Contribution of Trp63CreERT2-labeled cells to alveolar regeneration is independent of tuft cells.

Viral infection often causes severe damage to the lungs, leading to the appearance of ectopic basal cells (EBCs) and tuft cells in the lung parenchyma. Thus far, the roles of these ectopic epithelial cells in alveolar regeneration remain controversial. Here, we confirm that the ectopic tuft cells are originated from EBCs in mouse models and COVID-19 lungs. The differentiation of tuft cells from EBCs is promoted by Wnt inhibition while suppressed by Notch inhibition. Although progenitor functions have been suggested in other organs, pulmonary tuft cells don't proliferate or give rise to other cell lineages. Consistent with previous reports, Trp63CreERT2 and KRT5-CreERT2-labeled ectopic EBCs do not exhibit alveolar regeneration potential. Intriguingly, when tamoxifen was administrated post-viral infection, Trp63CreERT2 but not KRT5-CreERT2 labels islands of alveolar epithelial cells that are negative for EBC biomarkers. Furthermore, germline deletion of Trpm5 significantly increases the contribution of Trp63CreERT2-labeled cells to the alveolar epithelium. Although Trpm5 is known to regulate tuft cell development, complete ablation of tuft cell production fails to improve alveolar regeneration in Pou2f3-/- mice, implying that Trpm5 promotes alveolar epithelial regeneration through a mechanism independent of tuft cells.

FACT subunit SUPT16H associates with BRD4 and contributes to silencing of interferon signaling.

FACT (FAcilitates Chromatin Transcription) is a heterodimeric protein complex composed of SUPT16H and SSRP1, and a histone chaperone participating in chromatin remodeling during gene transcription. FACT complex is profoundly regulated, and contributes to both gene activation and suppression. Here we reported that SUPT16H, a subunit of FACT, is acetylated in both epithelial and natural killer (NK) cells. The histone acetyltransferase TIP60 contributes to the acetylation of SUPT16H middle domain (MD) at lysine 674 (K674). Such acetylation of SUPT16H is recognized by bromodomain protein BRD4, which promotes protein stability of SUPT16H in both epithelial and NK cells. We further demonstrated that SUPT16H-BRD4 associates with histone modification enzymes (HDAC1, EZH2), and further regulates their activation status and/or promoter association as well as affects the relevant histone marks (H3ac, H3K9me3 and H3K27me3). BRD4 is known to profoundly regulate interferon (IFN) signaling, while such function of SUPT16H has never been explored. Surprisingly, our results revealed that SUPT16H genetic knockdown via RNAi or pharmacological inhibition by using its inhibitor, curaxin 137 (CBL0137), results in the induction of IFNs and interferon-stimulated genes (ISGs). Through this mechanism, depletion or inhibition of SUPT16H is shown to efficiently inhibit infection of multiple viruses, including Zika, influenza, and SARS-CoV-2. Furthermore, we demonstrated that depletion or inhibition of SUPT16H also causes the remarkable activation of IFN signaling in NK cells, which promotes the NK-mediated killing of virus-infected cells in a co-culture system using human primary NK cells. Overall, our studies unraveled the previously un-appreciated role of FACT complex in coordinating with BRD4 and regulating IFN signaling in both epithelial and NK cells, and also proposed the novel application of the FACT inhibitor CBL0137 to treat viral infections.

VEGF receptor 2 (KDR) protects airways from mucus metaplasia through a Sox9-dependent pathway.

Mucus-secreting goblet cells are the dominant cell type in pulmonary diseases, e.g., asthma and cystic fibrosis (CF), leading to pathologic mucus metaplasia and airway obstruction. Cytokines including IL-13 are the major players in the transdifferentiation of club cells into goblet cells. Unexpectedly, we have uncovered a previously undescribed pathway promoting mucous metaplasia that involves VEGFa and its receptor KDR. Single-cell RNA sequencing analysis coupled with genetic mouse modeling demonstrates that loss of epithelial VEGFa, KDR, or MEK/ERK kinase promotes excessive club-to-goblet transdifferentiation during development and regeneration. Sox9 is required for goblet cell differentiation following Kdr inhibition in both mouse and human club cells. Significantly, airway mucous metaplasia in asthmatic and CF patients is also associated with reduced KDR signaling and increased SOX9 expression. Together, these findings reveal an unexpected role for VEGFa/KDR signaling in the defense against mucous metaplasia, offering a potential therapeutic target for this common airway pathology.

A molecular single-cell lung atlas of lethal COVID-19.

Respiratory failure is the leading cause of death in patients with severe SARS-CoV-2 infection1,2, but the host response at the lung tissue level is poorly understood. Here we performed single-nucleus RNA sequencing of about 116,000 nuclei from the lungs of nineteen individuals who died of COVID-19 and underwent rapid autopsy and seven control individuals. Integrated analyses identified substantial alterations in cellular composition, transcriptional cell states, and cell-to-cell interactions, thereby providing insight into the biology of lethal COVID-19. The lungs from individuals with COVID-19 were highly inflamed, with dense infiltration of aberrantly activated monocyte-derived macrophages and alveolar macrophages, but had impaired T cell responses. Monocyte/macrophage-derived interleukin-1ß and epithelial cell-derived interleukin-6 were unique features of SARS-CoV-2 infection compared to other viral and bacterial causes of pneumonia. Alveolar type 2 cells adopted an inflammation-associated transient progenitor cell state and failed to undergo full transition into alveolar type 1 cells, resulting in impaired lung regeneration. Furthermore, we identified expansion of recently described CTHRC1+ pathological fibroblasts3 contributing to rapidly ensuing pulmonary fibrosis in COVID-19. Inference of protein activity and ligand-receptor interactions identified putative drug targets to disrupt deleterious circuits. This atlas enables the dissection of lethal COVID-19, may inform our understanding of long-term complications of COVID-19 survivors, and provides an important resource for therapeutic development.

FACT subunit SUPT16H associates with BRD4 and contributes to silencing of antiviral interferon signaling.

FACT ( FA cilitates C hromatin T ranscription) is a heterodimeric protein complex composed of SUPT16H and SSRP1, and a histone chaperone participating in chromatin remodeling during gene transcription. FACT complex is profoundly regulated, and contributes to both gene activation and suppression. Here we reported that SUPT16H, a subunit of FACT, is acetylated at lysine 674 (K674) of middle domain (MD), which involves TIP60 histone acetyltransferase. Such acetylation of SUPT16H is recognized by bromodomain protein BRD4, which promotes protein stability of SUPT16H. We further demonstrated that SUPT16H-BRD4 associates with histone modification enzymes (EZH2, HDAC1) and affects histone marks (H3K9me3, H3K27me3 and H3ac). BRD4 is known to profoundly regulate interferon (IFN) signaling, while such function of SUPT16H has never been explored. Surprisingly, our results revealed that SUPT16H genetic knockdown via RNAi or pharmacological inhibition by using its inhibitor, curaxin 137 (CBL0137), results in the induction of IFNs and interferon-stimulated genes (ISGs). Through this mechanism, CBL0137 is shown to efficiently inhibit infection of multiple viruses, including Zika, influenza, and SARS-CoV-2. Furthermore, we demonstrated that CBL0137 also causes the remarkable activation of IFN signaling in natural killer (NK) cells, which promotes the NK-mediated killing of virus-infected cells in a co-culture system using human primary NK cells. Overall, our studies unraveled the previously un-appreciated role of FACT complex in regulating IFN signaling in both epithelial and NK cells, and also proposed the novel application of CBL0137 to treat viral infections.

Isl1 Regulation of Nkx2.1 in the Early Foregut Epithelium Is Required for Trachea-Esophageal Separation and Lung Lobation.

The esophagus and trachea arise from the dorsal and ventral aspects of the anterior foregut, respectively. Abnormal trachea-esophageal separation leads to the common birth defect esophageal atresia with or without trachea-esophageal fistula (EA/TEF). Yet the underlying cellular mechanisms remain unknown. Here, we combine Xenopus and mouse genetic models to identify that the transcription factor Isl1 orchestrates trachea-esophageal separation through modulating a specific epithelial progenitor cell population (midline epithelial cells [MECs], Isl1+ Nkx2.1+ Sox2+) located at the dorsal-ventral boundary of the foregut. Lineage tracing experiments show that MECs contribute to both tracheal and esophageal epithelium, and Isl1 is required for Nkx2.1 transcription in MECs. Deletion of the chromosomal region spanning the ISL1 gene has been found in patients with abnormal trachea-esophageal separation. Our studies thus provide definitive evidence that ISL1 is a critical player in the process of foregut morphogenesis, acting in a small progenitor population of boundary cells.

The cysteine-rich region of a baculovirus VP91 protein contributes to the morphogenesis of occlusion bodies.

The baculovirus core gene vp91 has been reported to be essential for nucleocapsid assembly and oral infection. Here, we studied the function of vp91 by analyzing its homologue, ha76, in Helicoverpa armigera nucleopolyhedrovirus (HearNPV). HA76 was expressed at the late stage of HearNPV infection; deletion of ha76 showed that the gene is required for budded virus production. A series of recombinants with truncated ha76 was constructed and analyzed in vitro and in vivo. The results showed that the region encoding the C-terminus of HA76 was essential for nucleocapsid assembly, whereas the N-terminal cysteine-rich region was responsible for oral infection. Electron microscope analyses further showed that the cysteine-rich region contributed to morphogenesis of occlusion bodies (OBs), with amino acids 136-223 of HA76 being critical for this function. The results revealed a novel function of VP91 and suggested that the impact on OB morphogenesis is partially related to oral infectivity.

A CRISPR/Cas9 screen identifies the histone demethylase MINA53 as a novel HIV-1 latency-promoting gene (LPG).

Although combination antiretroviral therapy is potent to block active replication of HIV-1 in AIDS patients, HIV-1 persists as transcriptionally inactive proviruses in infected cells. These HIV-1 latent reservoirs remain a major obstacle for clearance of HIV-1. Investigation of host factors regulating HIV-1 latency is critical for developing novel antiretroviral reagents to eliminate HIV-1 latent reservoirs. From our recently accomplished CRISPR/Cas9 sgRNA screens, we identified that the histone demethylase, MINA53, is potentially a novel HIV-1 latency-promoting gene (LPG). We next validated MINA53's function in maintenance of HIV-1 latency by depleting MINA53 using the alternative RNAi approach. We further identified that in vitro MINA53 preferentially demethylates the histone substrate, H3K36me3 and that in cells MINA53 depletion by RNAi also increases the local level of H3K36me3 at LTR. The effort to map the downstream effectors unraveled that H3K36me3 has the cross-talk with another epigenetic mark H4K16ac, mediated by KAT8 that recognizes the methylated H3K36 and acetylated H4K16. Removing the MINA53-mediated latency mechanisms could benefit the reversal of post-integrated latent HIV-1 proviruses for purging of reservoir cells. We further demonstrated that a pan jumonji histone demethylase inhibitor, JIB-04, inhibits MINA53-mediated demethylation of H3K36me3, and JIB-04 synergizes with other latency-reversing agents (LRAs) to reactivate latent HIV-1.

Curaxin CBL0137 has the potential to reverse HIV-1 latency.

A cure for human immunodeficiency virus type-1 (HIV-1) has been hampered by the limitation of current combination antiretroviral therapy (cART) to address the latent reservoirs in HIV-1 patients. One strategy proposed to eradicate these reservoirs is the "shock and kill" approach, where latency-reversing agents (LRAs) are used to reactivate and promote viral cell death and/or immune killing of reactivated cells. Here, we report that curaxin CBL0137, an antitumor compound, can potentiate tumor necrosis factor-α-mediated reactivation of latently infected HIV-1cell lines. Additionally, the single use of CBL0137 is sufficient to reactivate HIV-1 latent reservoirs in peripheral mononuclear cells (PBMCs) isolated from HIV-1 positive, cART-treated, aviremic patients. Thus, CBL0137 possesses capabilities as a LRA and could be considered for the "shock and kill" approach.

Curaxin CBL0100 Blocks HIV-1 Replication and Reactivation through Inhibition of Viral Transcriptional Elongation.

Despite combination antiretroviral therapy (cART), acquired immunodeficiency syndrome (AIDS), predominantly caused by the human immunodeficiency virus type 1 (HIV-1), remains incurable. The barrier to a cure lies in the virus' ability to establish a latent infection in HIV/AIDS patients. Unsurprisingly, efforts for a sterilizing cure have focused on the "shock and kill" strategy using latency-reversing agents (LRAs) to complement cART in order to eliminate these latent reservoirs. However, this method faces numerous challenges. Recently, the "block and lock" strategy has been proposed. It aims to reinforce a deep state of latency and prevent sporadic reactivation ("blip") of HIV-1 using latency-promoting agents (LPAs) for a functional cure. Our studies of curaxin 100 (CBL0100), a small-molecule targeting the facilitates chromatin transcription (FACT) complex, show that it blocks both HIV-1 replication and reactivation in in vitro and ex vivo models of HIV-1. Mechanistic investigation elucidated that CBL0100 preferentially targets HIV-1 transcriptional elongation and decreases the occupancy of RNA Polymerase II (Pol II) and FACT at the HIV-1 promoter region. In conclusion, CBL0100 is a newly identified inhibitor of HIV-1 transcription that can be used as an LPA in the "block and lock" cure strategy.

Screening of an FDA-approved compound library identifies levosimendan as a novel anti-HIV-1 agent that inhibits viral transcription.

Combination antiretroviral therapy (cART) has been proven to efficiently inhibit ongoing replication of human immunodeficiency virus type 1 (HIV-1), and significantly improve the health outcome in patients of acquired immune deficiency syndrome (AIDS). However, cART is unable to cure HIV-1/AIDS. Even in presence of cART there exists a residual viremia, contributed from the viral reservoirs of latently infected HIV-1 proviruses; this constitutes a major hurdle. Currently, there are multiple strategies aimed at eliminating or permanently silence these HIV-1 latent reservoirs being intensely explored. One such strategy, a recently emerged "block and lock" approach is appealing. For this approach, so-called HIV-1 latency-promoting agents (LPAs) are used to reinforce viral latency and to prevent the low-level or sporadic transcription of integrated HIV-1 proviruses. Although several LPAs have been reported, there is still a question of their suitability to be further developed as a safe and valid therapeutic agent for the clinical use. In this study, we aimed to identify new potential LPAs through the screening an FDA-approved compound library. A new and promising anti-HIV-1 inhibitor, levosimendan, was identified from these screens. Levosimendan is currently used to treat heart failure in clinics, but it demonstrates strong inhibition of TNFα-induced HIV-1 reactivation in multiple cell lines of HIV-1 latency through affecting the HIV-1 Tat-LTR transcriptional axis. Furthermore, we confirmed that in primary CD4+ T cells levosimendan inhibits both the acute HIV-1 replication and the reactivation of latent HIV-1 proviruses. As a summary, our studies successfully identify levosimendan as a novel and promising anti-HIV-1 inhibitor, which should be immediately investigated in vivo given that it is already an FDA-approved drug.

A Novel Bromodomain Inhibitor Reverses HIV-1 Latency through Specific Binding with BRD4 to Promote Tat and P-TEFb Association.

While combinatory antiretroviral therapy (cART) can effectively reduce HIV-1 viremia, it cannot eliminate HIV-1 infection. In the presence of cART, viral reservoirs remain latent, impeding the cure of HIV-1/AIDS. Recently, latency-reversing agents (LRAs) have been developed with the intent of purging latent HIV-1, providing an intriguing strategy for the eradication of the residual viral reservoirs. Our earlier studies show that the first-generation, methyl-triazolo bromodomain, and extra-terminal domain inhibitor (BETi), JQ1, facilitates the reversal of HIV-1 latency. BETis have emerged as a new class of compounds that are promising for this HIV-1 "shock and kill" eradication approach. However, when used as a single drug, JQ1 only modestly reverses HIV-1 latency, which complicates studying the underlining mechanisms. Meanwhile, it has been widely discussed that the induction of latent proviruses is stochastic (Ho et al., 2013). Thus, new BETis are currently under active development with focus on improving potency, ease of synthesis and structural diversity. Using fluorous-tagged multicomponent reactions, we developed a novel second-generation, 3,5-dimethylisoxazole BETi based on an imidazo[1,2-a] pyrazine scaffold, UMB-32. Furthermore, we screened 37 UMB-32 derivatives and identified that one, UMB-136, reactivates HIV-1 in multiple cell models of HIV-1 latency with better efficiency than either JQ1 or UMB-32. UMB-136 enhances HIV-1 transcription and increases viral production through the release of P-TEFb. Importantly, UMB-136 enhances the latency-reversing effects of PKC agonists (prostratin, bryostatin-1) in CD8-depleted PBMCs containing latent viral reservoirs. Our results illustrate that structurally improved BETis, such as UMB-136, may be useful as promising LRAs for HIV-1 eradication.

Identification of HIV-1 Tat-Associated Proteins Contributing to HIV-1 Transcription and Latency.

Human immunodeficiency virus type 1 (HIV-1) Tat is a virus-encoded trans-activator that plays a central role in viral transcription. We used our recently developed parallel analysis of in vitro translated open reading frames (ORFs) (PLATO) approach to identify host proteins that associate with HIV-1 Tat. From this proteomic assay, we identify 89 Tat-associated proteins (TAPs). We combine our results with other datasets of Tat or long terminal repeat (LTR)-associated proteins. For some of these proteins (NAT10, TINP1, XRCC5, SIN3A), we confirm their strong association with Tat. These TAPs also suppress Tat-mediated HIV-1 transcription. Removing suppression of HIV-1 transcription benefits the reversal of post-integrated, latent HIV-1 proviruses. We demonstrate that these transcriptionally suppressing TAPs contribute to HIV-1 latency in Jurkat latency (J-LAT) cells. Therefore, our proteomic analysis highlights the previously unappreciated TAPs that play a role in maintaining HIV-1 latency and can be further studied as potential pharmacological targets for the "shock and kill" HIV-1 cure strategy.

The Host Specificities of Baculovirus per os Infectivity Factors.

Baculoviruses are insect-specific pathogens with a generally narrow host ranges. Successful primary infection is initiated by the proper interaction of at least 8 conserved per os infectivity factors (PIFs) with the host's midgut cells, a process that remains largely a mystery. In this study, we investigated the host specificities of the four core components of the PIF complex, P74, PIF1, PIF2 and PIF3 by using Helicoverpa armigera nucleopolyhedrovirus (HearNPV) backbone. The four pifs of HearNPV were replaced by their counterparts from a group I Autographa californica multiple nucleopolyhedrovirus (AcMNPV) or a group II Spodoptera litura nucleopolyhedrovirus (SpltNPV). Transfection and infection assays showed that all the recombinant viruses were able to produce infectious budded viruses (BVs) and were lethal to H. armigera larvae via intrahaemocoelic injection. However, feeding experiments using very high concentration of occlusion bodies demonstrated that all the recombinant viruses completely lost oral infectivity except SpltNPV pif3 substituted pif3-null HearNPV (vHaBacΔpif3-Sppif3-ph). Furthermore, bioassay result showed that the median lethal concentration (LC50) value of vHaBacΔpif3-Sppif3-ph was 23-fold higher than that of the control virus vHaBacΔpif3-Hapif3-ph, indicating that SpltNPV pif3 can only partially substitute the function of HearNPV pif3. These results suggested that most of PIFs tested have strict host specificities, which may account, at least in part, for the limited host ranges of baculoviruses.

FACT Proteins, SUPT16H and SSRP1, Are Transcriptional Suppressors of HIV-1 and HTLV-1 That Facilitate Viral Latency.

Our functional genomic RNAi screens have identified the protein components of the FACT (facilitates chromatin transcription) complex, SUPT16H and SSRP1, as top host factors that negatively regulate HIV-1 replication. FACT interacts specifically with histones H2A/H2B to affect assembly and disassembly of nucleosomes, as well as transcription elongation. We further investigated the suppressive role of FACT proteins in HIV-1 transcription. First, depletion of SUPT16H or SSRP1 protein enhances Tat-mediated HIV-1 LTR (long terminal repeat) promoter activity. Second, HIV-1 Tat interacts with SUPT16H but not SSRP1 protein. However, both SUPT16H and SSRP1 are recruited to LTR promoter. Third, the presence of SUPT16H interferes with the association of Cyclin T1 (CCNT1), a subunit of P-TEFb, with the Tat-LTR axis. Removing inhibitory mechanisms to permit HIV-1 transcription is an initial and key regulatory step to reverse post-integrated latent HIV-1 proviruses for purging of reservoir cells. We therefore evaluated the role of FACT proteins in HIV-1 latency and reactivation. Depletion of SUPT16H or SSRP1 protein affects both HIV-1 transcriptional initiation and elongation and spontaneously reverses latent HIV-1 in U1/HIV and J-LAT cells. Similar effects were observed with a primary CD4+ T cell model of HIV-1 latency. FACT proteins also interfere with HTLV-1 Tax-LTR-mediated transcription and viral latency, indicating that they may act as general transcriptional suppressors for retroviruses. We conclude that FACT proteins SUPT16H and SSRP1 play a key role in suppressing HIV-1 transcription and promoting viral latency, which may serve as promising gene targets for developing novel HIV-1 latency-reversing agents.

IFI44 suppresses HIV-1 LTR promoter activity and facilitates its latency.

IFI44 is an interferon-alfa inducible protein, and is associated with infection of several viruses. However, IFI44 elicits minimal antiviral effects on these viruses, and its exact role is still unknown. Here we show that IFI44 inhibits HIV-1 replication in vitro. Through depletion of endogenous IFI44 or overexpression of IFI44 we confirm that IFI44 suppresses HIV-1 LTR promoter activity and affects viral transcription. Furthermore, we find that IFI44 localizes to nuclei and binds to the HIV-1 LTR promoter in HIV-1 infected cells. Removing suppression of HIV-1 transcription benefits reactivation of HIV-1 proviruses for purging latent reservoirs. We demonstrate that depletion of endogenous IFI44 in J-LAT cells induces reactivation of latent HIV-1. Based on these results, we propose a model in which IFI44 is recruited to the HIV-1 LTR, which may suppress viral transcription and prevent reactivation of latent HIV-1. Our study suggests a previously unrecognized anti-HIV phenomenon for interferon-stimulated proteins.

The ha72 core gene of baculovirus is essential for budded virus production and occlusion-derived virus embedding, and amino acid K22 plays an important role in its function.

ha72 of Helicoverpa armigera nucleopolyhedrovirus (a homologue of ac78) was identified as a conserved late baculovirus gene and characterized. HA72 localizes in the intranuclear ring zone. By generating mutants, we showed that HA72 is essential for budded virus (BD) production and occlusion-derived virus (ODV) embedding. HA72 also interacted with P33, a baculoviral sulfhydryl oxidase. A point mutation of amino acid 22 from lysine to glutamic acid curtailed BV production and precluded ODV occlusion as well as interaction with P33.