ABSTRACT
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ABSTRACT
Single-cell omics provide insight into cellular heterogeneity and function. Recent technological advances have accelerated single-cell analyses, but workflows remain expensive and complex. We present a method enabling simultaneous, ultra-high throughput single-cell barcoding of millions of cells for targeted analysis of proteins and RNAs. Quantum barcoding (QBC) avoids isolation of single cells by building cell-specific oligo barcodes dynamically within each cell. With minimal instrumentation (four 96-well plates and a multichannel pipette), cell-specific codes are added to each tagged molecule within cells through sequential rounds of classical split-pool synthesis. Here we show the utility of this technology in mouse and human model systems for as many as 50 antibodies to targeted proteins and, separately, >70 targeted RNA regions. We demonstrate that this method can be applied to multi-modal protein and RNA analyses. It can be scaled by expansion of the split-pool process and effectively renders sequencing instruments as versatile multi-parameter flow cytometers.
Subject(s)
Antibodies/analysis , High-Throughput Nucleotide Sequencing/methods , Proteins/analysis , RNA/analysis , Single-Cell Analysis/methods , Animals , Humans , Mice , Mice, Inbred C57BLABSTRACT
To identify critical host factors necessary for human immunodeficiency virus 1 (HIV-1) replication, large libraries of short-peptide-aptamers were expressed retrovirally. The target of one inhibitor peptide, Pep80, identified in this screen was determined to be Snapin, a protein associated with the soluble N-ethyl maleimide sensitive factor adaptor protein receptor (SNARE) complex that is critical for calcium-dependent exocytosis during neurotransmission. Pep80 inhibited Ca²âº release from intracellular stores and blocked downstream signaling by direct interruption of the association between Snapin and an intracellular calcium release channel, the ryanodine receptor (RyR). NFAT signaling was preferentially abolished by Pep80. Expression of Snapin overcame Pep80-mediated inhibition of Ca²âº/NFAT signaling and HIV-1 replication. Furthermore, Snapin induced HIV-1 replication in primary CD4⺠T cells. Thus, through its interaction with RyR, Snapin is a critical regulator of Ca²âº signaling and T cell activation. Use of the genetically selected intracellular aptamer inhibitors allowed us to define unique mechanisms important to HIV-1 replication and T cell biology.
Subject(s)
Calcium/metabolism , HIV-1/physiology , Ryanodine Receptor Calcium Release Channel/metabolism , T-Lymphocytes/metabolism , T-Lymphocytes/virology , Vesicular Transport Proteins/metabolism , Cell Line , Flow Cytometry , Humans , Immunoblotting , Immunoprecipitation , Jurkat Cells , Microscopy, Confocal , Protein Binding , Ryanodine Receptor Calcium Release Channel/genetics , Two-Hybrid System Techniques , Vesicular Transport Proteins/geneticsABSTRACT
To determine critical host factors involved in HIV-1 replication, a dominant effector genetics approach was developed to reveal signaling pathways on which HIV-1 depends for replication. A large library of short peptide aptamers was expressed via retroviral delivery in T cells. Peptides that interfered with T cell activation-dependent processes that might support HIV-1 replication were identified. One of the selected peptides altered signaling, lead to a difference in T cell activation status, and inhibited HIV-1 replication. The target of the peptide was JAB1/CSN5, a component of the signalosome complex. JAB1 expression overcame the inhibition of HIV-1 replication in the presence of peptide and also promoted HIV-1 replication in activated primary CD4(+) T cells. This peptide blocked physiological release of JAB1 from the accessory T cell surface protein LFA-1, downstream AP-1 dependent events, NFAT activation, and HIV-1 replication. Thus, genetic selection for intracellular aptamer inhibitors of host cell processes proximal to signals at the immunological synapse of T cells can define unique mechanisms important to HIV-1 replication.
Subject(s)
HIV-1/physiology , Intracellular Signaling Peptides and Proteins/metabolism , Lymphocyte Function-Associated Antigen-1/metabolism , Multiprotein Complexes/metabolism , Peptide Hydrolases/metabolism , Signal Transduction , T-Lymphocytes/virology , Virus Replication/physiology , Amino Acid Sequence , Aptamers, Peptide/chemistry , Aptamers, Peptide/pharmacology , CD4-Positive T-Lymphocytes/drug effects , CD4-Positive T-Lymphocytes/enzymology , CD4-Positive T-Lymphocytes/pathology , CD4-Positive T-Lymphocytes/virology , COP9 Signalosome Complex , HIV-1/drug effects , HIV-1/genetics , Humans , Intracellular Space/drug effects , Intracellular Space/metabolism , JNK Mitogen-Activated Protein Kinases/metabolism , Jurkat Cells , Lymphocyte Activation/drug effects , Models, Immunological , Molecular Sequence Data , NFATC Transcription Factors/metabolism , Peptide Library , Protein Binding/drug effects , Protein Transport/drug effects , Retroviridae/genetics , Signal Transduction/drug effects , T-Lymphocytes/drug effects , T-Lymphocytes/enzymology , T-Lymphocytes/pathology , Transcription Factor AP-1/metabolism , Transcription, Genetic/drug effects , Virus Replication/drug effectsABSTRACT
T cell activation is crucial for the productive HIV-1 infection of primary T cells; however, little is known about the host molecules involved in this process. We show that the host transcription factor NF-IL6 (also called C/EBPbeta) renders primary CD4(+) T cells highly permissive for HIV-1 replication. NF-IL6 facilitates reverse transcription of the virus by binding to and inhibiting the antiviral cytidine deaminase APOBEC3G. A mutation in NF-IL6 at Ser-288 weakened its binding to APOBEC3G and strongly inhibited HIV-1 replication. NF-IL6 also induced the replication of a Vif-deficient strain of HIV-1 in nonpermissive HUT78 cells. These data indicate that NF-IL6 is a natural inhibitor of APOBEC3G that facilitates HIV-1 replication. Host factors, such as NF-IL6, that are involved in early HIV-1 replication are potential targets for anti-HIV-1 therapy. Our findings shed light on the activation of HIV-1 replication by T cell host molecules and reveal a unique regulation of DNA deamination by APOBEC3G and NF-IL6.
