One of the most explored therapeutic approaches aimed at eradicating HIV-1 reservoirs is the "shock and kill" strategy which is based on HIV-1 reactivation in latently-infected cells ("shock" phase) ...while maintaining antiretroviral therapy (ART) in order to prevent spreading of the infection by the neosynthesized virus. This kind of strategy allows for the "kill" phase, during which latently-infected cells die from viral cytopathic effects or from host cytolytic effector mechanisms following viral reactivation. Several latency reversing agents (LRAs) with distinct mechanistic classes have been characterized to reactivate HIV-1 viral gene expression. Some LRAs have been tested in terms of their potential to purge latent HIV-1
in clinical trials, showing that reversing HIV-1 latency is possible. However, LRAs alone have failed to reduce the size of the viral reservoirs. Together with the inability of the immune system to clear the LRA-activated reservoirs and the lack of specificity of these LRAs, the heterogeneity of the reservoirs largely contributes to the limited success of clinical trials using LRAs. Indeed, HIV-1 latency is established in numerous cell types that are characterized by distinct phenotypes and metabolic properties, and these are influenced by patient history. Hence, the silencing mechanisms of HIV-1 gene expression in these cellular and tissue reservoirs need to be better understood to rationally improve this cure strategy and hopefully reach clinical success.
The persistence of latently infected cells in patients under combinatory antiretroviral therapy (cART) is a major hurdle to HIV-1 eradication. Strategies to purge these reservoirs are needed and ...activation of viral gene expression in latently infected cells is one promising strategy. Bromodomain and Extraterminal (BET) bromodomain inhibitors (BETi) are compounds able to reactivate latent proviruses in a positive transcription elongation factor b (P-TEFb)-dependent manner. In this study, we tested the reactivation potential of protein kinase C (PKC) agonists (prostratin, bryostatin-1 and ingenol-B), which are known to activate NF-κB signaling pathway as well as P-TEFb, used alone or in combination with P-TEFb-releasing agents (HMBA and BETi (JQ1, I-BET, I-BET151)). Using in vitro HIV-1 post-integration latency model cell lines of T-lymphoid and myeloid lineages, we demonstrated that PKC agonists and P-TEFb-releasing agents alone acted as potent latency-reversing agents (LRAs) and that their combinations led to synergistic activation of HIV-1 expression at the viral mRNA and protein levels. Mechanistically, combined treatments led to higher activations of P-TEFb and NF-κB than the corresponding individual drug treatments. Importantly, we observed in ex vivo cultures of CD8+-depleted PBMCs from 35 cART-treated HIV-1+ aviremic patients that the percentage of reactivated cultures following combinatory bryostatin-1+JQ1 treatment was identical to the percentage observed with anti-CD3+anti-CD28 antibodies positive control stimulation. Remarkably, in ex vivo cultures of resting CD4+ T cells isolated from 15 HIV-1+ cART-treated aviremic patients, the combinations bryostatin-1+JQ1 and ingenol-B+JQ1 released infectious viruses to levels similar to that obtained with the positive control stimulation. The potent effects of these two combination treatments were already detected 24 hours post-stimulation. These results constitute the first demonstration of LRA combinations exhibiting such a potent effect and represent a proof-of-concept for the co-administration of two different types of LRAs as a potential strategy to reduce the size of the latent HIV-1 reservoirs.
IFN-I is the key regulatory component activating and modulating the response of innate and adaptive immune system to bacterial as well as viral pathogens. IFN-I promotes the expression of IFN-induced ...genes (ISG) and, consequently, the production of chemokines, e.g., CXCL10. Those chemokines control migration and localization of immune cells in tissues, and, thus, are critical to the function of the innate immune system during infection. Consequently, the regulation of IFN-I signaling is essential for the proper induction of an immune response. Our previous study has shown that E3 ubiquitin ligase Pellino3 positively regulates IFNβ expression and secretion. Herein, we examined the role of Pellino3 ligase in regulating
expression in response to IFNβ stimulation. Our experiments were carried out on murine macrophage cell line (BMDM) and human monocytes cell line (THP-1) using IFNβ as a IFNAR ligand. We demonstrate that Pellino3 is important for IFNβ-induced phosphorylation and nuclear translocation of STAT1/STAT2/IRF9 complex which interacts with
promoter and enhances its expression. In this study, we characterize a novel molecular mechanism allowing Pellino3-dependent modulation of the IFNβ-induced response in BMDM and THP-1 cell lines.
Highlights ► HIV-1 Rev activity on viral RNAs depends on host factors. ► PSF and MATR3 are cellular host factors that bind viral RNA and promote Rev activity. ► PSF and Rev associate with nascent RNA ...that is released to MATR3. ► PSF commits viral RRE containing RNAs to Rev mediated export through MATR3.
Averaged estimates of RNA polymerase II (RNAPII) elongation rates in mammalian cells have been shown to range between 1.3 and 4.3 kb min−1. In this work, nascent RNAs from an integrated human ...immunodeficiency virus type 1‐derived vector were detectable at the single living cell level by fluorescent RNA tagging. At steady state, a constant number of RNAs was measured corresponding to a minimal density of polymerases with negligible fluctuations over time. Recovery of fluorescence after photobleaching was complete within seconds, indicating a high rate of RNA biogenesis. The calculated transcription rate above 50 kb min−1 points towards a wide dynamic range of RNAPII velocities in living cells.
RNA polymerase II elongation has been estimated to occur at a rate of 1–5 kb min−1 in mammalian cells. The authors now find that the rate of elongation can surpass 50 kb min−1 and propose that RNA polymerase II displays a wider than expected dynamic range of velocities during transcription.
Central to the fully competent replication cycle of the human immunodeficiency virus type 1 (HIV-1) is the nuclear export of unspliced and partially spliced RNAs mediated by the Rev ...posttranscriptional activator and the Rev response element (RRE).
Here, we introduce a novel method to explore the proteome associated with the nuclear HIV-1 RNAs. At the core of the method is the generation of cell lines harboring an integrated provirus carrying RNA binding sites for the MS2 bacteriophage protein. Flag-tagged MS2 is then used for affinity purification of the viral RNA. By this approach we found that the viral RNA is associated with the host nuclear matrix component MATR3 (Matrin 3) and that its modulation affected Rev activity. Knockdown of MATR3 suppressed Rev/RRE function in the export of unspliced HIV-1 RNAs. However, MATR3 was able to associate with Rev only through the presence of RRE-containing viral RNA.
In this work, we exploited a novel proteomic method to identify MATR3 as a cellular cofactor of Rev activity. MATR3 binds viral RNA and is required for the Rev/RRE mediated nuclear export of unspliced HIV-1 RNAs.
We report the case of a successful complex percutaneous intervention in a patient with Fontan circulation and severe heart failure. The patient presented with cyanosis; Fontan conduit stenosis was ...detected, and the fenestration was patent. The complex interventional procedure allowed for a long‐term stabilization of the patient's condition.
Angiogram at the end of a successful complex percutaneous intervention in a patient with Fontan circulation. Visible Fontan conduit after stenting and vascular plug in the main pulmonary artery. RPA, right pulmonary artery; LPA, left pulmonary artery.
Currently, there are four seasonal coronaviruses associated with relatively mild respiratory tract disease in humans. However, there is also a plethora of animal coronaviruses which have the ...potential to cross the species border. This regularly results in the emergence of new viruses in humans. In 2002, severe acute respiratory syndrome coronavirus (SARS-CoV) emerged and rapidly disappeared in May 2003. In 2012, Middle East respiratory syndrome coronavirus (MERS-CoV) was identified as a possible threat to humans, but its pandemic potential so far is minimal, as human-to-human transmission is ineffective. The end of 2019 brought us information about severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emergence, and the virus rapidly spread in 2020, causing an unprecedented pandemic. At present, studies on the virus are carried out using a surrogate system based on the immortalized simian Vero E6 cell line. This model is convenient for diagnostics, but it has serious limitations and does not allow for understanding of the biology and evolution of the virus. Here, we show that fully differentiated human airway epithelium cultures constitute an excellent model to study infection with the novel human coronavirus SARS-CoV-2. We observed efficient replication of the virus in the tissue, with maximal replication at 2 days postinfection. The virus replicated in ciliated cells and was released apically.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged by the end of 2019 and rapidly spread in 2020. At present, it is of utmost importance to understand the biology of the virus, rapidly assess the treatment potential of existing drugs, and develop new active compounds. While some animal models for such studies are under development, most of the research is carried out in Vero E6 cells. Here, we propose fully differentiated human airway epithelium cultures as a model for studies on SARS-CoV-2.
Posttranscriptional regulation of HIV-1 replication is finely controlled by viral and host factors. Among the former, Rev controls the export of partially spliced and unspliced viral RNAs from the ...nucleus and their translation in the cytoplasm or incorporation into new virions as genomic viral RNA. To investigate the functional role of the Rev cofactor MATR3 in the context of HIV infection, we modulated its expression in Jurkat cells and primary peripheral blood lymphocytes (PBLs). We confirmed that MATR3 is a positive regulator of HIV-1 acting at a posttranscriptional level. By applying the same approach to J-lat cells, a well-established model for the study of HIV-1 latency, we observed that MATR3 depletion did not affect transcriptional reactivation of the integrated provirus, but caused a reduction of Gag production. Following these observations, we hypothesized that MATR3 could be involved in the establishment of HIV-1 posttranscriptional latency. Indeed, mechanisms acting at the posttranscriptional level have been greatly overlooked in favor of transcriptional pathways. MATR3 was almost undetectable in resting PBLs, but could be promptly upregulated upon cellular stimulation with PHA. However, HIV latency-reversing agents were poor inducers of MATR3 levels, providing a rationale for their inability to fully reactivate the virus. These data have been confirmed
in cells derived from patients under suppressive ART. Finally, in the context of MATR3-depleted J-lat cells, impaired reactivation by SAHA could be fully rescued by MATR3 reconstitution, demonstrating a direct role of MATR3 in the posttranscriptional regulation of HIV-1 latency.
The life cycle of HIV-1 requires integration of a DNA copy into the genome of the host cell. Transcription of the viral genes generates RNAs that are exported to the cytoplasm with the contribution of viral and cellular factors to get translated or incorporated in the newly synthesized virions. It has been observed that highly effective antiretroviral therapy, which is able to reduce circulating virus to undetectable levels, cannot fully eradicate the virus from cellular reservoirs that harbor a transcriptionally latent provirus. Thus, persistence of latently infected cells is the major barrier to a cure for HIV-1 infection. In order to purge these reservoirs of latently infected cells, it has been proposed to activate transcription to stimulate the virus to complete its life cycle. This strategy is believed to unmask these reservoirs, making them vulnerable to the immune system. However, limited successes of this approach may indicate additional posttranscriptional restrictions that need to be overcome for full virus reactivation. In this work we identify the cellular protein MATR3 as an essential cofactor of viral RNA processing. Reactivation of HIV-1 transcription
is not sufficient to allow completion of a full life cycle of the virus if MATR3 is depleted. Furthermore, MATR3 is poorly expressed in quiescent CD4
T lymphocytes that are the major reservoir of latent HIV-1. Cells derived from aviremic HIV-1 patients under antiretroviral therapy didn't express MATR3, and most importantly, latency-reversing agents proposed for the rescue of latent provirus were ineffective for MATR3 upregulation. To conclude, our work identifies a cellular factor required for full HIV-1 reactivation and points to the revision of the current strategies for purging viral reservoirs that focus only on transcription.
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