The antiviral factor APOBEC3G enhances the recognition of HIV-infected primary T cells by natural killer cells.

APOBEC3G (A3G) is an intrinsic antiviral factor that inhibits the replication of human immunodeficiency virus (HIV) by deaminating cytidine residues to uridine. This causes guanosine-to-adenosine hypermutation in the opposite strand and results in inactivation of the virus. HIV counteracts A3G through the activity of viral infectivity factor (Vif), which promotes degradation of A3G. We report that viral protein R (Vpr), which interacts with a uracil glycosylase, also counteracted A3G by diminishing the incorporation of uridine. However, this process resulted in activation of the DNA-damage-response pathway and the expression of natural killer (NK) cell-activating ligands. Our results show that pathogen-induced deamination of cytidine and the DNA-damage response to virus-mediated repair of the incorporation of uridine enhance the recognition of HIV-infected cells by NK cells.

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.

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.

CD4 is expressed on a heterogeneous subset of hematopoietic progenitors, which persistently harbor CXCR4 and CCR5-tropic HIV proviral genomes in vivo.

Latent HIV infection of long-lived cells is a barrier to viral clearance. Hematopoietic stem and progenitor cells are a heterogeneous population of cells, some of which are long-lived. CXCR4-tropic HIVs infect a broad range of HSPC subtypes, including hematopoietic stem cells, which are multi-potent and long-lived. However, CCR5-tropic HIV infection is limited to more differentiated progenitor cells with life spans that are less well understood. Consistent with emerging data that restricted progenitor cells can be long-lived, we detected persistent HIV in restricted HSPC populations from optimally treated people. Further, genotypic and phenotypic analysis of amplified env alleles from donor samples indicated that both CXCR4- and CCR5-tropic viruses persisted in HSPCs. RNA profiling confirmed expression of HIV receptor RNA in a pattern that was consistent with in vitro and in vivo results. In addition, we characterized a CD4high HSPC sub-population that was preferentially targeted by a variety of CXCR4- and CCR5-tropic HIVs in vitro. Finally, we present strong evidence that HIV proviral genomes of both tropisms can be transmitted to CD4-negative daughter cells of multiple lineages in vivo. In some cases, the transmitted proviral genomes contained signature deletions that inactivated the virus, eliminating the possibility that coincidental infection explains the results. These data support a model in which both stem and non-stem cell progenitors serve as persistent reservoirs for CXCR4- and CCR5-tropic HIV proviral genomes that can be passed to daughter cells.

Quiescence Promotes Latent HIV Infection and Resistance to Reactivation from Latency with Histone Deacetylase Inhibitors.

Human immunodeficiency virus type 1 (HIV-1) establishes transcriptionally silent latent infections in resting memory T cells and hematopoietic stem and progenitor cells (HSPCs), which allows the virus to persist in infected individuals despite antiretroviral therapy. Developing in vitro models of HIV-1 latency that recapitulate the characteristics of latently infected cells in vivo is crucial to identifying and developing effective latency-reversing therapies. HSPCs exist in a quiescent state in vivo, and quiescence is correlated with latent infections in T cells. However, current models for culturing HSPCs and for infecting T cells in vitro require that the cells be maintained in an actively proliferating state. Here we describe a novel culture system in which primary human HSPCs cultured under hypothermic conditions are maintained in a quiescent state. We show that these quiescent HSPCs are susceptible to predominantly latent infection with HIV-1, while actively proliferating and differentiating HSPCs obtain predominantly active infections. Furthermore, we demonstrate that the most primitive quiescent HSPCs are more resistant to spontaneous reactivation from latency than more differentiated HSPCs and that quiescent HSPCs are resistant to reactivation by histone deacetylase inhibitors or P-TEFb activation but are susceptible to reactivation by protein kinase C (PKC) agonists. We also demonstrate that inhibition of HSP90, a known regulator of HIV transcription, recapitulates the quiescence and latency phenotypes of hypothermia, suggesting that hypothermia and HSP90 inhibition may regulate these processes by similar mechanisms. In summary, these studies describe a novel model for studying HIV-1 latency in human primary cells maintained in a quiescent state.IMPORTANCE Human immunodeficiency virus type 1 (HIV-1) establishes a persistent infection for which there remains no feasible cure. Current approaches are unable to clear the virus despite decades of therapy due to the existence of latent reservoirs of integrated HIV-1, which can reactivate and contribute to viral rebound following treatment interruption. Previous clinical attempts to reactivate the latent reservoirs in an individual so that they can be eliminated by the immune response or viral cytopathic effect have failed, indicating the need for a better understanding of the processes regulating HIV-1 latency. Here we characterize a novel in vitro model of HIV-1 latency in primary hematopoietic stem and progenitor cells isolated from human cord blood that may better recapitulate the behavior of latently infected cells in vivo This model can be used to study mechanisms regulating latency and potential therapeutic approaches to reactivate latent infections in quiescent cells.

Vpr Promotes Macrophage-Dependent HIV-1 Infection of CD4+ T Lymphocytes.

Vpr is a conserved primate lentiviral protein that promotes infection of T lymphocytes in vivo by an unknown mechanism. Here we demonstrate that Vpr and its cellular co-factor, DCAF1, are necessary for efficient cell-to-cell spread of HIV-1 from macrophages to CD4+ T lymphocytes when there is inadequate cell-free virus to support direct T lymphocyte infection. Remarkably, Vpr functioned to counteract a macrophage-specific intrinsic antiviral pathway that targeted Env-containing virions to LAMP1+ lysosomal compartments. This restriction of Env also impaired virological synapses formed through interactions between HIV-1 Env on infected macrophages and CD4 on T lymphocytes. Treatment of infected macrophages with exogenous interferon-alpha induced virion degradation and blocked synapse formation, overcoming the effects of Vpr. These results provide a mechanism that helps explain the in vivo requirement for Vpr and suggests that a macrophage-dependent stage of HIV-1 infection drives the evolutionary conservation of Vpr.

Children's school-breakfast reports and school-lunch reports (in 24-h dietary recalls): conventional and reporting-error-sensitive measures show inconsistent accuracy results for retention interval and breakfast location.

Validation-study data were analysed to investigate retention interval (RI) and prompt effects on the accuracy of fourth-grade children's reports of school-breakfast and school-lunch (in 24-h recalls), and the accuracy of school-breakfast reports by breakfast location (classroom; cafeteria). Randomly selected fourth-grade children at ten schools in four districts were observed eating school-provided breakfast and lunch, and were interviewed under one of eight conditions created by crossing two RIs ('short'--prior-24-hour recall obtained in the afternoon and 'long'--previous-day recall obtained in the morning) with four prompts ('forward'--distant to recent, 'meal name'--breakfast, etc., 'open'--no instructions, and 'reverse'--recent to distant). Each condition had sixty children (half were girls). Of 480 children, 355 and 409 reported meals satisfying criteria for reports of school-breakfast and school-lunch, respectively. For breakfast and lunch separately, a conventional measure--report rate--and reporting-error-sensitive measures--correspondence rate and inflation ratio--were calculated for energy per meal-reporting child. Correspondence rate and inflation ratio--but not report rate--showed better accuracy for school-breakfast and school-lunch reports with the short RI than with the long RI; this pattern was not found for some prompts for each sex. Correspondence rate and inflation ratio showed better school-breakfast report accuracy for the classroom than for cafeteria location for each prompt, but report rate showed the opposite. For each RI, correspondence rate and inflation ratio showed better accuracy for lunch than for breakfast, but report rate showed the opposite. When choosing RI and prompts for recalls, researchers and practitioners should select a short RI to maximise accuracy. Recommendations for prompt selections are less clear. As report rates distort validation-study accuracy conclusions, reporting-error-sensitive measures are recommended.

Epigenetic silencing of engineered L1 retrotransposition events in human embryonic carcinoma cells.

Long interspersed element-1 (LINE-1 or L1) retrotransposition continues to affect human genome evolution. L1s can retrotranspose in the germline, during early development and in select somatic cells; however, the host response to L1 retrotransposition remains largely unexplored. Here we show that reporter genes introduced into the genome of various human embryonic carcinoma-derived cell lines (ECs) by L1 retrotransposition are rapidly and efficiently silenced either during or immediately after their integration. Treating ECs with histone deacetylase inhibitors rapidly reverses this silencing, and chromatin immunoprecipitation experiments revealed that reactivation of the reporter gene was correlated with changes in chromatin status at the L1 integration site. Under our assay conditions, rapid silencing was also observed when reporter genes were delivered into ECs by mouse L1s and a zebrafish LINE-2 element, but not when similar reporter genes were delivered into ECs by Moloney murine leukaemia virus or human immunodeficiency virus, suggesting that these integration events are silenced by distinct mechanisms. Finally, we demonstrate that subjecting ECs to culture conditions that promote differentiation attenuates the silencing of reporter genes delivered by L1 retrotransposition, but that differentiation, in itself, is not sufficient to reactivate previously silenced reporter genes. Thus, our data indicate that ECs differ from many differentiated cells in their ability to silence reporter genes delivered by L1 retrotransposition.

Effectiveness of Prompts on Fourth-Grade Children's Dietary Recall Accuracy Depends on Retention Interval and Varies by Gender.

BACKGROUND: Dietary recall accuracy is related to retention interval (RI) (i.e., time between to-be-reported meals and the interview), and possibly to prompts. To the best of our knowledge, no study has evaluated their combined effect. OBJECTIVE: The combined influence of RI and prompts on children's recall accuracy was investigated in this study. Two RIs [short (prior-24-h recall obtained in afternoon) and long (previous-day recall obtained in morning)] were crossed with 4 prompts [forward (distant-to-recent), meal-name (breakfast, lunch, etc.), open (no instructions), and reverse (recent-to-distant)], creating 8 conditions. METHODS: Fourth-grade children (n = 480; 50% girls) were randomly selected from consenting children at 10 schools in 4 districts in a southern state during 3 school years (2011-2012, 2012-2013, and 2013-2014). Each child was observed eating school-provided breakfast and lunch, and interviewed one time under 1 of the 8 conditions. Condition assignment was constrained so that each had 60 children (30 girls). Accuracy measures were food-item omission and intrusion rates, and energy correspondence rate and inflation ratio. For each measure, linear models determined effects of RI, prompt, gender, and interactions (2-way, 3-way); race/ethnicity, school year, and district were control variables. RESULTS: RI (P values < 0.015) and prompt (P values < 0.005) were significant for all 4 accuracy measures. RI × prompt (P values < 0.001) was significant for 3 accuracy measures (not intrusion rate). Prompt × gender (P = 0.005) was significant for omission rate. RI × prompt × gender was significant for intrusion rate and inflation ratio (P values < 0.001). For the short vs. long RI across prompts and genders, accuracy was better by 33-50% for each accuracy measure. CONCLUSIONS: To obtain the most accurate recalls possible from children, studies should be designed to use a short rather than long RI. Prompts affect children's recall accuracy, although the effectiveness of different prompts depends on RI and varies by gender: at a short RI, the choice of prompts has little systematic effect on accuracy, whereas at a long RI, reverse prompts may elicit the most accurate recalls.

Children's Social Desirability: Effects of Test Assessment Mode.

This study examined a recently developed short version of the Children's Social Desirability (CSD-S) scale with 157 fourth-grade children. Of interest was a) whether one-month test-retest reliability would vary as a function of test assessment mode (interview or classroom), gender, race, SES, and BMI percentile, and b) whether the degree of social desirability would vary as a function of these same variables. The CSD-S scale showed good test-retest reliability for both interview and classroom assessment modes (.85 and .83, respectively). Internal consistency also was good (first interview administration = .84; first classroom administration = .81). Reliability was good and did not vary significantly over assessment mode or any child subgroup variables, suggesting that the CSD-S scale is appropriate for general use. The interview mode elicited significantly more socially desirable answers than did the classroom mode. Social desirability did not differ across child subgroups. Some of these findings were examined, and replicated, on another sample. Thus, the CSD-S scale may be used with diverse groups of children to a) reliably assess a social desirability bias that may systematically bias other self-reports of interest to researchers and b) examine individual differences in degree of social desirability.

Adaptor protein 1 promotes cross-presentation through the same tyrosine signal in major histocompatibility complex class I as that targeted by HIV-1.

Certain antigen-presenting cells (APCs) process and present extracellular antigen with major histocompatibility complex class I (MHC-I) molecules to activate naive CD8(+) T cells in a process termed cross-presentation. We used insights gained from HIV immune evasion strategies to demonstrate that the clathrin adaptor protein adaptor protein 1 (AP-1) is necessary for cross-presentation by MHC-I molecules containing a cytoplasmic tail tyrosine signal (murine MHC-I molecules, human MHC-I HLA-A and HLA-B allotypes). In contrast, AP-1 activity was not needed for cross-presentation by MHC-I molecules containing a human MHC-I HLA-C cytoplasmic tail, which does not contain a tyrosine signal. AP-1 activity was also dispensable for presentation of endogenous antigens by MHC-I via the classical pathway. In APCs, we show that HIV Nef disrupts cross-presentation by MHC-I containing the tyrosine signal but does not affect cross-presentation by MHC-I containing the HLA-C cytoplasmic tail. Thus, we provide evidence for two separable cross-presentation pathways, only one of which is targeted by HIV.

CD133+ hematopoietic progenitor cells harbor HIV genomes in a subset of optimally treated people with long-term viral suppression.

BACKGROUND: Hematopoietic progenitor cells (HPCs) in the bone marrow of human immunodeficiency virus (HIV)-infected individuals have been proposed as a persistent reservoir of virus. However, some studies have suggested that HIV genomes detected in HPCs arise from T-cell contamination. METHODS: CD133-sorted HPCs and CD133-depleted bone marrow cells were purified from bone marrow specimens obtained from 11 antiretroviral-treated donors in whom the HIV load had been <48 copies/mL for at least 6 months. CD133 and CD3 expression on the cells was assessed by flow cytometry. HIV DNA was quantified by real-time polymerase chain reaction analysis. RESULTS: HIV genomes were detected in CD133-sorted samples from 6 donors, including 2 in whom viral loads were undetectable for >8 years. CD3(+) T cells represented <1% of cells in all CD133-sorted samples. For 5 of 6 CD133-sorted samples with detectable HIV DNA, the HIV genomes could not be explained by contaminating CD3(+) T cells. Donors with detectable HIV DNA in HPCs received their diagnosis significantly more recently than the remaining donors but had had undetectable viral loads for similar periods. CONCLUSIONS: HIV genomes can be detected in CD133-sorted cells from a subset of donors with long-term viral suppression and, in most cases, cannot be explained by contamination with CD3(+) T cells.

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.

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.

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.

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.

Latent HIV-1 infection occurs in multiple subsets of hematopoietic progenitor cells and is reversed by NF-κB activation.

The ability of HIV-1 to establish a latent infection presents a barrier to curing HIV. The best-studied reservoir of latent virus in vivo is resting memory CD4(+) T cells, but it has recently been shown that CD34(+) hematopoietic progenitor cells (HPCs) can also become latently infected by HIV-1 in vitro and in vivo. CD34(+) cells are not homogenous, however, and it is not yet known which types of CD34(+) cells support a latent infection. Furthermore, the mechanisms through which latency is established in this cell type are not yet known. Here we report the development of a primary cell model for latent HIV-1 infection in HPCs. We demonstrate that in this model, latent infection can be established in all subsets of HPCs examined, including HPCs with cell surface markers consistent with immature hematopoietic stem and progenitor cells. We further show that the establishment of latent infection in these cells can be reversed by tumor necrosis factor alpha (TNF-α) through an NF-κB-dependent mechanism. In contrast, we do not find evidence for a role of positive transcription elongation factor b (P-TEFb) in the establishment of latent infection in HPCs. Finally, we demonstrate that prostratin and suberoylanilide hydroxamic acid (SAHA), but not hexamethylene bisacetamide (HMBA) or 5-aza-2'-deoxycytidine (Aza-CdR), reactivate latent HIV-1 in HPCs. These findings illuminate the mechanisms through which latent infection can be established in HPCs and suggest common pathways through which latent virus could be reactivated in both HPCs and resting memory T cells to eliminate latent reservoirs of HIV-1.

Down-modulation of CD8αß is a fundamental activity of primate lentiviral Nef proteins.

It is well established that the Nef proteins of human and simian immunodeficiency viruses (HIV and SIV) modulate major histocompatibility complex class I (MHC-I) cell surface expression to protect infected cells against lysis by cytotoxic T lymphocytes (CTLs). Recent data supported the observation that Nef also manipulates CTLs directly by down-modulating CD8αß (J. A. Leonard, T. Filzen, C. C. Carter, M. Schaefer, and K. L. Collins, J. Virol. 85:6867-6881, 2011), but it remained unknown whether this Nef activity is conserved between different lineages of HIV and SIV. In this study, we examined a total of 42 nef alleles from 16 different primate lentiviruses representing most major lineages of primate lentiviruses, as well as nonpandemic HIV-1 strains and the direct precursors of HIV-1 (SIVcpz and SIVgor). We found that the vast majority of these nef alleles strongly down-modulate CD8ß in human T cells. Primate lentiviral Nefs generally interacted specifically with the cytoplasmic tail of CD8ß, and down-modulation of this receptor was dependent on the conserved dileucine-based motif and two adjacent acidic residues (DD/E) in the C-terminal flexible loop of SIV Nef proteins. Both of these motifs are known to be important for the interaction of HIV-1 Nef with AP-2, and they were also shown to be critical for down-modulation of CD4 and CD28, but not MHC-I, by SIV Nefs. Our results show that down-modulation of CD4, CD8ß, and CD28 involves largely overlapping (but not identical) domains and is most likely dependent on conserved interactions of primate lentiviral Nefs with cellular adaptor proteins. Furthermore, our data demonstrate that Nef-mediated down-modulation of CD8αß is a fundamental property of primate lentiviruses and suggest that direct manipulation of CD8+ T cells plays a relevant role in viral immune evasion.

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 ).

HIV-1 Nef disrupts intracellular trafficking of major histocompatibility complex class I, CD4, CD8, and CD28 by distinct pathways that share common elements.

The Nef protein is an important HIV virulence factor that promotes the degradation of host proteins to augment virus production and facilitate immune evasion. The best-characterized targets of Nef are major histocompatibility complex class I (MHC-I) and CD4, but Nef also has been reported to target several other proteins, including CD8ß, CD28, CD80, CD86, and CD1d. To compare and contrast the effects of Nef on each protein, we constructed a panel of chimeric proteins in which the extracellular and transmembrane regions of the MHC-I allele HLA-A2 were fused to the cytoplasmic tails of CD4, CD28, CD8ß, CD80, CD86, and CD1d. We found that Nef coprecipitated with and disrupted the expression of molecules with cytoplasmic tails from MHC-I HLA-A2, CD4, CD8ß, and CD28, but Nef did not bind to or alter the expression of molecules with cytoplasmic tails from CD80, CD86, and CD1d. In addition, we used short interfering RNA (siRNA) knockdown and coprecipitation experiments to implicate AP-1 as a cellular cofactor for Nef in the downmodulation of both CD28 and CD8ß. The interaction with AP-1 required for CD28 and CD8ß differed from the AP-1 interaction required for MHC-I downmodulation in that it was mediated through the dileucine motif within Nef (LL(164,165)AA) and did not require the tyrosine binding pocket of the AP-1 µ subunit. In addition, we demonstrate a requirement for ß-COP as a cellular cofactor for Nef that was necessary for the degradation of targeted molecules HLA-A2, CD4, and CD8. These studies provide important new information on the similarities and differences with which Nef affects intracellular trafficking and help focus future research on the best potential pharmaceutical targets.