ER-associated degradation adapter Sel1L is required for CD8+ T cell function and memory formation following acute viral infection.

The maintenance of antigen-specific CD8+ T cells underlies the efficacy of vaccines and immunotherapies. Pathways contributing to CD8+ T cell loss are not completely understood. Uncovering the pathways underlying the limited persistence of CD8+ T cells would be of significant benefit for developing novel strategies of promoting T cell persistence. Here, we demonstrate that murine CD8+ T cells experience endoplasmic reticulum (ER) stress following activation and that the ER-associated degradation (ERAD) adapter Sel1L is induced in activated CD8+ T cells. Sel1L loss limits CD8+ T cell function and memory formation following acute viral infection. Mechanistically, Sel1L is required for optimal bioenergetics and c-Myc expression. Finally, we demonstrate that human CD8+ T cells experience ER stress upon activation and that ER stress is negatively associated with improved T cell functionality in T cell-redirecting therapies. Together, these results demonstrate that ER stress and ERAD are important regulators of T cell function and persistence.

Structure-Activity Relationships of Natural and Semisynthetic Plecomacrolides Suggest Distinct Pathways for HIV-1 Immune Evasion and Vacuolar ATPase-Dependent Lysosomal Acidification.

The human immunodeficiency virus (HIV)-encoded accessory protein Nef enhances pathogenicity by reducing major histocompatibility complex I (MHC-I) cell surface expression, protecting HIV-infected cells from immune recognition. Nef-dependent downmodulation of MHC-I can be reversed by subnanomolar concentrations of concanamycin A (1), a well-known inhibitor of vacuolar ATPase, at concentrations below those that interfere with lysosomal acidification or degradation. We conducted a structure-activity relationship study that assessed 76 compounds for Nef inhibition, 24 and 72 h viability, and lysosomal neutralization in Nef-expressing primary T cells. This analysis demonstrated that the most potent compounds were natural concanamycins and their derivatives. Comparison against a set of new, semisynthetic concanamycins revealed that substituents at C-8 and acylation of C-9 significantly affected Nef potency, target cell viability, and lysosomal neutralization. These findings provide important progress toward understanding the mechanism of action of these compounds and the identification of an advanced lead anti-HIV Nef inhibitory compound.

HIV-1 Vpr combats the PU.1-driven antiviral response in primary human macrophages.

HIV-1 Vpr promotes efficient spread of HIV-1 from macrophages to T cells by transcriptionally downmodulating restriction factors that target HIV-1 Envelope protein (Env). Here we find that Vpr induces broad transcriptomic changes by targeting PU.1, a transcription factor necessary for expression of host innate immune response genes, including those that target Env. Consistent with this, we find silencing PU.1 in infected macrophages lacking Vpr rescues Env. Vpr downmodulates PU.1 through a proteasomal degradation pathway that depends on physical interactions with PU.1 and DCAF1, a component of the Cul4A E3 ubiquitin ligase. The capacity for Vpr to target PU.1 is highly conserved across primate lentiviruses. In addition to impacting infected cells, we find that Vpr suppresses expression of innate immune response genes in uninfected bystander cells, and that virion-associated Vpr can degrade PU.1. Together, we demonstrate Vpr counteracts PU.1 in macrophages to blunt antiviral immune responses and promote viral spread.

Multi-omic single-cell velocity models epigenome-transcriptome interactions and improves cell fate prediction.

Multi-omic single-cell datasets, in which multiple molecular modalities are profiled within the same cell, offer an opportunity to understand the temporal relationship between epigenome and transcriptome. To realize this potential, we developed MultiVelo, a differential equation model of gene expression that extends the RNA velocity framework to incorporate epigenomic data. MultiVelo uses a probabilistic latent variable model to estimate the switch time and rate parameters of chromatin accessibility and gene expression and improves the accuracy of cell fate prediction compared to velocity estimates from RNA only. Application to multi-omic single-cell datasets from brain, skin and blood cells reveals two distinct classes of genes distinguished by whether chromatin closes before or after transcription ceases. We also find four types of cell states: two states in which epigenome and transcriptome are coupled and two distinct decoupled states. Finally, we identify time lags between transcription factor expression and binding site accessibility and between disease-associated SNP accessibility and expression of the linked genes. MultiVelo is available on PyPI, Bioconda and GitHub ( https://github.com/welch-lab/MultiVelo ).

Data we can trust.

"On behalf of all authors of the submission, I warrant that the work is original and scientifically accurate ..." If you've submitted a manuscript to the Journal of Clinical Investigation or JCI Insight, this phrase should sound familiar. This statement is the very first thing that we ask authors to verify for every new submission. While this may seem like a simple formality or just another screen to click through, certifying the accuracy of information presented to the journal is essential to the publishing process and scientific integrity. Data accuracy forms the foundation of the scientific enterprise, and without it, the enterprise risks crumbling.

Hematopoietic Stem and Progenitor Cells (HSPCs).

Cord blood is a readily available source of hematopoietic stem and progenitor cells (HSPCs) which can be infected with HIV-1 in vitro to produce inducible latently infected cells for reactivation studies. Infected HSPCs can also be found in the setting of clinically undetectable viremia in vivo. Here we describe an in vitro infection model utilizing cord blood derived HSPCs, as well as methods for isolating and characterizing provirus from bone marrow HSPCs from suppressed patients.

Concanamycin A counteracts HIV-1 Nef to enhance immune clearance of infected primary cells by cytotoxic T lymphocytes.

Nef is an HIV-encoded accessory protein that enhances pathogenicity by down-regulating major histocompatibility class I (MHC-I) expression to evade killing by cytotoxic T lymphocytes (CTLs). A potent Nef inhibitor that restores MHC-I is needed to promote immune-mediated clearance of HIV-infected cells. We discovered that the plecomacrolide family of natural products restored MHC-I to the surface of Nef-expressing primary cells with variable potency. Concanamycin A (CMA) counteracted Nef at subnanomolar concentrations that did not interfere with lysosomal acidification or degradation and were nontoxic in primary cell cultures. CMA specifically reversed Nef-mediated down-regulation of MHC-I, but not CD4, and cells treated with CMA showed reduced formation of the Nef:MHC-I:AP-1 complex required for MHC-I down-regulation. CMA restored expression of diverse allotypes of MHC-I in Nef-expressing cells and inhibited Nef alleles from divergent clades of HIV and simian immunodeficiency virus, including from primary patient isolates. Lastly, we found that restoration of MHC-I in HIV-infected cells was accompanied by enhanced CTL-mediated clearance of infected cells comparable to genetic deletion of Nef. Thus, we propose CMA as a lead compound for therapeutic inhibition of Nef to enhance immune-mediated clearance of HIV-infected cells.

Vpr Is a VIP: HIV Vpr and Infected Macrophages Promote Viral Pathogenesis.

HIV infects several cell types in the body, including CD4+ T cells and macrophages. Here we review the role of macrophages in HIV infection and describe complex interactions between viral proteins and host defenses in these cells. Macrophages exist in many forms throughout the body, where they play numerous roles in healthy and diseased states. They express pattern-recognition receptors (PRRs) that bind viral, bacterial, fungal, and parasitic pathogens, making them both a key player in innate immunity and a potential target of infection by pathogens, including HIV. Among these PRRs is mannose receptor, a macrophage-specific protein that binds oligosaccharides, restricts HIV replication, and is downregulated by the HIV accessory protein Vpr. Vpr significantly enhances infection in vivo, but the mechanism by which this occurs is controversial. It is well established that Vpr alters the expression of numerous host proteins by using its co-factor DCAF1, a component of the DCAF1-DDB1-CUL4 ubiquitin ligase complex. The host proteins targeted by Vpr and their role in viral replication are described in detail. We also discuss the structure and function of the viral protein Env, which is stabilized by Vpr in macrophages. Overall, this literature review provides an updated understanding of the contributions of macrophages and Vpr to HIV pathogenesis.

Truth and transparency in a time of crisis.

Lessons from history underline the importance of having direct lines of communication to and from public health officials, who must remain free from policital bias in times of crisis.

Changing the editorial process at JCI and JCI Insight in response to the COVID-19 pandemic.

The editors of JCI and JCI Insight are revisiting our editorial processes in light of the strain that the COVID-19 pandemic places on the worldwide scientific community. Here, we discuss adjustments to our decision framework in light of restrictions placed on laboratory working conditions for many of our authors.

Mannose receptor is an HIV restriction factor counteracted by Vpr in macrophages.

HIV-1 Vpr is necessary for maximal HIV infection and spread in macrophages. Evolutionary conservation of Vpr suggests an important yet poorly understood role for macrophages in HIV pathogenesis. Vpr counteracts a previously unknown macrophage-specific restriction factor that targets and reduces the expression of HIV Env. Here, we report that the macrophage mannose receptor (MR), is a restriction factor targeting Env in primary human monocyte-derived macrophages. Vpr acts synergistically with HIV Nef to target distinct stages of the MR biosynthetic pathway and dramatically reduce MR expression. Silencing MR or deleting mannose residues on Env rescues Env expression in HIV-1-infected macrophages lacking Vpr. However, we also show that disrupting interactions between Env and MR reduces initial infection of macrophages by cell-free virus. Together these results reveal a Vpr-Nef-Env axis that hijacks a host mannose-MR response system to facilitate infection while evading MR's normal role, which is to trap and destroy mannose-expressing pathogens.

Human cells have defense mechanisms against viral infection known as restriction factors. These are proteins that break down parts of a virus including its DNA or proteins. To evade these defenses, viruses in turn make proteins that block or break down restriction factors. This battle between human and viral proteins determines which types of cells are infected and how quickly a virus can multiply and spread to new cells. HIV produces a protein called Vpr that counteracts a restriction factor found in immune cells called macrophages. However, the identity of the restriction factor targeted by Vpr is a mystery. When Vpr is missing, this unknown restriction factor breaks down a virus protein called Env. Env is a glycoprotein, which is a protein with sugars attached. When Env levels are low, HIV cannot spread to other cells and multiply. Identifying the restriction factor that breaks down Env may lead to new ways of treating and preventing HIV infections. Now, Lubow et al. reveal that the unknown restriction factor in macrophages is a protein called the mannose receptor. This protein binds and destroys proteins containing mannose, a type of sugar found on bacteria and some viruses. The experiments revealed that the mannose receptor grabs mannose on the HIV protein Env. This causes Env to be broken down and stops HIV from spreading. Lubow et al. also find that Vpr works with another protein produced by HIV called Nef to reduce the number of mannose receptors on macrophages. The two proteins do this by targeting different steps in the assembly of mannose receptors, allowing the virus to multiply and spread more efficiently. The experiments suggest that drugs that simultaneously block Vpr and Nef might prevent or suppress HIV infections. More studies are needed to develop and test potential HIV-treatments targeting Vpr and Nef.

The Impact of Cellular Proliferation on the HIV-1 Reservoir.

Human immunodeficiency virus (HIV) is a chronic infection that destroys the immune system in infected individuals. Although antiretroviral therapy is effective at preventing infection of new cells, it is not curative. The inability to clear infection is due to the presence of a rare, but long-lasting latent cellular reservoir. These cells harboring silent integrated proviral genomes have the potential to become activated at any moment, making therapy necessary for life. Latently-infected cells can also proliferate and expand the viral reservoir through several methods including homeostatic proliferation and differentiation. The chromosomal location of HIV proviruses within cells influences the survival and proliferative potential of host cells. Proliferating, latently-infected cells can harbor proviruses that are both replication-competent and defective. Replication-competent proviral genomes contribute to viral rebound in an infected individual. The majority of available techniques can only assess the integration site or the proviral genome, but not both, preventing reliable evaluation of HIV reservoirs.

Hematopoietic Stem and Progenitor Cells Are a Distinct HIV Reservoir that Contributes to Persistent Viremia in Suppressed Patients.

Long-lived reservoirs of persistent HIV are a major barrier to a cure. CD4+ hematopoietic stem and progenitor cells (HSPCs) have the capacity for lifelong survival, self-renewal, and the generation of daughter cells. Recent evidence shows that they are also susceptible to HIV infection in vitro and in vivo. Whether HSPCs harbor infectious virus or contribute to plasma virus (PV) is unknown. Here, we provide strong evidence that clusters of identical proviruses from HSPCs and their likely progeny often match residual PV. A higher proportion of these sequences match residual PV than proviral genomes from bone marrow and peripheral blood mononuclear cells that are observed only once. Furthermore, an analysis of near-full-length genomes isolated from HSPCs provides evidence that HSPCs harbor functional HIV proviral genomes that often match residual PV. These results support the conclusion that HIV-infected HSPCs form a distinct and functionally significant reservoir of persistent HIV in infected people.

The National Health and Nutrition Examination Survey's Food Insecurity Questionnaire Completed by Children: Effects of Assessment Mode (Classroom versus Interview).

The National Health and Nutrition Examination Survey's food insecurity questionnaire was administered to 155 children (77 African American, 65 White, 13 "Other" [7 Hispanic; 6 mixed races]) in grade 4 twice, 28-32 days apart. Test-retest reliabilities were modest and somewhat similar for assessment mode (classroom, interview) and subgroup variables (gender, race, socioeconomic status, academic achievement, body mass index percentile, social desirability). As academic achievement increased, White and Other children reported less food insecurity, and African-American children reported slightly less. As social desirability increased, White and African-American children reported slightly more food insecurity, and Other children reported substantially more. Although the questionnaire may be acceptable for use with diverse groups of children in grade 4, validation is needed.

Class 1-Selective Histone Deacetylase (HDAC) Inhibitors Enhance HIV Latency Reversal while Preserving the Activity of HDAC Isoforms Necessary for Maximal HIV Gene Expression.

Combinations of drugs that affect distinct mechanisms of HIV latency aim to induce robust latency reversal leading to cytopathicity and elimination of the persistent HIV reservoir. Thus far, attempts have focused on combinations of protein kinase C (PKC) agonists and pan-histone deacetylase inhibitors (HDIs) despite the knowledge that HIV gene expression is regulated by class 1 histone deacetylases. We hypothesized that class 1-selective HDIs would promote more robust HIV latency reversal in combination with a PKC agonist than pan-HDIs because they preserve the activity of proviral factors regulated by non-class 1 histone deacetylases. Here, we show that class 1-selective agents used alone or with the PKC agonist bryostatin-1 induced more HIV protein expression per infected cell. In addition, the combination of entinostat and bryostatin-1 induced viral outgrowth, whereas bryostatin-1 combinations with pan-HDIs did not. When class 1-selective HDIs were used in combination with pan-HDIs, the amount of viral protein expression and virus outgrowth resembled that of pan-HDIs alone, suggesting that pan-HDIs inhibit robust gene expression induced by class 1-selective HDIs. Consistent with this, pan-HDI-containing combinations reduced the activity of NF-κB and Hsp90, two cellular factors necessary for potent HIV protein expression, but did not significantly reduce overall cell viability. An assessment of viral clearance from in vitro cultures indicated that maximal protein expression induced by class 1-selective HDI treatment was crucial for reservoir clearance. These findings elucidate the limitations of current approaches and provide a path toward more effective strategies to eliminate the HIV reservoir.IMPORTANCE Despite effective antiretroviral therapy, HIV evades eradication in a latent form that is not affected by currently available drug regimens. Pharmacologic latency reversal that leads to death of cellular reservoirs has been proposed as a strategy for reservoir elimination. Because histone deacetylases (HDACs) promote HIV latency, HDAC inhibitors have been a focus of HIV cure research. However, many of these inhibitors broadly affect multiple classes of HDACs, including those that promote HIV gene expression (class 1 HDACs). Here, we demonstrate that targeted treatment with class 1-selective HDAC inhibitors induced more potent HIV latency reversal than broadly acting agents. Additionally, we provide evidence that broadly acting HDIs are limited by inhibitory effects on non-class 1 HDACs that support the activity of proviral factors. Thus, our work demonstrates that the use of targeted approaches to induce maximum latency reversal affords the greatest likelihood of reservoir elimination.