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People with HIV exhibit persistent inflammation that correlates with HIV-associated comorbidities including accelerated aging, increased risk of cardiovascular disease, and neuroinflammation. Mechanisms that perpetuate chronic inflammation in people with HIV undergoing antiretroviral treatments are poorly understood. One hypothesis is that the persistent low-level expression of HIV proviruses, including RNAs generated from defective proviral genomes, drives the immune dysfunction that is responsible for chronic HIV pathogenesis. We explore factors during HIV infection that contribute to the generation of a pool of defective proviruses as well as how HIV-1 mRNA and proteins alter immune function in people living with HIV. [ABSTRACT FROM AUTHOR]

Highlights • HIV-1 utilizes several forms of cell-to-cell transmission that influence viral dissemination, survival and pathogenesis. • HIV-1 cell-to-cell transmission mediates the generation of latent infection in CD4 + T cells. • HIV-1 cell-to-cell transmission affects the regulation of HIV-1 latency, challenging the development of curative therapies. Abstract HIV-1 spreads through both the release of cell-free particles and by cell-to-cell transmission. Mounting evidence indicates that cell-to-cell transmission is more efficient than cell-free transmission of particles and likely influences the pathogenesis of HIV-1 infection. This mode of viral transmission also influences the generation and maintenance of the latent reservoir, which represents the main obstacle for curing the infection. In this review we will discuss general cell contact-dependent mechanisms that HIV-1 utilizes for its spread and the evidence pointing to cell-to-cell transmission as a mechanism for the establishment and maintenance of latent infection. [ABSTRACT FROM AUTHOR]

Long-lived latent HIV-infected cells lead to the rebound of virus replication following antiretroviral treatment interruption and present a major barrier to eliminating HIV infection. These latent reservoirs, which include quiescent memory T cells and tissueresident macrophages, represent a subset of cells with decreased or inactive proviral transcription. HIV proviral transcription is regulated at multiple levels including transcription initiation, polymerase recruitment, transcription elongation, and chromatin organization. How these biochemical processes are coordinated and their potential role in repressing HIV transcription along with establishing and maintaining latency are reviewed. [ABSTRACT FROM AUTHOR]

The article presents a phylogenetic study that revealed Licuala as not monophyletic. The study resolved a group of species from Licuala and the rest of the Livistoninae and cited its relation to Joahnnesteijsmannia. It conducted a survey of Licuala specimens from several herbaria indicating eight species should be recognized within this genus.

Abstract: The multifunctional HIV-1 protein Nef possesses several motifs that interact with signaling molecules in infected T cells. In order to determine whether Nef influences T cell activation, cells were infected with Nef-positive and Nef-negative clones of HIV. CD28 expression and changes in tyrosine phosphorylation were monitored. We observed no Nef-dependent changes in CD28 expression or function. However, infection with Nef-positive virus led to changes in tyrosine phosphorylation. This Nef-induced phosphorylation was observed in unstimulated cells, and c-Cbl was identified as one of the proteins whose phosphorylation was upregulated by Nef. Furthermore, Lck is required for Nef-mediated c-Cbl tyrosine phosphorylation. These results suggest that Nef modifies T cell signaling in the absence of T cell receptor engagement and co-stimulation. [Copyright &y& Elsevier]

Previous systematic treatments of the neotropical palm genus Hyospathe have recognized from two to 18 species. An explicit, quantitative, repeatable sequence of operations for delimiting and testing groups of specimens and applying species concepts is carried out. Multivariate statistical analysis of morphological data is used to delimit and test groups of specimens. Cluster analysis is used to distinguish between characters and traits. Analysis of qualitative and quantitative characters reveals six groups of specimens, and the Phylogenetic Species Concept is applied to these groups. Two species, H. peruviana Henderson and H. frontinensis Henderson, are described as new. One of the specimen groups is large and widespread, and six geographically separate subgroups can be recognized within it. These subgroups can be distinguished by one or more significantly different quantitative characters. A Phylogenetic Subspecies Concept is applied to these subgroups. Three subspecies, H. elegans subsp. costaricensis Henderson, H. elegans subsp. sanblasensis Henderson, and H. elegans subsp. tacarcunensis Henderson are described as new, and two new combinations are made: H. elegans subsp. sodiroi (Dammer ex Burret) Henderson and H. elegans subsp, concinna (H. E. Moore) Henderson. One subspecies occurring in the Amazon region is complex morphologically and is not resolved by the methods used here. [ABSTRACT FROM AUTHOR]

Chemotaxis is the consequence of environmental factors engaging their receptors to initiate signaling cascades. However, the biochemical mechanisms controlling this phenomenon are not clear. We employed an in vitro modified Boyden 48-well chemotaxis migration system to characterize the role of signal transducers in type IV collagen (CIV) induced A2058 human melanoma cell migration. Using specific pharmacological inhibitors and a series of dominant-negative and constitutively active signaling proteins, we show that Ras and Rac GTPases, PI-3K, and PKC participate in cell migration mediated by β1 integrins. Collagen also induces a time- dependent degradation of IκB-α and an increase in nuclear translocation of NF-κB which is dependent on PKC pathway. More importantly, collagen-stimulated melanoma cell migration directly correlated with an increase in NF-κB transactivation. Furthermore, CIV induced an increase in β1 integrin mRNA levels. Specific NF-κB inhibitors Helenalin and SN-50 inhibited melanoma cell migration to collagen, indicating a novel requirement for NF-κB transactivation in cell chemotaxis mediated by β1 integrin signals. These results describe signal transduction events that are initiated by type IV collagen through β1 integrins and demonstrate an important role for NF-κB in regulating melanoma chemotaxis.Oncogene (2003) 22, 98–108. doi:10.1038/sj.onc.1206059 [ABSTRACT FROM AUTHOR]

Analyzes the phenetic and phylogeny of Reinhardtia using cluster analysis and principal component analysis. Success of the classification of the specimen into six specie using discriminant analysis; Characteristics of the species; Decrease in sizes of leaves and inflorescence.

Defective HIV-1 proviruses represent a population of viral genomes that are selected for by immune pressures, and clonally expanded to dominate the persistent HIV-1 proviral genome landscape. There are examples of RNA and protein expression from these compromised genomes which are generated by a variety of mechanisms. Despite the evidence that these proviruses are transcribed and translated, their role in HIV pathogenesis has not been fully explored. The potential for these genomes to participate in immune stimulation is particularly relevant considering the accumulation of cells harboring these defective proviruses over the course of antiretroviral therapy in people living with HIV. The expression of defective proviruses in different cells and tissues could drive innate sensing mechanisms and inflammation. They may also alter antiviral T cell responses and myeloid cell functions that directly contribute to HIV-1 associated chronic comorbidities. Understanding the impact of these defective proviruses needs to be considered as we advance cure strategies that focus on targeting the diverse population of HIV-1 proviral genomes. [ABSTRACT FROM AUTHOR]

Of the 12 million people who inject drugs worldwide, 13% live with HIV. Whether opioid use impacts HIV pathogenesis and latency is an outstanding question. To gain insight into whether opioid use influences the proviral landscape and latent HIV reservoir, we performed intact proviral DNA assays (IPDA) on peripheral blood mononuclear cells (PBMCs) from antiretroviral therapy (ART)-suppressed people living with HIV (PWH) with or without current opioid use. No differences were observed between PWH with and without opioid use in the frequency of HIV intact and defective proviral genomes. To evaluate the latent reservoir, we activated PBMCs from ART-suppressed PWH with or without opioid use and assessed the induction of HIV RNA. PWH using opioids had diminished responses to ex vivo HIV reactivation, suggesting a smaller reversible reservoir of HIV-1 latently infected cells. However, in vitro studies using primary CD4+ T cells treated with morphine showed no effect of opioids on HIV-1 infection, replication or latency establishment. The discrepancy in our results from in vitro and clinical samples suggests that while opioids may not directly impact HIV replication, latency and reactivation in CD4+ T cells, opioid use may indirectly shape the HIV reservoir in vivo by modulating general immune functions. [ABSTRACT FROM AUTHOR]

Chromatin influences Human Immunodeficiency Virus (HIV) integration and replication. This review highlights critical host factors that influence chromatin structure and organization and that also impact HIV integration, transcriptional regulation and latency. Furthermore, recent attempts to target chromatin associated factors to reduce the HIV proviral load are discussed. [ABSTRACT FROM AUTHOR]

The development of therapies to eliminate the latent HIV-1 reservoir is hampered by our incomplete understanding of the biomolecular mechanism governing HIV-1 latency. To further complicate matters, recent single-cell RNA sequencing (scRNAseq) studies reported extensive heterogeneity between latently HIV-1-infected primary T cells, implying that latent HIV-1 infection can persist in greatly differing host cell environments. We show here that transcriptomic heterogeneity is also found between latently infected T cell lines, which allowed us to study the underlying mechanisms of intercell heterogeneity at high signal resolution. Latently infected T cells exhibited a dedifferentiated phenotype, characterized by the loss of T cell-specific markers and gene regulation profiles reminiscent of hematopoietic stem cells (HSC). These changes had functional consequences. As reported for stem cells, latently HIV-1-infected T cells efficiently forced lentiviral superinfections into a latent state and favored glycolysis. As a result, metabolic reprogramming or cell redifferentiation destabilized latent infection. Guided by these findings, data mining of single-cell RNA-seq data of latently HIV-1-infected primary T cells from patients revealed the presence of similar dedifferentiation motifs. More than 20% of the highly detectable genes that were differentially regulated in latently infected cells were associated with hematopoietic lineage development (e.g., HUWE1, IRF4, PRDM1, BATF3, TOX, ID2, IKZF3, and CDK6) or were hematopoietic markers (SRGN; hematopoietic proteoglycan core protein). The data add to evidence that the biomolecular phenotype of latently HIV-1-infected cells differs from that of normal T cells and strategies to address their differential phenotype need to be considered in the design of therapeutic cure interventions. IMPORTANCE HIV-1 persists in a latent reservoir in memory CD4 T cells for the lifetime of a patient. Understanding the biomolecular mechanisms used by the host cells to suppress viral expression will provide essential insights required to develop curative therapeutic interventions. Unfortunately, our current understanding of these control mechanisms is still limited. By studying gene expression profiles, we demonstrated that latently HIV-1-infected T cells have a dedifferentiated T cell phenotype. Software-based data integration allowed the identification of drug targets that would redifferentiate viral host cells and, by extension, destabilize latent HIV-1 infection events. The importance of the presented data lies within the clear demonstration that HIV-1 latency is a host cell phenomenon. As such, therapeutic strategies must first restore proper host cell functionality to accomplish efficient HIV-1 reactivation. [ABSTRACT FROM AUTHOR]

Expressed on tissue-resident macrophages, the receptor tyrosine kinase, recepteur d'orgine nantais (RON), functions to maintain inflammation homeostasis by activating genes that promote wound repair and resolve inflammation while repressing genes that perpetuate tissue damage and cell death. Chronic HIV-1 infection is associated with dysregulated inflammation, and we hypothesize that diminished RON expression contributes to the development of end organ diseases such as HIV-1-associated CNS disease. To explore RON function in vivo, we used CNS tissue from a well-characterized SIV macaque model and examined the temporal regulation of RON in the brain during the course of infection. Following prolonged SIV infection, RON expression was inversely correlated with the development of CNS disease; RON was maintained in animals that did not develop CNS lesions and was reduced in SIV-infected macaques that demonstrated moderate to severe inflammatory lesions. Arginase-1 expression was reduced in the brain during late infection, whereas expression of the inflammatory genes, IL-12p40 and TNF-α, was elevated. To validate a role for RON in regulating HIV-1 in primary cells, we used human tissue-resident macrophages isolated from tonsil as a tractable cell model. RON signaling in tissue-resident macrophages, both ligand dependent and independent, limited HIV-1 replication. Furthermore, prolonged HIV-1 infection in vitro resulted in downregulation of RON. We propose a model in which, following chronic HIV-1 infection in the brain, RON expression is decreased, genes that quell inflammation are repressed, and inflammatory mediators are induced to promote tissue inflammation. [ABSTRACT FROM AUTHOR]

Broad-scale patterns of species diversity have received much attention in the literature, yet the mechanisms behind their formation may not explain species richness disparities across small spatial scales. Few taxa display high species diversity on either side of Wallace's Line and our understanding of the processes causing this biogeographical pattern remains limited, particularly in plant lineages. To understand the evolution of this biogeographical pattern, a time-calibrated molecular phylogeny of Livistoninae palms (Arecaceae) was used to infer the colonization history of the Sahul tectonic plate region and to test for disparities in diversification rates across taxa and across each side of Wallace's Line. Our analyses allowed us to examine how timing, migration history, and shifts in diversification rates have contributed to shape the biogeographical pattern observed in Livistoninae. We inferred that each of the three genera found in Sahul crossed Wallace's Line only once and relatively recently. In addition, at least two of the three dispersing genera underwent an elevation in their diversification rate leading to high species richness on each side of Wallacea. The correspondence of our results with Southeast Asian geologic and climatic history show how palms emerge as excellent models for understanding the historical formation of fine-scale biogeographic patterns in a phylogenetic framework. [ABSTRACT FROM AUTHOR]

HIV-1 establishes a persistent proviral reservoir by integrating into the genome of infected host cells. Current antiretroviral treatments do not target this persistent population of proviruses which include latently infected cells that upon treatment interruption can be reactivated to contribute to HIV-1 rebound. Deep sequencing of persistent HIV proviruses has revealed that greater than 90% of integrated HIV genomes are defective and unable to produce infectious virions. We hypothesized that intragenic elements in the HIV genome support transcription of aberrant HIV-1 RNAs from defective proviruses that lack long terminal repeats (LTRs). Using an intact provirus detection assay, we observed that resting CD4+ T cells and monocyte-derived macrophages (MDMs) are biased towards generating defective HIV-1 proviruses. Multiplex reverse transcription droplet digital PCR identified env and nef transcripts which lacked 5' untranslated regions (UTR) in acutely infected CD4+ T cells and MDMs indicating transcripts are generated that do not utilize the promoter within the LTR. 5'UTR-deficient env transcripts were also identified in a cohort of people living with HIV (PLWH) on ART, suggesting that these aberrant RNAs are produced in vivo. Using 5' rapid amplification of cDNA ends (RACE), we mapped the start site of these transcripts within the Env gene. This region bound several cellular transcription factors and functioned as a transcriptional regulatory element that could support transcription and translation of downstream HIV-1 RNAs. These studies provide mechanistic insights into how defective HIV-1 proviruses are persistently expressed to potentially drive inflammation in PLWH. Author summary: People living with HIV establish a persistent reservoir which includes latently infected cells that fuel viral rebound upon treatment interruption. However, the majority of HIV-1 genomes in these persistently infected cells are defective. Whether these defective HIV genomes are expressed and whether they contribute to HIV associated diseases including accelerated aging, neurodegenerative symptoms, and cardiovascular diseases are still outstanding questions. In this paper, we demonstrate that acute infection of macrophages and resting T cells is biased towards generating defective viruses which are expressed by DNA regulatory elements in the HIV genome. These studies describe an alternative mechanism for chronic expression of HIV genomes. [ABSTRACT FROM AUTHOR]

Biorepositories provide a critical resource for gaining knowledge of emerging infectious diseases and offer a mechanism to rapidly respond to outbreaks; the emergence of the novel coronavirus, SARS-CoV-2, has proved their importance. During the COVID-19 pandemic, the absence of centralized, national biorepository efforts meant that the onus fell on individual institutions to establish sample repositories. As a safety-net hospital, Boston Medical Center (BMC) recognized the importance of creating a COVID-19 biorepository to both support critical science at BMC and ensure representation in research for its urban patient population, most of whom are from underserved communities. This article offers a realistic overview of the authors' experience in establishing this biorepository at the onset of the COVID-19 pandemic during the height of the first surge of cases in Boston, Massachusetts, with the hope that the challenges and solutions described are useful to other institutions. Going forward, funders, policymakers, and infectious disease and public health communities must support biorepository implementation as an essential element of future pandemic preparedness. [ABSTRACT FROM AUTHOR]

Activation through the T-cell receptor and the costimulatory receptor CD28 supports efficient HIV transcription as well as reactivation of latent provirus. To characterize critical signals associated with CD28 that regulate HIV-1 transcription, we generated a library of chimeric CD28 receptors that harbored different combinations of key tyrosine residues in the cytoplasmic tail, Tyr-173, Tyr-188, Tyr-191, and Tyr-200. We found that Tyr-191 and Tyr-200 induce HIV-1 transcription via the activation of NF-κB and its recruitment to the HIV-long terminal repeat. Tyr-188 modifies positive and negative signals associated with CD28. Importantly, signaling through Tyr-188, Tyr-191, and Tyr-200 is required to overcome the inhibition posed by Tyr-173. CD28 also regulates P-TEFb activity, which is necessary for HIV-1 transcription processivity, by limiting the release of P-TEFb from the HEXIM1-7SK inhibitory complex in response to T-cell receptor signaling. Our studies reveal that CD28 regulates HIV-1 provirus transcription through a complex interplay of positive and negative signals that may be manipulated to control HIV-1 transcription and replication. [ABSTRACT FROM AUTHOR]

Controlling the HIV/AIDS epidemic remains a major challenge, with approximately 5 million new HIV infections annually. Cyclopentenone prostaglandins (CyPG), such as 15-deoxy-Δ12,14-PGJ2 (15d-PGJ2), are arachidonic acid-derived endogenous electrophiles that possess anti-HIV activity by an unknown mechanism. Given that the reactive α,β-unsaturated ketone in the cyclopentenone ring of 15d-PGJ2 covalently modifies key Cys thiols in select proteins, we hypothesized that 15d-PGJ2 inhibits HIV transcription and replication by targeting Cys thiols in HIV-1 Tat. Tat is a potent transactivator of viral gene expression required for HIV transcriptional elongation and replication. Our studies indicate that 15d-PGJ2 treatment of cells inhibits Tat-dependent transcription and replication of HIV-1, while 9,10-dihydro-15d-PGJ2, PGE2, PGF2α, or PGD2 that lack the reactive α,β-unsaturated ketone were ineffective. The inhibition of Tat activity by 15d-PGJ2 was dose-dependent, with an IC50 of 1.2 µM and independent of NF-κB pathway. Furthermore, using a biotinylated derivative of 15d-PGJ2, we demonstrate that 15d-PGJ2 modifies free Cys-thiols in Tat to form covalent Michael adducts and that the interaction was further increased on reduction of Tat. 15d-PGJ2-modified Tat was unable to transactivate the HIV long terminal repeat in U937 human macrophages. These data demonstrate that Tat acts as a molecular target of CyPG leading to the inhibition of transcription and also surest a novel therapeutic approach to complement current antiretroviral strategies for HIV/AIDS. [ABSTRACT FROM AUTHOR]

Abstract: Inflammation facilitates tumor progression including metastasis. Interleukin-8 (IL-8) is a chemokine that regulates polymorphonuclear neutrophil (PMN) mobilization and activity and we hypothesize that this cytokine influences tumor behavior. We have demonstrated that IL-8 is crucial for PMN-mediated melanoma extravasation under flow conditions. In addition, IL-8 is up-regulated in PMNs upon co-culturing with melanoma cells. Melanoma cells induce IκB-α degradation in PMNs indicating that NF-κB signaling is active in PMNs. Furthermore, the production of IL-8 in PMNs is NF-κB dependent. We have further identified that interleukin-6 (IL-6) and interleukin-1β (IL-1β) from PMN-melanoma co-cultures synergistically contribute to IκB-α degradation and IL-8 synthesis in PMNs. Taken together, these findings show that melanoma cells induce PMNs to secrete IL-8 through activation of NF-κB and suggest a model in which this interaction promotes a microenvironment that is favorable for metastasis. [Copyright &y& Elsevier]

Background: Exposure to Staphylococcal Enterotoxin B (SEB), a bacterial superantigen secreted by the Gram-positive bacteria Staphyloccocus aureus, results in the expansion and eventual clonal deletion and anergy of Vβ 8+ T cells, as well as massive cytokine release, including Interleukin-2 (IL- 2). This IL-2 is rapidly secreted following exposure to SEB and may contribute to the symptoms seen following exposure to this bacterial toxin. The Tec family kinase ITK has been shown to be important for the production of IL-2 by T cells stimulated in vitro and may represent a good target for blocking the production of this cytokine in vivo. In order to determine if ITK represents such a target, mice lacking ITK were analyzed for their response to SEB exposure. Results: It was found that T cells from mice lacking ITK exhibited significantly reduced proliferative responses to SEB exposure in vitro, as well as in vivo. Examination of IL-2 production revealed that ITK null mice produced reduced levels of this cytokine in vitro, and more dramatically, in vivo. In vivo analysis of c-jun phosphorylation, previously shown to be critical for regulating IL-2 production, revealed that this pathway was specifically activated in SEB reactive Vβ 8+ (but not non-reactive Vβ 6+) T cells from WT mice, but not in Vβ 8+ T cells from ITK null mice. However, toxicity analysis indicated that both WT and ITK null animals were similarly affected by SEB exposure. Conclusion: These data show that ITK is required for IL-2 production induced by SEB in vivo, and may regulate signals leading IL-2 production, in part by regulating phosphorylation of c-jun. The data also suggest that perturbing T cell activation pathways leading to IL-2 does not necessarily lead to improved responses to SEB toxicity. [ABSTRACT FROM AUTHOR]

The molecular networks involved in the regulation of HIV replication, transcription, and latency remain incompletely defined. To expand our understanding of these networks, we performed an unbiased high-throughput yeast one-hybrid screen, which identified 42 human transcription factors and 85 total protein-DNA interactions with HIV-1 and HIV-2 long terminal repeats. We investigated a subset of these transcription factors for transcriptional activity in cell-based models of infection. KLF2 and KLF3 repressed HIV-1 and HIV-2 transcription in CD4+ T cells, whereas PLAGL1 activated transcription of HIV-2 through direct protein-DNA interactions. Using computational modeling with interacting proteins, we leveraged the results from our screen to identify putative pathways that define intrinsic transcriptional networks. Overall, we used a high-throughput functional screen, computational modeling, and biochemical assays to identify and confirm several candidate transcription factors and biochemical processes that influence HIV-1 and HIV-2 transcription and latency. [ABSTRACT FROM AUTHOR]

Antimicrobial stewardship (AMS) is well established in Australian hospitals. Electronic medical record (EMR) implementation has lagged in Australia, with two Healthcare Information and Management Systems Society (HIMSS) Stage 6 hospitals and one Stage 7 hospital as of September 2020. Specific barriers faced by AMS teams with paper-based prescribing and medical records include real-time identification of antimicrobials orders; the ability to prospectively monitor antimicrobial use; and the integration of fundamental point of prescribing AMS principles into routine clinical practice. There are few local guidelines to assist Australian hospitals and AMS teams beyond "out of the box" EMR functionality. EMR implementation has enormous potential to positively impact AMS teams through more efficient workflows and the ability to expand the reach and coverage of AMS activities. There are inevitable limitations associated with EMR implementation that must be considered. In this paper, four Australian hospitals share their experience with EMR roll out, AMS customisation and how they have overcome specific barriers in local AMS practice. [ABSTRACT FROM AUTHOR]

Background Mycobacterium tuberculosis (Mtb) and human immunodeficiency virus (HIV) coinfection increases mortality, accelerates progression to acquired immune deficiency syndrome, and exacerbates tuberculosis disease. However, the impact of pre-existing Mtb infection on subsequent HIV infection has not been fully explored. We hypothesized that Mtb infection creates an immunological environment that influences the course of HIV infection, and we investigated whether pre-existing Mtb infection impacts the susceptibility of CD4+ T cells to HIV-1 infection. Methods Plasma and blood CD4+ T cells isolated from HIV-negative individuals across the Mtb infection spectrum and non-Mtb-infected control individuals were analyzed for inflammation markers and T-cell phenotypes. CD4+ T cells were infected with HIV-1 in vitro and were monitored for viral replication. Results We observed differences in proinflammatory cytokines and the relative proportion of memory T-cell subsets depending on Mtb infection status. CD4+ T cells derived from individuals with latent Mtb infection supported more efficient HIV-1 transcription, release, and replication. Enhanced HIV-1 replication correlated with higher percentages of CD4+ TEM and TTD cells. Conclusions Pre-existing Mtb infection creates an immunological environment that reflects Mtb infection status and influences the susceptibility of CD4+ T cells to HIV-1 replication. These findings provide cellular and molecular insights into how pre-existing Mtb infection influences HIV-1 pathogenesis. [ABSTRACT FROM AUTHOR]

Background: Mycobacterium tuberculosis (Mtb) and human immunodeficiency virus (HIV) coinfection increases mortality, accelerates progression to acquired immune deficiency syndrome, and exacerbates tuberculosis disease. However, the impact of pre-existing Mtb infection on subsequent HIV infection has not been fully explored. We hypothesized that Mtb infection creates an immunological environment that influences the course of HIV infection, and we investigated whether pre-existing Mtb infection impacts the susceptibility of CD4+ T cells to HIV-1 infection.Methods: Plasma and blood CD4+ T cells isolated from HIV-negative individuals across the Mtb infection spectrum and non-Mtb-infected control individuals were analyzed for inflammation markers and T-cell phenotypes. CD4+ T cells were infected with HIV-1 in vitro and were monitored for viral replication.Results: We observed differences in proinflammatory cytokines and the relative proportion of memory T-cell subsets depending on Mtb infection status. CD4+ T cells derived from individuals with latent Mtb infection supported more efficient HIV-1 transcription, release, and replication. Enhanced HIV-1 replication correlated with higher percentages of CD4+ TEM and TTD cells.Conclusions: Pre-existing Mtb infection creates an immunological environment that reflects Mtb infection status and influences the susceptibility of CD4+ T cells to HIV-1 replication. These findings provide cellular and molecular insights into how pre-existing Mtb infection influences HIV-1 pathogenesis. [ABSTRACT FROM AUTHOR]

A major barrier to curing HIV-1 is the long-lived latent reservoir that supports re-emergence of HIV-1 upon treatment interruption. Targeting this reservoir will require mechanistic insights into the establishment and maintenance of HIV-1 latency. Whether T cell signaling at the time of HIV-1 infection influences productive replication or latency is not fully understood. We used a panel of chimeric antigen receptors (CARs) with different ligand binding affinities to induce a range of signaling strengths to model differential T cell receptor signaling at the time of HIV-1 infection. Stimulation of T cell lines or primary CD4+ T cells expressing chimeric antigen receptors supported HIV-1 infection regardless of affinity for ligand; however, only signaling by the highest affinity receptor facilitated HIV-1 expression. Activation of chimeric antigen receptors that had intermediate and low binding affinities did not support provirus transcription, suggesting that a minimal signal is required for optimal HIV-1 expression. In addition, strong signaling at the time of infection produced a latent population that was readily inducible, whereas latent cells generated in response to weaker signals were not easily reversed. Chromatin immunoprecipitation showed HIV-1 transcription was limited by transcriptional elongation and that robust signaling decreased the presence of negative elongation factor, a pausing factor, by more than 80%. These studies demonstrate that T cell signaling influences HIV-1 infection and the establishment of different subsets of latently infected cells, which may have implications for targeting the HIV-1 reservoir. [ABSTRACT FROM AUTHOR]

Viral protein R (Vpr) is an HIV-1 accessory protein whose function remains poorly understood. In this report, we sought to determine the requirement of Vpr for facilitating HIV-1 infection of monocyte-derived dendritic cells (MDDCs), one of the first cell types to encounter virus in the peripheral mucosal tissues. In this report, we characterize a significant restriction of Vpr-deficient virus replication and spread in MDDCs alone and in cell-to-cell spread in MDDC-CD4+ T cell cocultures. This restriction of HIV-1 replication in MDDCs was observed in a single round of virus replication and was rescued by the expression of Vpr in trans in the incoming virion. Interestingly, infections of MDDCs with viruses that encode Vpr mutants unable to interact with either the DCAF1/DDB1 E3 ubiquitin ligase complex or a host factor hypothesized to be targeted for degradation by Vpr also displayed a significant replication defect. While the extent of proviral integration in HIV-1-infected MDDCs was unaffected by the absence of Vpr, the transcriptional activity of the viral long terminal repeat (LTR) from Vpr-deficient proviruses was significantly reduced. Together, these results characterize a novel postintegration restriction of HIV-1 replication in MDDCs and show that the interaction of Vpr with the DCAF1/DDB1 E3 ubiquitin ligase complex and the yet-to-be-identified host factor might alleviate this restriction by inducing transcription from the viral LTR. Taken together, these findings identify a robust in vitro cell culture system that is amenable to addressing mechanisms underlying Vpr-mediated enhancement of HIV-1 replication. [ABSTRACT FROM AUTHOR]

Despite advances in antiretroviral therapy, HIV-1 infection remains incurable in patients and continues to present a significant public health burden worldwide. While a number of factors contribute to persistent HIV-1 infection in patients, the presence of a stable, long-lived reservoir of latent provirus represents a significant hurdle in realizing an effective cure. One potential strategy to eliminate HIV-1 reservoirs in patients is reactivation of latent provirus with latency reversing agents in combination with antiretroviral therapy, a strategy termed “shock and kill”. This strategy has shown limited clinical effectiveness thus far, potentially due to limitations of the few therapeutics currently available. We have identified a novel class of benzazole compounds effective at inducing HIV-1 expression in several cellular models. These compounds do not act via histone deacetylase inhibition or T cell activation, and show specificity in activating HIV-1 in vitro. Initial exploration of structure-activity relationships and pharmaceutical properties indicates that these compounds represent a potential scaffold for development of more potent HIV-1 latency reversing agents. [ABSTRACT FROM AUTHOR]

Periodontal infections contribute to HIV-associated co-morbidities in the oral cavity and provide a model to interrogate the dysregulation of macrophage function, inflammatory disease progression, and HIV replication during co-infections. We investigated the effect of Porphyromonas gingivalis on the establishment of HIV infection in monocyte-derived macrophages. HIV replication in macrophages was significantly repressed in the presence of P. gingivalis . This diminished viral replication was due partly to a decrease in the expression of integrated HIV provirus. HIV repression depended upon signaling through TLR4 as knock-down of TLR4 with siRNA rescued HIV expression. Importantly, HIV expression was reactivated upon removal of P. gingivalis. Our observations suggest that exposure of macrophages to Gram-negative bacteria influence the establishment and maintenance of HIV persistence in macrophages through a TLR4-dependent mechanism. [ABSTRACT FROM AUTHOR]

Since HIV-1 has a propensity to integrate into actively expressed genes, transcriptional interference from neighboring host promoters has been proposed to contribute to the establishment and maintenance HIV-1 latency. To gain insights into how endogenous promoters influence HIV-1 transcription we utilized a set of inducible T cell lines and characterized whether there were correlations between expression of endogenous genes, provirus and long terminal repeat architecture. We show that neighboring promoters are active but have minimal impact on HIV-1 transcription, in particular, expression of the endogenous gene did not prevent expression of HIV-1 following induction of latent provirus. We also demonstrate that releasing paused RNAP II by diminishing negative elongation factor (NELF) is sufficient to reactivate transcriptionally repressed HIV-1 provirus regardless of the integration site and orientation of the provirus suggesting that NELF-mediated RNAP II pausing is a common mechanism of maintaining HIV-1 latency. [ABSTRACT FROM AUTHOR]

CD4+ T cell subsets differentially support HIV-1 replication. For example, quiescent CD4+ memory T cells are susceptible to HIV-1 infection but do not support robust HIV-1 transcription and have been implicated as the primary reservoir of latent HIV-1. T cell transcription factors that regulate maturation potentially limit HIV-1 transcription and mediate the establishment and maintenance of HIV-1 latency. We report that B lymphocyte-induced maturation protein-1 (Blimp-1), a critical regulator of B and T cell differentiation, is highly expressed in memory CD4+ T cells compared with naive CD4+ T cells and represses basal and Tat-mediated HIV-1 transcription. Blimp-1 binds an IFN-stimulated response element within HIV-1 provirus, and it is displaced following T cell activation. Reduction of Blimp-1 in infected primary T cells including CD4+ memory T cells increases RNA polymerase II processivity, histone acetylation, and baseline HIV-1 transcription. Therefore, the transcriptional repressor, Blimp-1, is an intrinsic factor that predisposes CD4+ memory T cells to latent HIV-1 infection. [ABSTRACT FROM AUTHOR]

A barrier to eradicating HIV infection is targeting and eliminating latently infected cells. Events that contribute to HIV transcriptional latency include repressive chromatin structure, transcriptional interference, the inability of Tat to recruit positive transcription factor b, and poor processivity of RNA polymerase II (RNAP II). In this study, we investigated mechanisms by which negative elongation factor (NELF) establishes and maintains HIV latency. Negative elongation factor (NELF) induces RNAP II promoter proximal pausing and limits provirus expression in HIV-infected primary CD4+ T cells. Decreasing NELF expression overcomes RNAP II pausing to enhance HIV transcription elongation in infected primary T cells, demonstrating the importance of pausing in repressing HIV transcription. We also show that RNAP II pausing is coupled to premature transcription termination and chromatin remodeling. NELF interacts with Pcf11, a transcription termination factor, and diminishing Pcf11 in primary CD4+ T cells induces HIV transcription elongation. In addition, we identify NCoR1-GPS2-HDAC3 as a NELF-interacting corepressor complex that is associated with repressed HIV long terminal repeats. We propose a model in which NELF recruits Pcf11 and NCoR1-GPS2-HDAC3 to paused RNAP II, reinforcing repression of HIV transcription and establishing a critical checkpoint for HIV transcription and latency. [ABSTRACT FROM AUTHOR]

Abstract: Interleukin 2-inducible T cell kinase (ITK) influences T cell signaling by coordinating actin polymerization and polarization as well as recruitment of kinases and adapter proteins. ITK regulates multiple steps of HIV-1 replication, including virion assembly and release. Fluorescent microscopy was used to examine the functional interactions between ITK and HIV-1 Gag during viral particle release. ITK and Gag colocalized at the plasma membrane and were concentrated at sites of F-actin accumulation and membrane lipid rafts in HIV-1 infected T cells. There was polarized staining of ITK, Gag, and actin towards sites of T cell conjugates. Small molecule inhibitors of ITK disrupted F-actin capping, perturbed Gag-ITK colocalization, inhibited virus like particle release, and reduced HIV replication in primary human CD4+ T cells. These data provide insight as to how ITK influences HIV-1 replication and suggest that targeting host factors that regulate HIV-1 egress provides an innovative strategy for controlling HIV infection. [Copyright &y& Elsevier]

Many elongation factors in eukaryotes promote gene expression by increasing the processivity of RNA polymerase II (Pol II). However, the stability of RNA Pol II elongation complexes suggests that such complexes are not inherently prone to prematurely terminating transcription, particularly at physiological nucleotide concentrations. We show that the termination factor, Pcf11, causes premature termination on an HIV provirus. The transcription that occurs when Pcf11 is depleted from cells or an extract is no longer sensitive to 6-dichloro-1-β-D-ribofuranosylbenzimidazole (DRB), a compound that causes premature termination. Hence, Pcf11 can act as a negative elongation factor to repress RNA Pol II gene expression in eukaryotic cells. [ABSTRACT FROM AUTHOR]

Abstract: Current drugs used for antiretroviral therapy against human immunodeficiency virus (HIV) have a narrow spectrum of activity and, more often, have associated toxicities and severe side effects in addition to developing resistance. Thus, there is a need to develop new therapeutic strategies against HIV/AIDS to complement the already existing ones. Surprisingly, transactivator of transcription (Tat), an early virus-encoded protein required for the efficient transcription of the HIV genome, has not been developed as a target for small molecular therapeutics. We have previously described the ability of an endogenous Michael acceptor electrophile (MAE), 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2), to inhibit Tat-dependent transcription by targeting its cysteine (Cys)-rich domain. In an effort to identify other MAEs possessing inhibitory activity against HIV-1 Tat, we tested a collection of plant-derived compounds with electrophilic properties, including curcumin, rosmarinic acid, and gambogic acid, for their ability to inhibit Tat. Celastrol (Cel), a triterpenoid MAE isolated from Tripterygium wilfordii, exhibited the highest inhibitory activity against Tat. Using biochemical techniques, we demonstrate that Cel, by covalently modifying the cysteine thiols, inhibits Tat transactivation function. Using circular dichroism spectroscopy, we show that alkylation of Tat brought about a change in the secondary structure of Tat, which inhibited the transcription elongation of the HIV proviral genome by effecting mechanisms other than Tat–TAR (transactivation-responsive region) interaction. Our results demonstrate the underlying mechanism of antiretroviral activity of the plant-derived MAEs and suggest that Cel could serve as a lead compound to develop novel antiviral therapeutics. [Copyright &y& Elsevier]

Epidemiological studies suggest a correlation between severity of acquired immunodeficiency syndrome (AIDS) and selenium deficiency, indicating a protective role for this anti-oxidant during HIV infection. Here we demonstrate that thioredoxin reductase-1 (TR1), a selenium-containing pyridine nucleotide-disulfide oxidoreductase that reduces protein disulfides to free thiols, negatively regulates the activity of the HIV-1 encoded transcriptional activator, Tat, in human macrophages. We used a small interfering RNA approach as well as a high affinity substrate of TR1, ebselen, to demonstrate that Tat-dependent transcription and HIV-1 replication were significantly increased in human macrophages when TR1 activity was reduced. The increase in HIV-1 replication in TR1 small interfering RNA-treated cells was independent of the redox-sensitive transcription factor, NF-κB. These studies indicate that TR-1 acts as a negative regulator of Tat-dependent transcription. Furthermore, in vitro biochemical assays with recombinant Tat protein confirmed that TR1 targets two disulfide bonds within the Cys-rich motif required for efficient HIV-1 transactivation. Increasing TR1 expression along with other selenoproteins by supplementing with selenium suggests a potential inexpensive adjuvant therapy for HIV/AIDS patients. [ABSTRACT FROM AUTHOR]

Treatment for HIV has relied on the use of antiretroviral agents that can be subject to the development of resistant viruses. The study of inhibitors directed against cellular proteins required for HIV replication is therefore of growing interest. Inducible T cell kinase (ITK) is a Tec family tyrosine kinase that regulates T cell receptor (TCR)-induced activation of PLCγ-1, Ca2+ mobilization and transcription factor activation, and actin rearrangement downstream of both TCR and chemokine receptors. Because productive infection of T cells with HIV requires T cell activation, chemokine receptors and actin reorganization, we asked whether ITK affects HIV infection using ITK-specific siRNA. a kinase-inactive ITK mutant or an ITK inhibitor. We demonstrate that loss of ITK function resulted in marked reductions in intracellular p24 levels upon HIV infection. Loss of ITK function after establishment of HIV infection also decreased virus spread within the culture. Inhibition of ITK did not affect expression of the HIV coreceptors CD4 or CXCR4 but partially blocked HIV viral entry, an effect that correlated with decreased actin polarization to gp120. Additionally, ITK was required for efficient HIV transcription, and overexpression of ITK increased both viral transcription and virus-like particle formation. Our data suggest that inhibition of ITK blocks HIV infection by affecting multiple steps of HIV replication. [ABSTRACT FROM AUTHOR]

Human immunodeficiency virus (HIV) transcription requires virally encoded Tat and the P-TEFb protein complex, which together associate with the Tat-activating region, a structured region in the nascent transcript. P-TEFb phosphorylates proteins in the transcription elongation complex, including RNA polymerase H (pot H), to stimulate elongation and to overcome premature termination. However, the status of the elongation complex on the HIV long terminal repeat (LTR) in a repressed state is not known. Chromatin immunoprecipitation demonstrated that NELF, a negative transcription elongation factor, was associated with the LTR. Depleting NELF increased processive HIV transcription and replication. Mapping pol II on the LTR showed that pot II was paused and that NELF depletion released pol II. Decreasing NELF also correlated with displacement of a positioned nucleosome and increased acetylation of histone H4, suggesting coupling of transcription elongation and chromatin remodeling. Previous work has indicated that the Tat-activating region plays a critical role in regulating transcription from the LTR. Our results reveal an earlier stage, mediated by NELF, when repression occurs at the HIV LTR. [ABSTRACT FROM AUTHOR]

Mumps virus (MuV), a rubulavirus of the paramyxovirus family, causes acute infections in humans. MuV has seven genes including a small hydrophobic (SH) gene, which encodes a type I membrane protein of 57 amino acid residues. The function of the SH protein is not clear, although its expression is not necessary for growth of MuV in tissue culture cells. It is speculated that MuV SH plays a role in viral pathogenesis. Simian virus 5 (SV5), a closely related rubulavirus, encodes a 44-amino-acid-residue SH protein. Recombinant SV5 lacking the SH gene (rSV5ASH) is viable and has no growth defect in tissue culture cells. However, rSV5ASH induces apoptosis in tissue culture cells and is attenuated in vivo. Neutralizing antibodies against tumor necrosis factor alpha (TNF-α) and TNF-α receptor 1 block rSV5ΔSH-induced apoptosis, suggesting that SV5 SH plays an essential role in blocking the TNF-α-mediated apoptosis pathway. Because MuV is closely related to SV5, we hypothesize that the SH protein of MuV has a function similar to that of SV5, even though there is no sequence homology between them. To test this hypothesis and to study the function of MuV SH, we have replaced the open reading frame (ORF) of SV5 SH with the ORF of MuV SH in a SV5 genome background. The recombinant SV5 (rSV5ΔSH+MuV-SH) was analyzed in comparison with SV5. It was found that rSV5ΔSH+MuV-SH was viable and behaved like wild-type SV5, suggesting that MuV SH has a function similar to that of SV5 SH. Furthermore, both ectopically expressed SV5 SH and MuV SH blocked activation of NF-κB by TNF-α in a reporter gene assay, suggesting that both SH proteins can inhibit TNF-α signaling. [ABSTRACT FROM AUTHOR]

Activation of HIV-1-infected T cells through the T cell receptor and costimulatory molecule CD28 induces proviral transcription; however, the mechanism behind this enhanced virus expression is unknown. Jurkat T cells and primary CD4[sup +] T cells expressing a CD8α/CD28 chimeric receptor containing a mutation at tyrosine 200 in the cytoplasmic tall were unable to fully induce HIV-1 proviral transcription in response to CD8α/28 receptor cross-linking in comparison to CD28 costimulation. The loss of transactivation seen with the mutant chimeric receptor correlated with a decrease in Vav tyrosine phosphorylation. CD28-dependent activation of HIV-1 transcription also required the GTPase activity of Rac1, which was not activated during costimulation with the mutated receptor. Furthermore, the mutated receptor was unable to induce NF-κB DNA binding or transactivation, as demonstrated by electromobility shift assays and HIV-1 long terminal repeat and NF-κB-dependent reporter constructs. These studies show that signaling events initiated by tyrosine 200 of CD28 are required for efficient expression of HIV-1 transcription in activated T cells. [ABSTRACT FROM AUTHOR]

CCAAT/enhancer binding proteins (C/EBP) have been shown to be required for HIV-1 transcription and replication in macrophages. However, whether these transcription factors influence the ability of virus to establish infection by altering cytokine or receptor expression or primarily regulate HIV-1 transcription has not been determined. By inhibiting endogenous C/EBP activity with a dominant-negative protein, we demonstrate that functional C/EBPs are not required for HIV-1 infection and that these factors influence replication by a transcriptional mechanism. C/EBPβ recruits coactivators to the HIV-1 long-terminal repeat (LTR) and physically interacts with histone acetyltransferase (HAT) complexes, suggesting that C/EBPs participate in remodeling the chromatin organization of the HIV-1 provirus. Furthermore, overexpression of a C/EBP dominant-negative inhibits displacement of nucleosomes located at the HIV-1 transcriptional start site. These results provide insight into the general mechanisms by which C/EBPs regulate macrophage-restricted HIV-1 transcription. [Copyright &y& Elsevier]

The major barrier to HIV-1 cure is the persistence of latent provirus, which is not eradicated by antiretroviral therapy. The "shock and kill" approach entails stimulating viral production with latency-reversing agents followed by the killing of cells actively producing the virus by immune clearance. However, this approach does not induce all intact proviruses, leaving a residual reservoir. CRISPR/Cas9 has been utilized to excise integrated Human Immunodeficiency Virus (HIV) DNA from infected cells in an RNA-guided, sequence-specific manner. Here, we seek to epigenetically silence the proviral DNA by introducing nuclease-deficient disabled Cas9 (dCas9) coupled with a transcriptional repressor domain derived from Kruppel-associated box (KRAB). We show that specific guide RNAs (gRNAs) and dCas9-KRAB repress HIV-1 transcription and reactivation of latent HIV-1 provirus. This repression is correlated with chromatin changes, including decreased H3 histone acetylation and increased histone H3 lysine 9 trimethylation, histone marks that are associated with transcriptional repression. dCas9-KRAB-mediated inhibition of HIV-1 transcription suggests that CRISPR can be engineered as a tool for block-and-lock strategies. [ABSTRACT FROM AUTHOR]