Differential susceptibility of cells infected with defective and intact HIV proviruses to killing by obatoclax and other small molecules.

OBJECTIVES: Some drugs that augment cell-intrinsic defenses or modulate cell death/survival pathways have been reported to selectively kill cells infected with HIV or Simian Immunodeficiency Virus (SIV), but comparative studies are lacking. We hypothesized that these drugs may differ in their ability to kill cells infected with intact and defective proviruses. DESIGN: To investigate this hypothesis, drugs were tested ex vivo on peripheral blood mononuclear cells (PBMC) from nine antiretroviral therapy (ART)-suppressed individuals. METHODS: We tested drugs currently in clinical use or human trials, including auranofin (p53 modulator), interferon alpha2A, interferon gamma, acitretin (RIG-I inducer), GS-9620/vesatolimod (TLR7 agonist), nivolumab (PD-1 blocker), obatoclax (Bcl-2 inhibitor), birinapant [inhibitor of apoptosis proteins (IAP) inhibitor], bortezomib (proteasome inhibitor), and INK128/sapanisertib [mammalian target of rapamycin mTOR] [c]1/2 inhibitor). After 6 days of treatment, we measured cell counts/viabilities and quantified levels of total, intact, and defective HIV DNA by droplet digital PCR (Intact Proviral DNA Assay). RESULTS: Obatoclax reduced intact HIV DNA [median = 27-30% of dimethyl sulfoxide control (DMSO)] but not defective or total HIV DNA. Other drugs showed no statistically significant effects. CONCLUSION: Obatoclax and other Bcl-2 inhibitors deserve further study in combination therapies aimed at reducing the intact HIV reservoir in order to achieve a functional cure and/or reduce HIV-associated immune activation.

Changes in adolescents' daily-life solitary experiences during the COVID-19 pandemic: an experience sampling study.

BACKGROUND: Adolescent solitude was drastically impacted by the COVID-19 pandemic. As solitude is crucial for adolescent development through its association with both positive and negative developmental outcomes, it is critical to understand how adolescents' daily-life solitary experiences changed as a result of the pandemic. METHODS: Using three waves of Experience Sampling Method data from a longitudinal study, we compared adolescents' daily-life solitary experiences in the early (nT1=100; MAge=16.1; SDAge=1.9; 93% girls) and mid-pandemic (nT2=204; MAge=16.5; SDAge=2.0; 79% girls) to their pre-pandemic experiences. RESULTS: We found that adolescents with lower levels of pre-pandemic social support and social skills reported wanting to be alone less and feeling like an outsider more at both time points during the pandemic. In the mid-pandemic wave, adolescents with higher levels of pre-pandemic social support and social skills reported decreases in positive affect compared to the pre-pandemic wave. CONCLUSION: This study shows that adolescents' daily-life solitary experiences worsened throughout the COVID-19 pandemic. There should be continued concern for the wellbeing of all adolescents, not only those already at risk, as effects of the pandemic on mental health might only manifest later.

Lifetime and Current Self-Harm Thoughts and Behaviors and Their Relationship to Parent and Peer Attachment.

Background: Previous research suggests attachment is a vulnerability factor for self-harm thoughts and behaviors in adults. Yet, few studies have investigated this relationship during adolescence, although adolescence is a critical period for changes in attachment relationships and self-harm onset. Whether and how attachment relates to self-harm thoughts and behaviors as measured in daily life is also unknown. Aims: To investigate whether and how paternal, maternal, and peer attachment are associated with lifetime and current adolescent self-harm thoughts and behaviors. Additionally, to examine how different attachment bonds interact in relation to lifetime and current adolescent self-harm thoughts and behaviors. Method: Pre-existing data from N = 1,913 adolescents of the SIGMA study were used. Attachment and lifetime history of self-harm thoughts and behaviors were measured via retrospective questionnaires. Current self-harm thoughts and behaviors were assessed 10 times per day for 6 days using the experience sampling method (ESM). Results: Paternal and maternal attachments were associated with lifetime self-harm thoughts and behaviors and current self-harm thoughts. No significant associations were found between peer attachment and self-harm outcomes. Limitations: Some analyses were underpowered. Conclusion: Our results highlight the importance of parent-child attachment relationships, which may be intervention targets for prevention and treatment of adolescent self-harm.

Single-Cell Imaging Shows That the Transcriptional State of the HIV-1 Provirus and Its Reactivation Potential Depend on the Integration Site.

Current antiretroviral treatment fails to cure HIV-1 infection since latent provirus resides in long-lived cellular reservoirs, rebounding whenever therapy is discontinued. The molecular mechanisms underlying HIV-1 latency are complex where the possible link between integration and transcription is poorly understood. HIV-1 integration is targeted toward active chromatin by the direct interaction with a host protein, lens epithelium-derived growth factor (LEDGF/p75). LEDGINs are small-molecule inhibitors of the LEDGF/p75-integrase (IN) interaction that effectively inhibit and retarget HIV-1 integration out of preferred integration sites, resulting in residual provirus that is more latent. Here, we describe a single-cell branched DNA imaging method for simultaneous detection of viral DNA and RNA. We investigated how treatment with LEDGINs affects the location, transcription, and reactivation of HIV-1 in both cell lines and primary cells. This approach demonstrated that LEDGIN-mediated retargeting hampered the baseline transcriptional state and the transcriptional reactivation of the provirus, evidenced by the reduction in viral RNA expression per residual copy. Moreover, treatment of primary cells with LEDGINs induced an enrichment of provirus in deep latency. These results corroborate the impact of integration site selection for the HIV-1 transcriptional state and support block-and-lock functional cure strategies in which the latent reservoir is permanently silenced after retargeting. IMPORTANCE A longstanding question exists on the impact of the HIV-1 integration site on viral gene expression. This unsolved question has significant implications for the search toward an HIV-1 cure, as eradication strategies set up to reactivate and eliminate HIV-1 depend on the site where the provirus is integrated. The main determinant for integration site selection is the interaction of the HIV-1 integrase (IN) and the host chromatin targeting factor, LEDGF/p75. LEDGINs are small-molecule inhibitors of the LEDGF/p75-IN interaction that inhibit and retarget HIV-1 integration out of preferred integration sites. Using both LEDGINs and branched DNA (bDNA) imaging, we now investigated, in much detail, the impact of integration site selection on the three-dimensional location of the provirus, HIV-1 transcription, and reactivation. Our results provide evidence for a "block-and-lock" functional cure strategy that aims to permanently silence HIV-1 by LEDGIN-mediated retargeting to sites that are less susceptible to reactivation after treatment interruption.

CRISPR/Cas9-Induced Mutagenesis Corroborates the Role of Transportin-SR2 in HIV-1 Nuclear Import.

To infect nondividing cells, HIV-1 needs to cross the nuclear membrane. The importin transportin-SR2 (TRN-SR2 or transportin-3) has been proposed to mediate HIV-1 nuclear import, but the detailed mechanism remains unresolved. The direct interaction of TRN-SR2 with HIV-1 integrase (IN) has been proposed to drive HIV-1 nuclear import. Alternatively, TRN-SR2 may play an indirect role by mediating nuclear import of cleavage and polyadenylation specificity factor 6 (CPSF6). To unravel the role of TRN-SR2, we designed CRISPR/Cas9 guide RNAs targeting different exons of TNPO3. Although this approach failed to generate full knockouts, monoallelic knockout clones were generated with indel mutations. HIV-1 replication was hampered in those clones at the level of HIV-1 nuclear import without an effect on the cellular distribution of the TRN-SR2 cargoes CPSF6 or alternative splicing factor1/pre-mRNA splicing factor SF2 (ASF/SF2). Recombinant ΔV105 TRN-SR2 expressed in clone 15.15 was 2-fold impaired for interaction with HIV-1 IN and classified as an interaction mutant. Our data support a model whereby TRN-SR2 acts as a cofactor of HIV-1 nuclear import without compromising the nuclear import of cellular cargoes. CRISPR/Cas9-induced mutagenesis can be used as a method to generate interface mutants to characterize host factors of human pathogens. IMPORTANCE Combination antiretroviral therapy (cART) effectively controls HIV-1 by reducing viral loads, but it does not cure the infection. Lifelong treatment with cART is a prerequisite for sustained viral suppression. The rapid emergence of drug-resistant viral strains drives the necessity to discover new therapeutic targets. The nuclear import of HIV-1 is crucial in the HIV-1 replication cycle, but the detailed mechanism remains incompletely understood. This study provides evidence that TRN-SR2 directly mediates HIV-1 nuclear import via the interaction with HIV-1 integrase. The interaction between those proteins is therefore a promising target toward a rational drug design which could lead to new therapeutic strategies due to the bottleneck nature of HIV-1 nuclear import.

The Claiming Costs Scale: A new instrument for measuring the costs potential beneficiaries face when claiming social benefits.

It is a well-known feature of social protection systems that not all persons who are entitled to social benefits also claim these benefits. The costs people face when claiming benefits is considered an important cause of this phenomenon of non-take-up. In this paper, we developed and examined the psychometric properties of a new scale, the Claiming Cost Scale (CCS), which measures three dimensions of costs associated with claiming benefits. A multi-phase instrument development method was performed to develop the instrument. The item pool was generated based on a literature review, and presented to academic experts (n = 9) and experts by experience (n = 5) to assess content and face validity. In a second stage, centrality and dispersion, construct validity, convergent and divergent validity, and internal reliability of the instrument were tested. These analyses were based on two samples (n = 141 and n = 1265) of individuals living in low-income households in Belgium. Nine items were retained, which represent three factors (Information costs, Process costs and Stigma). The confirmatory factor analysis proved adequate model fitness. Both convergent and divergent validity were good, and internal consistency was adequate, with Cronbach's alpha ranging between .73 and .87. The findings showed that the CCS is a valid and reliable instrument for assessing the costs potential beneficiaries face when claiming benefits. Consisting of only nine items, the scale can be easily implemented in large-scale survey research or used in day-to-day work of service providers who are interested in understanding non-take-up of their service.

The Impact of COVID-19 on Adolescents' Daily Lives: The Role of Parent-Child Relationship Quality.

COVID-19 lockdown measures have profoundly impacted adolescent' daily life, with research suggesting an increase in irritability, stress, loneliness, and family conflict. A potential protective factor is parent-child relationship quality; however, no studies have investigated this. We used data from SIGMA, a longitudinal, experience sampling cohort study, in which N = 173 adolescents aged 11 to 20 were tested before and during COVID-19. Multilevel analyses showed decreased daily-life irritability and increased loneliness from pre- to mid-pandemic. Daily-life stress levels were unchanged. Relationship quality was negatively associated with irritability and loneliness and buffered against the increase in loneliness. Effect sizes were small and do not support a strong effect of the first lockdown on irritability, stress, loneliness, and family conflict in adolescents.

LEDGINs, Inhibitors of the Interaction Between HIV-1 Integrase and LEDGF/p75, Are Potent Antivirals with a Potential to Cure HIV Infection.

A permanent cure remains the greatest challenge in the field of HIV research. In order to reach this goal, a profound understanding of the molecular mechanisms controlling HIV integration and transcription is needed. Here we provide an overview of recent advances in the field. Lens epithelium-derived growth factor p75 (LEDGF/p75), a transcriptional coactivator, tethers and targets the HIV integrase into transcriptionally active regions of the chromatin through an interaction with the epigenetic mark H3K36me2/3. This finding prompted us to propose a "block-and-lock" strategy to retarget HIV integration into deep latency. A decade ago, we pioneered protein-protein interaction inhibitors for HIV and discovered LEDGINs. LEDGINs are small molecule inhibitors of the interaction between the integrase binding domain (IBD) of LEDGF/p75 and HIV integrase. They modify integration site selection and therefore might be molecules with a "block-and-lock" mechanism of action. Here we will describe how LEDGINs may become part in the future functional cure strategies.

Role of Transportin-SR2 in HIV-1 Nuclear Import.

The HIV replication cycle depends on the interaction of viral proteins with proteins of the host. Unraveling host-pathogen interactions during the infection is of great importance for understanding the pathogenesis and the development of antiviral therapies. To date HIV uncoating and nuclear import are the most debated steps of the HIV-1 replication cycle. Despite numerous studies during past decades, there is still much controversy with respect to the identity and the role of viral and host factors involved in these processes. In this review, we provide a comprehensive overview on the role of transportin-SR2 as a host cell factor during active nuclear transport.

Towards a Functional Cure of HIV-1: Insight Into the Chromatin Landscape of the Provirus.

Despite potent combination antiretroviral therapy, HIV-1 infection persists due to irreversible integration of the virus in long-living cells of the immune system. The main focus of HIV-1 cure strategies has been on HIV-1 eradication, yet without great success so far. Therefore, HIV-1 remission or a functional cure, whereby the virus is silenced rather than eradicated, is considered as an alternative strategy. Elite controllers, individuals who spontaneously control HIV-1, may point us the way toward a functional HIV-1 cure. In order to achieve such a cure, a profound understanding of the mechanisms controlling HIV-1 expression and silencing is needed. In recent years, evidence has grown that the site of integration as well as the chromatin landscape surrounding the integration site affects the transcriptional state of the provirus. Still, at present, the impact of integration site selection on the establishment and maintenance of the HIV-1 reservoirs remains poorly understood. The discovery of LEDGF/p75 as a binding partner of HIV-1 integrase has led to a better understanding of integration site selection. LEDGF/p75 is one of the important determinants of integration site selection and targets integration toward active genes. In this review, we will provide an overview of the most important determinants of integration site selection. Secondly, we will discuss the chromatin landscape at the integration site and its implications on HIV-1 gene expression and silencing. Finally, we will discuss how interventions that affect integration site selection or modifications of the chromatin could yield a functional cure of HIV-1 infection.

Block-And-Lock Strategies to Cure HIV Infection.

Today HIV infection cannot be cured due to the presence of a reservoir of latently infected cells inducing a viral rebound upon treatment interruption. Hence, the latent reservoir is considered as the major barrier for an HIV cure. So far, efforts to completely eradicate the reservoir via a shock-and-kill approach have proven difficult and unsuccessful. Therefore, more research has been done recently on an alternative block-and-lock functional cure strategy. In contrast to the shock-and-kill strategy that aims to eradicate the entire reservoir, block-and-lock aims to permanently silence all proviruses, even after treatment interruption. HIV silencing can be achieved by targeting different factors of the transcription machinery. In this review, we first describe the underlying mechanisms of HIV transcription and silencing. Next, we give an overview of the different block-and-lock strategies under investigation.

Insight in HIV Integration Site Selection Provides a Block-and-Lock Strategy for a Functional Cure of HIV Infection.

Despite significant improvements in therapy, the HIV/AIDS pandemic remains an important threat to public health. Current treatments fail to eradicate HIV as proviral DNA persists in long-living cellular reservoirs, leading to viral rebound whenever treatment is discontinued. Hence, a better understanding of viral reservoir establishment and maintenance is required to develop novel strategies to destroy latently infected cells, and/or to durably silence the latent provirus in infected cells. Whereas the mechanism of integration has been well studied from a catalytic point of view, it remains unknown how integration site selection and transcription are linked. In recent years, evidence has grown that lens epithelium-derived growth factor p75 (LEDGF/p75) is the main determinant of HIV integration site selection and that the integration site affects the transcriptional state of the provirus. LEDGINs have been developed as small molecule inhibitors of the interaction between LEDGF/p75 and integrase. Recently, it was shown that LEDGIN treatment in cell culture shifts the residual integrated provirus towards the inner nuclear compartment and out of transcription units in a dose dependent manner. This LEDGIN-mediated retargeting increased the proportion of provirus with a transcriptionally silent phenotype and the residual reservoir proved refractory to reactivation in vitro. LEDGINs provide us with a research tool to study the link between integration and transcription, a quintessential question in retrovirology. LEDGIN-mediated retargeting of the residual reservoirs provides a novel potential "block-and-lock" strategy as a functional cure of HIV infection.