediated virus entry and was not observed in CD4+ T cells. Investigations into the mechanism governing these sex-dependent differences revealed that the host restriction factor SAMHD1 exists in a hyperphosphorylated, less active state in male-derived macrophages. In addition, the major kinase responsible for SAMHD1 phosphorylation, CDK1, exhibited lower levels of expression in female-derived macrophages in all tested donor pairs. The sex-dependent differences in viral restriction imposed by SAMHD1 were abrogated upon its depletion. CONCLUSIONS: We conclude that SAMHD1 is an essential modulator of infectivity in a sex-dependent manner in macrophages, constituting a novel component of sex differences in innate immune control of HIV-1.

T-cell responses targeting HIV Nef uniquely correlate with infected cell frequencies after long-term antiretroviral therapy.

HIV-specific CD8+ T-cell responses limit viral replication in untreated infection. After the initiation of antiretroviral therapy (ART), these responses decay and the infected cell population that remains is commonly considered to be invisible to T-cells. We hypothesized that HIV antigen recognition may persist in ART-treated individuals due to low-level or episodic protein expression. We posited that if persistent recognition were occurring it would be preferentially directed against the early HIV gene products Nef, Tat, and Rev as compared to late gene products, such as Gag, Pol, and Env, which have higher barriers to expression. Using a primary cell model of latency, we observed that a Nef-specific CD8+ T-cell clone exhibited low-level recognition of infected cells prior to reactivation and robust recognition shortly thereafter. A Gag-specific CD8+ T-cell clone failed to recognized infected cells under these conditions, corresponding with a lack of detectable Gag expression. We measured HIV-specific T-cell responses in 96 individuals who had been suppressed on ART for a median of 7 years, and observed a significant, direct correlation between cell-associated HIV DNA levels and magnitudes of IFN-γ-producing Nef/Tat/Rev-specific T-cell responses. This correlation was confirmed in an independent cohort (n = 18). Correlations were not detected between measures of HIV persistence and T-cell responses to other HIV antigens. The correlation with Nef/Tat/Rev-specific T-cells was attributable to Nef-specific responses, the breadth of which also correlated with HIV DNA levels. These results suggest that ongoing Nef expression in ART-treated individuals drives preferential maintenance and/or expansion of T-cells reactive to this protein, implying sensing of infected cells by the immune system. The direct correlation, however, suggests that recognition does not result in efficient elimination of infected cells. These results raise the possibility that enhancing the cytolytic activity of Nef-specific T-cells may lead to reductions in infected cell frequencies, even in the absence of therapeutic latency reversal.

Transcriptomic Analysis Implicates the p53 Signaling Pathway in the Establishment of HIV-1 Latency in Central Memory CD4 T Cells in an In Vitro Model.

The search for an HIV-1 cure has been greatly hindered by the presence of a viral reservoir that persists despite antiretroviral therapy (ART). Studies of HIV-1 latency in vivo are also complicated by the low proportion of latently infected cells in HIV-1 infected individuals. A number of models of HIV-1 latency have been developed to examine the signaling pathways and viral determinants of latency and reactivation. A primary cell model of HIV-1 latency, which incorporates the generation of primary central memory CD4 T cells (TCM), full-length virus infection (HIVNL4-3) and ART to suppress virus replication, was used to investigate the establishment of HIV latency using RNA-Seq. Initially, an investigation of host and viral gene expression in the resting and activated states of this model indicated that the resting condition was reflective of a latent state. Then, a comparison of the host transcriptome between the uninfected and latently infected conditions of this model identified 826 differentially expressed genes, many of which were related to p53 signaling. Inhibition of the transcriptional activity of p53 by pifithrin-α during HIV-1 infection reduced the ability of HIV-1 to be reactivated from its latent state by an unknown mechanism. In conclusion, this model may be used to screen latency reversing agents utilized in shock and kill approaches to cure HIV, to search for cellular markers of latency, and to understand the mechanisms by which HIV-1 establishes latency.

HIV Integration Site Analysis of Cellular Models of HIV Latency with a Probe-Enriched Next-Generation Sequencing Assay.

UNLABELLED: Antiretroviral therapy (ART) is successful in the suppression of HIV but cannot target and eradicate the latent proviral reservoir. The location of retroviral integration into the human genome is thought to play a role in the clonal expansion of infected cells and HIV persistence. We developed a high-throughput targeted sequence capture assay that uses a pool of HIV-specific probes to enrich Illumina libraries prior to deep sequencing. Using an expanded clonal population of ACH-2 cells, we demonstrate that this sequence capture assay has an extremely low false-positive rate. This assay assessed four cellular models commonly used to study HIV latency and latency-reversing agents: ACH-2 cells, J-Lat cells, the Bcl-2-transduced primary CD4(+)model, and the cultured TCM(central memory) CD4(+)model. HIV integration site characteristics and genes were compared between these cellular models and to previously reported patient data sets. Across these cellular models, there were significant differences in integration site characteristics, including orientation relative to that of the host gene, the proportion of clonally expanded sites, and the proportion located within genic regions and exons. Despite a greater diversity of minority integration sites than expected in ACH-2 cells, their integration site characteristics consistently differed from those of the other models and from the patient samples. Gene ontology analysis of highly represented genes from the patient samples found little overlap with HIV-containing genes from the cell lines. These findings show that integration site differences exist among the commonly used cellular models of HIV latency and in comparison to integration sites found in patient samples. IMPORTANCE: Despite the success of ART, currently there is no successful therapy to eradicate integrated proviruses. Cellular models of HIV latency are used to test the efficacy of latency-reversing agents, but it is unclear how well these models reflect HIV integration into the human genome in vivo We have developed a novel probe-based sequence enrichment assay to sequence and analyze integrated HIV. We compared HIV integration site characteristics between four cellular models and to previously described patient data sets. Significant differences were detected in the distribution of HIV integration sites between cellular models of HIV latency and compared to data sets from patient samples. The results from this study have implications for how well these cellular models of HIV infection truly reflect HIV integration in vivo and their applicability in drug discovery for novel latency-reversing agents.

Janus kinase inhibition suppresses PKC-induced cytokine release without affecting HIV-1 latency reversal ex vivo.

BACKGROUND: Despite the durable viral suppression afforded by antiretroviral therapy, HIV-1 eradication will require strategies to target latently infected cells that persist in infected individuals. Protein kinase C (PKC) activation is a promising strategy to reactivate latent proviruses and allow for subsequent recognition and clearance of infected cells by the immune system. Ingenol derivatives are PKC agonists that induce latency reversal but also lead to T cell activation and the release of pro-inflammatory cytokines, which would be undesirable in vivo. In this work, we sought to identify compounds that would suppress pro-inflammatory cytokine production in the context of PKC activation. DESIGN AND METHODS: We performed an in vitro screen to identify compounds that could dampen pro-inflammatory cytokine release associated with T cell activation, using IL-6 as a model cytokine. We then tested the ability of the most promising screening hit, the FDA-approved Janus Kinase (JAK) inhibitor ruxolitinib, to diminish release of multiple cytokines and its effect on latency reversal using cells from HIV-1-positive, aviremic participants. RESULTS: We demonstrate that co-administration of ruxolitinib with ingenol-3,20-dibenzoate significantly reduces pro-inflammatory cytokine release without impairing latency reversal ex vivo. CONCLUSION: The combination of ingenol compounds and JAK inhibition represents a novel strategy for HIV-1 eradication.

HIV-1 Vpu utilizes both cullin-RING ligase (CRL) dependent and independent mechanisms to downmodulate host proteins.

BACKGROUND: Hijacking of the cullin-RING E3 ubiquitin ligase (CRL) machinery is a common mechanism employed by diverse groups of viruses for the efficient counteraction and degradation of host proteins. In particular, HIV-1 Vpu usurps the SCF(ß-TrCP) E3 ubiquitin ligase complex to mark CD4 for degradation by the 26S proteasome. Vpu also interacts with and downmodulates a number of other host proteins, including the restriction factor BST-2. However, whether Vpu primarily relies on a cullin-dependent or -independent mechanism to antagonize its cellular targets has not been fully elucidated. RESULTS: We utilized a sulphamate AMP analog, MLN4924, to effectively block the activation of CRLs within infected primary CD4(+) T cells. MLN4924 treatment, in a dose dependent manner, efficiently relieved surface downmodulation and degradation of CD4 by NL4-3 Vpu. MLN4924 inhibition was highly specific, as this inhibitor had no effect on Nef's ability to downregulate CD4, which is accomplished by a CRL-independent mechanism. In contrast, NL4-3 Vpu's capacity to downregulate BST-2, NTB-A and CCR7 was not inhibited by the drug. Vpu's from both a transmitted founder (T/F) and chronic carrier (CC) virus preserved the ability to downregulate BST-2 in the presence of MLN4924. Finally, depletion of cellular pools of cullin 1 attenuated Vpu's ability to decrease CD4 but not BST-2 surface levels. CONCLUSIONS: We conclude that Vpu employs both CRL-dependent and CRL-independent modes of action against host proteins. Notably, we also establish that Vpu-mediated reduction of BST-2 from the cell surface is independent of ß-TrCP and the CRL- machinery and this function is conserved by Vpu's from primary isolates. Therefore, potential therapies aimed at antagonizing the activities of Vpu may need to address these distinct mechanisms of action in order to achieve a maximal effect.

Ex Vivo Bioactivity and HIV-1 Latency Reversal by Ingenol Dibenzoate and Panobinostat in Resting CD4(+) T Cells from Aviremic Patients.

The human immunodeficiency virus type 1 (HIV-1) latent reservoir in resting CD4(+) T cells represents a major barrier to viral eradication. Small compounds capable of latency reversal have not demonstrated uniform responses across in vitro HIV-1 latency cell models. Characterizing compounds that demonstrate latency-reversing activity in resting CD4(+) T cells from aviremic patients ex vivo will help inform pilot clinical trials aimed at HIV-1 eradication. We have optimized a rapid ex vivo assay using resting CD4(+) T cells from aviremic HIV-1(+) patients to evaluate both the bioactivity and latency-reversing potential of candidate latency-reversing agents (LRAs). Using this assay, we characterize the properties of two candidate compounds from promising LRA classes, ingenol 3,20-dibenzoate (a protein kinase C agonist) and panobinostat (a histone deacetylase inhibitor), in cells from HIV-1(+) antiretroviral therapy (ART)-treated aviremic participants, including the effects on cellular activation and cytotoxicity. Ingenol induced viral release at levels similar to those of the positive control (CD3/28 receptor stimulation) in cells from a majority of participants and represents an exciting LRA candidate, as it combines a robust viral reactivation potential with a low toxicity profile. At concentrations that blocked histone deacetylation, panobinostat displayed a wide range of potency among participant samples and consistently induced significant levels of apoptosis. The protein kinase C agonist ingenol 3,20-dibenzoate demonstrated significant promise in a rapid ex vivo assay using resting CD4(+) T cells from treated HIV-1-positive patients to measure latent HIV-1 reactivation.

Kinase control of latent HIV-1 infection: PIM-1 kinase as a major contributor to HIV-1 reactivation.

Despite the clinical relevance of latent HIV-1 infection as a block to HIV-1 eradication, the molecular biology of HIV-1 latency remains incompletely understood. We recently demonstrated the presence of a gatekeeper kinase function that controls latent HIV-1 infection. Using kinase array analysis, we here expand on this finding and demonstrate that the kinase activity profile of latently HIV-1-infected T cells is altered relative to that of uninfected T cells. A ranking of altered kinases generated from these kinome profile data predicted PIM-1 kinase as a key switch involved in HIV-1 latency control. Using genetic and pharmacologic perturbation strategies, we demonstrate that PIM-1 activity is indeed required for HIV-1 reactivation in T cell lines and primary CD4 T cells. The presented results thus confirm that kinases are key contributors to HIV-1 latency control. In addition, through mutational studies we link the inhibitory effect of PIM-1 inhibitor IV (PIMi IV) on HIV-1 reactivation to an AP-1 motif in the CD28-responsive element of the HIV-1 long terminal repeat (LTR). The results expand our conceptual understanding of the dynamic interactions of the host cell and the latent HIV-1 integration event and position kinome profiling as a research tool to reveal novel molecular mechanisms that can eventually be targeted to therapeutically trigger HIV-1 reactivation.

An in-depth comparison of latent HIV-1 reactivation in multiple cell model systems and resting CD4+ T cells from aviremic patients.

The possibility of HIV-1 eradication has been limited by the existence of latently infected cellular reservoirs. Studies to examine control of HIV latency and potential reactivation have been hindered by the small numbers of latently infected cells found in vivo. Major conceptual leaps have been facilitated by the use of latently infected T cell lines and primary cells. However, notable differences exist among cell model systems. Furthermore, screening efforts in specific cell models have identified drug candidates for "anti-latency" therapy, which often fail to reactivate HIV uniformly across different models. Therefore, the activity of a given drug candidate, demonstrated in a particular cellular model, cannot reliably predict its activity in other cell model systems or in infected patient cells, tested ex vivo. This situation represents a critical knowledge gap that adversely affects our ability to identify promising treatment compounds and hinders the advancement of drug testing into relevant animal models and clinical trials. To begin to understand the biological characteristics that are inherent to each HIV-1 latency model, we compared the response properties of five primary T cell models, four J-Lat cell models and those obtained with a viral outgrowth assay using patient-derived infected cells. A panel of thirteen stimuli that are known to reactivate HIV by defined mechanisms of action was selected and tested in parallel in all models. Our results indicate that no single in vitro cell model alone is able to capture accurately the ex vivo response characteristics of latently infected T cells from patients. Most cell models demonstrated that sensitivity to HIV reactivation was skewed toward or against specific drug classes. Protein kinase C agonists and PHA reactivated latent HIV uniformly across models, although drugs in most other classes did not.

An HIV-encoded antisense long noncoding RNA epigenetically regulates viral transcription.

The abundance of long noncoding RNAs (lncRNAs) and their wide range of functional roles in human cells are fast becoming realized. Importantly, lncRNAs have been identified as epigenetic modulators and consequently play a pivotal role in the regulation of gene expression. A human immunodeficiency virus-encoded antisense RNA transcript has recently been reported and we sought to characterize this RNA and determine its potential role in viral transcription regulation. The intrinsic properties of this human immunodeficiency virus-expressed lncRNA were characterized and the data presented here suggest that it functions as an epigenetic brake to modulate viral transcription. Suppression of this long antisense transcript with small single-stranded antisense RNAs resulted in the activation of viral gene expression. This lncRNA was found to localize to the 5' long-term repeats (LTR) and to usurp components of endogenous cellular pathways that are involved in lncRNA directed epigenetic gene silencing. Collectively, we find that this viral expressed antisense lncRNA is involved in modulating human immunodeficiency virus gene expression and that this regulatory effect is due to an alteration in the epigenetic landscape at the viral promoter.