ABSTRACT
3' untranslated region (3'UTR) variants are strongly associated with human traits and diseases, yet few have been causally identified. We developed the massively parallel reporter assay for 3'UTRs (MPRAu) to sensitively assay 12,173 3'UTR variants. We applied MPRAu to six human cell lines, focusing on genetic variants associated with genome-wide association studies (GWAS) and human evolutionary adaptation. MPRAu expands our understanding of 3'UTR function, suggesting that simple sequences predominately explain 3'UTR regulatory activity. We adapt MPRAu to uncover diverse molecular mechanisms at base pair resolution, including an adenylate-uridylate (AU)-rich element of LEPR linked to potential metabolic evolutionary adaptations in East Asians. We nominate hundreds of 3'UTR causal variants with genetically fine-mapped phenotype associations. Using endogenous allelic replacements, we characterize one variant that disrupts a miRNA site regulating the viral defense gene TRIM14 and one that alters PILRB abundance, nominating a causal variant underlying transcriptional changes in age-related macular degeneration.
Subject(s)
3' Untranslated Regions/genetics , Biological Evolution , Disease/genetics , Genome-Wide Association Study , Algorithms , Alleles , Gene Expression Regulation , Genes, Reporter , Genetic Variation , Humans , Phenotype , Polymorphism, Single Nucleotide/genetics , Polyribosomes/metabolism , Quantitative Trait Loci/genetics , RNA/geneticsABSTRACT
The SARS-CoV-2 spike (S) protein variant D614G supplanted the ancestral virus worldwide, reaching near fixation in a matter of months. Here we show that D614G was more infectious than the ancestral form on human lung cells, colon cells, and on cells rendered permissive by ectopic expression of human ACE2 or of ACE2 orthologs from various mammals, including Chinese rufous horseshoe bat and Malayan pangolin. D614G did not alter S protein synthesis, processing, or incorporation into SARS-CoV-2 particles, but D614G affinity for ACE2 was reduced due to a faster dissociation rate. Assessment of the S protein trimer by cryo-electron microscopy showed that D614G disrupts an interprotomer contact and that the conformation is shifted toward an ACE2 binding-competent state, which is modeled to be on pathway for virion membrane fusion with target cells. Consistent with this more open conformation, neutralization potency of antibodies targeting the S protein receptor-binding domain was not attenuated.
Subject(s)
Betacoronavirus/physiology , Betacoronavirus/ultrastructure , Spike Glycoprotein, Coronavirus/physiology , Spike Glycoprotein, Coronavirus/ultrastructure , Angiotensin-Converting Enzyme 2 , Animals , Antibodies, Monoclonal/immunology , Antibodies, Viral/immunology , Betacoronavirus/pathogenicity , COVID-19 , Cells, Cultured , Coronavirus Infections/virology , Female , Genetic Variation , HEK293 Cells , Humans , Male , Models, Molecular , Pandemics , Peptidyl-Dipeptidase A/metabolism , Pneumonia, Viral/virology , Protein Conformation , Protein Processing, Post-Translational , Receptors, Coronavirus , Receptors, Virus/metabolism , SARS-CoV-2 , Species SpecificityABSTRACT
Antisense Piwi-interacting RNAs (piRNAs) guide silencing of established transposons during germline development, and sense piRNAs drive ping-pong amplification of the antisense pool, but how the germline responds to genome invasion is not understood. The KoRV-A gammaretrovirus infects the soma and germline and is sweeping through wild koalas by a combination of horizontal and vertical transfer, allowing direct analysis of retroviral invasion of the germline genome. Gammaretroviruses produce spliced Env mRNAs and unspliced transcripts encoding Gag, Pol, and the viral genome, but KoRV-A piRNAs are almost exclusively derived from unspliced genomic transcripts and are strongly sense-strand biased. Significantly, selective piRNA processing of unspliced proviral transcripts is conserved from insects to placental mammals. We speculate that bypassed splicing generates a conserved molecular pattern that directs proviral genomic transcripts to the piRNA biogenesis machinery and that this "innate" piRNA response suppresses transposition until antisense piRNAs are produced, establishing sequence-specific adaptive immunity.
Subject(s)
Gammaretrovirus/genetics , Phascolarctidae/genetics , RNA, Small Interfering/genetics , Animals , DNA Transposable Elements , Gammaretrovirus/metabolism , Gammaretrovirus/pathogenicity , Gene Products, env/genetics , Gene Products, env/metabolism , Gene Products, gag/genetics , Gene Products, gag/metabolism , Gene Products, pol/genetics , Gene Products, pol/metabolism , Genome , Germ Cells/metabolism , Germ Cells/virology , Male , Mice , Mice, Inbred C57BL , Phascolarctidae/virology , RNA Splicing , RNA, Antisense/genetics , RNA, Antisense/metabolism , RNA, Small Interfering/metabolismABSTRACT
Human immunodeficiency virus 1 (HIV-1) infection is associated with heightened inflammation and excess risk of cardiovascular disease, cancer and other complications. These pathologies persist despite antiretroviral therapy. In two independent cohorts, we found that innate lymphoid cells (ILCs) were depleted in the blood and gut of people with HIV-1, even with effective antiretroviral therapy. ILC depletion was associated with neutrophil infiltration of the gut lamina propria, type 1 interferon activation, increased microbial translocation and natural killer (NK) cell skewing towards an inflammatory state, with chromatin structure and phenotype typical of WNT transcription factor TCF7-dependent memory T cells. Cytokines that are elevated during acute HIV-1 infection reproduced the ILC and NK cell abnormalities ex vivo. These results show that inflammatory cytokines associated with HIV-1 infection irreversibly disrupt ILCs. This results in loss of gut epithelial integrity, microbial translocation and memory NK cells with heightened inflammatory potential, and explains the chronic inflammation in people with HIV-1.
Subject(s)
Cytokines/blood , HIV-1/immunology , HIV-1/pathogenicity , Immunity, Innate , Killer Cells, Natural/immunology , Lymphocytes/immunology , T Cell Transcription Factor 1/immunology , Gene Expression Regulation , HIV Infections/genetics , HIV Infections/immunology , HIV Infections/virology , Homeostasis/immunology , Humans , Immunologic Memory , In Vitro Techniques , Inflammation/genetics , Inflammation/immunology , Inflammation/virology , T Cell Transcription Factor 1/genetics , Wnt Signaling Pathway/immunologyABSTRACT
The magnitude of the 2013-2016 Ebola virus disease (EVD) epidemic enabled an unprecedented number of viral mutations to occur over successive human-to-human transmission events, increasing the probability that adaptation to the human host occurred during the outbreak. We investigated one nonsynonymous mutation, Ebola virus (EBOV) glycoprotein (GP) mutant A82V, for its effect on viral infectivity. This mutation, located at the NPC1-binding site on EBOV GP, occurred early in the 2013-2016 outbreak and rose to high frequency. We found that GP-A82V had heightened ability to infect primate cells, including human dendritic cells. The increased infectivity was restricted to cells that have primate-specific NPC1 sequences at the EBOV interface, suggesting that this mutation was indeed an adaptation to the human host. GP-A82V was associated with increased mortality, consistent with the hypothesis that the heightened intrinsic infectivity of GP-A82V contributed to disease severity during the EVD epidemic.
Subject(s)
Ebolavirus/genetics , Ebolavirus/pathogenicity , Hemorrhagic Fever, Ebola/virology , Viral Envelope Proteins/chemistry , Viral Envelope Proteins/genetics , Africa, Western/epidemiology , Amino Acid Substitution , Animals , Callithrix , Carrier Proteins/chemistry , Carrier Proteins/metabolism , Cheirogaleidae , Cytoplasm/virology , Ebolavirus/physiology , Hemorrhagic Fever, Ebola/epidemiology , Humans , Intracellular Signaling Peptides and Proteins , Membrane Glycoproteins/chemistry , Membrane Glycoproteins/metabolism , Niemann-Pick C1 Protein , Protein Conformation, alpha-Helical , Viral Envelope Proteins/metabolism , Virion/chemistry , Virion/pathogenicity , VirulenceABSTRACT
Innate lymphoid cells (ILCs) are a diverse population of cells that include NK cells and contribute to tissue homeostasis and repair, inflammation, and provide protection from infection. The interplay between human blood ILCs, as well as their responses to HIV-1 infection, remains poorly understood. This study used transcriptional and chromatin profiling to explore these questions. Transcriptional profiling and flow cytometry analysis support that there are four main ILC subsets found in human blood. Unlike in mice, human NK cells expressed the tissue repair protein amphiregulin (AREG). AREG production was induced by TCF7/WNT, IL-2, and IL-15, and inhibited by TGFB1, a cytokine increased in people living with HIV-1. In HIV-1 infection, the percentage of AREG+ NK cells correlated positively with the numbers of ILCs and CD4+ T cells but negatively with the concentration of inflammatory cytokine IL-6. NK-cell knockout of the TGFB1-stimulated WNT antagonist RUNX3 increased AREG production. Antiviral gene expression was increased in all ILC subsets from HIV-1 viremic people, and anti-inflammatory gene MYDGF was increased in an NK-cell subset from HIV-1-infected people whose viral load was undetectable in the absence of antiretroviral therapy. The percentage of defective NK cells in people living with HIV-1 correlated inversely with ILC percentage and CD4+ T-cell counts. CD4+ T cells and their production of IL-2 prevented the loss of NK-cell function by activating mTOR. These studies clarify how ILC subsets are interrelated and provide insight into how HIV-1 infection disrupts NK cells, including an uncharacterized homeostatic function in NK cells.
Subject(s)
HIV Infections , HIV-1 , Humans , Mice , Animals , Immunity, Innate , Lymphocytes/metabolism , HIV-1/metabolism , Interleukin-2/metabolism , Chromatin , Killer Cells, Natural , Cytokines , HIV Infections/geneticsABSTRACT
Intron-containing RNA expressed from the HIV-1 provirus activates type 1 interferon in primary human blood cells, including CD4+ T cells, macrophages, and dendritic cells. To identify the innate immune receptor required for detection of intron-containing RNA expressed from the HIV-1 provirus, a loss-of-function screen was performed with short hairpin RNA-expressing lentivectors targeting twenty-one candidate genes in human monocyte-derived dendritic cells. Among the candidate genes tested, only knockdown of XPO1 (CRM1), IFIH1 (MDA5), or MAVS prevented activation of the interferon-stimulated gene ISG15. The importance of IFIH1 protein was demonstrated by rescue of the knockdown with nontargetable IFIH1 coding sequence. Inhibition of HIV-1-induced ISG15 by the IFIH1-specific Nipah virus V protein, and by IFIH1-transdominant 2-CARD domain-deletion or phosphomimetic point mutations, indicates that IFIH1 (MDA5) filament formation, dephosphorylation, and association with MAVS are all required for innate immune activation in response to HIV-1 transduction. Since both IFIH1 (MDA5) and DDX58 (RIG-I) signal via MAVS, the specificity of HIV-1 RNA detection by IFIH1 was demonstrated by the fact that DDX58 knockdown had no effect on activation. RNA-Seq showed that IFIH1 knockdown in dendritic cells globally disrupted the induction of IFN-stimulated genes by HIV-1. Finally, specific enrichment of unspliced HIV-1 RNA by IFIH1 (MDA5), over two orders of magnitude, was revealed by formaldehyde cross-linking immunoprecipitation (f-CLIP). These results demonstrate that IFIH1 is the innate immune receptor for intron-containing RNA from the HIV-1 provirus and that IFIH1 potentially contributes to chronic inflammation in people living with HIV-1, even in the presence of effective antiretroviral therapy.
Subject(s)
Dendritic Cells , HIV-1 , Immunity, Innate , Interferon-Induced Helicase, IFIH1 , Introns , Proviruses , RNA, Viral , Humans , HIV-1/genetics , HIV-1/immunology , Interferon-Induced Helicase, IFIH1/genetics , Interferon-Induced Helicase, IFIH1/metabolism , Proviruses/genetics , Dendritic Cells/immunology , Dendritic Cells/virology , Dendritic Cells/metabolism , Introns/genetics , RNA, Viral/genetics , RNA, Viral/immunology , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Adaptor Proteins, Signal Transducing/immunology , HIV Infections/immunology , HIV Infections/virology , HIV Infections/genetics , Karyopherins/genetics , Karyopherins/metabolismABSTRACT
Interaction between the Ebola virus envelope glycoprotein (GP) and the endosomal membrane is an essential step during virus entry into the cell. Acidic pH and Ca2+ have been implicated in mediating the GP-membrane interaction. However, the molecular mechanism by which these environmental factors regulate the conformational changes that enable engagement of GP with the target membrane is unknown. Here, we apply fluorescence correlation spectroscopy (FCS) and single-molecule Förster resonance energy transfer (smFRET) imaging to elucidate how the acidic pH, Ca2+ and anionic phospholipids in the late endosome promote GP-membrane interaction, thereby facilitating virus entry. We find that bis(monoacylglycero)phosphate (BMP), which is specific to the late endosome, is especially critical in determining the Ca2+-dependence of the GP-membrane interaction. Molecular dynamics (MD) simulations suggested residues in GP that sense pH and induce conformational changes that make the fusion loop available for insertion into the membrane. We similarly confirm residues in the fusion loop that mediate GP's interaction with Ca2+, which likely promotes local conformational changes in the fusion loop and mediates electrostatic interactions with the anionic phospholipids. Collectively, our results provide a mechanistic understanding of how the environment of the late endosome regulates the timing and efficiency of virus entry.
Subject(s)
Ebolavirus , Hemorrhagic Fever, Ebola , Humans , Ebolavirus/physiology , Calcium/metabolism , Viral Envelope Proteins/metabolism , Endosomes/metabolism , Protein Conformation , Virus Internalization , Membrane Fusion , Hydrogen-Ion ConcentrationABSTRACT
Prime editing systems have enabled the incorporation of precise edits within a genome without introducing double strand breaks. Previous studies defined an optimal primer binding site (PBS) length for the pegRNA of â¼13 nucleotides depending on the sequence composition. However, optimal PBS length characterization has been based on prime editing outcomes using plasmid or lentiviral expression systems. In this study, we demonstrate that for prime editor (PE) ribonucleoprotein complexes, the auto-inhibitory interaction between the PBS and the spacer sequence affects pegRNA binding efficiency and target recognition. Destabilizing this auto-inhibitory interaction by reducing the complementarity between the PBS-spacer region enhances prime editing efficiency in multiple prime editing formats. In the case of end-protected pegRNAs, a shorter PBS length with a PBS-target strand melting temperature near 37°C is optimal in mammalian cells. Additionally, a transient cold shock treatment of the cells post PE-pegRNA delivery further increases prime editing outcomes for pegRNAs with optimized PBS lengths. Finally, we show that prime editor ribonucleoprotein complexes programmed with pegRNAs designed using these refined parameters efficiently correct disease-related genetic mutations in patient-derived fibroblasts and efficiently install precise edits in primary human T cells and zebrafish.
Subject(s)
Cold Temperature , Gene Editing , Zebrafish , Animals , Humans , Binding Sites , Cold-Shock Response , CRISPR-Cas Systems , Mammals , Ribonucleoproteins , Zebrafish/geneticsABSTRACT
The HIV-1 genome encodes a small number of proteins with structural, enzymatic, regulatory, and accessory functions. These viral proteins interact with a number of host factors to promote the early and late stages of HIV-1 infection. During the early stages of infection, interactions between the viral proteins and host factors enable HIV-1 to enter the target cell, traverse the cytosol, dock at the nuclear pore, gain access to the nucleus, and integrate into the host genome. Similarly, the viral proteins recruit another set of host factors during the late stages of infection to orchestrate HIV-1 transcription, translation, assembly, and release of progeny virions. Among the host factors implicated in HIV-1 infection, Cyclophilin A (CypA) was identified as the first host factor to be packaged within HIV-1 particles. It is now well established that CypA promotes HIV-1 infection by directly binding to the viral capsid. Mechanistic models to pinpoint CypA's role have spanned from an effect in the producer cell to the early steps of infection in the target cell. In this review, we will describe our understanding of the role(s) of CypA in HIV-1 infection, highlight the current knowledge gaps, and discuss the potential role of this host factor in the post-nuclear entry steps of HIV-1 infection.
Subject(s)
Cyclophilin A , HIV Infections , HIV-1 , Humans , Capsid Proteins/genetics , Cell Nucleus/metabolism , Cyclophilin A/genetics , Cyclophilin A/metabolism , HIV Infections/metabolism , HIV-1/genetics , HIV-1/metabolism , Viral Proteins/metabolism , Host-Pathogen InteractionsABSTRACT
The Ebola virus (EBOV) envelope glycoprotein (GP) is a membrane fusion machine required for virus entry into cells. Following endocytosis of EBOV, the GP1 domain is cleaved by cellular cathepsins in acidic endosomes, removing the glycan cap and exposing a binding site for the Niemann-Pick C1 (NPC1) receptor. NPC1 binding to cleaved GP1 is required for entry. How this interaction translates to GP2 domain-mediated fusion of viral and endosomal membranes is not known. Here, using a bulk fluorescence dequenching assay and single-molecule Förster resonance energy transfer (smFRET)-imaging, we found that acidic pH, Ca2+, and NPC1 binding synergistically induce conformational changes in GP2 and permit virus-liposome lipid mixing. Acidic pH and Ca2+ shifted the GP2 conformational equilibrium in favor of an intermediate state primed for NPC1 binding. Glycan cap cleavage on GP1 enabled GP2 to transition from a reversible intermediate to an irreversible conformation, suggestive of the postfusion 6-helix bundle; NPC1 binding further promoted transition to the irreversible conformation. Thus, the glycan cap of GP1 may allosterically protect against inactivation of EBOV by premature triggering of GP2.
Subject(s)
Ebolavirus/physiology , Membrane Fusion , Viral Envelope Proteins/chemistry , Viral Envelope Proteins/metabolism , Allosteric Regulation , Calcium/metabolism , Ebolavirus/chemistry , Ebolavirus/genetics , Ebolavirus/metabolism , Fluorescence Resonance Energy Transfer , HEK293 Cells , Humans , Hydrogen-Ion Concentration , Intracellular Signaling Peptides and Proteins/metabolism , Niemann-Pick C1 Protein , Polysaccharides/metabolism , Protein Binding , Protein Conformation , Protein Domains , Viral Envelope Proteins/genetics , Virus InternalizationABSTRACT
BACKGROUND & AIMS: The molecular checkpoints driving T cell activation and cytokine responses in ulcerative colitis (UC) are incompletely understood. Here, we studied the Tec kinase ITK in UC. METHODS: We analyzed patients with inflammatory bowel disease (n = 223) and evaluated ITK activity as well as the functional effects of cyclosporine-A (CsA). In addition, 3 independent murine colitis models were used to investigate the functional role of ITK. Finally, the activity of ITK was blocked via pharmacological inhibitors and genetically engineered mice. Readout parameters were mini-endoscopy, histopathology, mucosal T cell apoptosis, and cytokine production. RESULTS: We found an expansion of pITK-expressing mucosal CD4+ T cells in UC rather than Crohn's disease that correlated with disease severity. CsA suppressed activation of ITK in cultured CD4+ T cells and calcineurin-containing microclusters adjacent to the T cell receptor signaling complex. Functionally, the capacity of CsA to suppress activity of experimental colitis was critically dependent on ITK. Genetic inactivation of Itk via gene targeting or induction of allele-sensitive Itk mutants prevented experimental colitis in 3 colitis models, and treatment with pharmacological ITK blockers suppressed established colitis. In addition, ITK controlled apoptosis and activation of mucosal Th2 and Th17 lymphocytes via NFATc2 signaling pathways. CONCLUSIONS: ITK activation was detected in UC and could be down-regulated in cultured T cells by CsA administration. Selective targeting of ITK emerges as an attractive approach for treatment of chronic intestinal inflammation and potentially UC by driving resolution of mucosal inflammation.
Subject(s)
Anti-Inflammatory Agents/pharmacology , Colitis, Ulcerative/prevention & control , Colon/drug effects , Intestinal Mucosa/drug effects , Intraepithelial Lymphocytes/drug effects , Protein Kinase Inhibitors/pharmacology , Protein-Tyrosine Kinases/antagonists & inhibitors , Animals , Apoptosis/drug effects , Cells, Cultured , Colitis, Ulcerative/enzymology , Colitis, Ulcerative/immunology , Colitis, Ulcerative/pathology , Colon/enzymology , Colon/immunology , Colon/pathology , Cyclosporine/pharmacology , Cytokines/metabolism , Disease Models, Animal , Humans , Intestinal Mucosa/enzymology , Intestinal Mucosa/immunology , Intestinal Mucosa/pathology , Intraepithelial Lymphocytes/enzymology , Intraepithelial Lymphocytes/immunology , Intraepithelial Lymphocytes/pathology , Mice, Knockout , Molecular Targeted Therapy , Phosphorylation , Protein-Tyrosine Kinases/genetics , Protein-Tyrosine Kinases/metabolism , Signal TransductionABSTRACT
The HIV-1 capsid protein makes up the core of the virion and plays a critical role in early steps of HIV replication. Due to its exposure in the cytoplasm after entry, HIV capsid is a target for host cell factors that act directly to block infection such as TRIM5α and MxB. Several host proteins also play a role in facilitating infection, including in the protection of HIV-1 capsid from recognition by host cell restriction factors. Through an unbiased screening approach, called HIV-CRISPR, we show that the CPSF6-binding deficient, N74D HIV-1 capsid mutant is sensitive to restriction mediated by human TRIM34, a close paralog of the well-characterized HIV restriction factor TRIM5α. This restriction occurs at the step of reverse transcription, is independent of interferon stimulation, and limits HIV-1 infection in key target cells of HIV infection including CD4+ T cells and monocyte-derived dendritic cells. TRIM34 can also restrict some SIV capsids. TRIM34 restriction requires TRIM5α as knockout or knockdown of TRIM5α results in a loss of antiviral activity. Through immunofluorescence studies, we show that TRIM34 and TRIM5α colocalize to cytoplasmic bodies and are more frequently observed to be associated with infecting N74D capsids than with WT HIV-1 capsids. Our results identify TRIM34 as an HIV-1 CA-targeting restriction factor and highlight the potential role for heteromultimeric TRIM interactions in contributing to restriction of HIV-1 infection in human cells.
Subject(s)
Capsid Proteins/metabolism , Carrier Proteins/metabolism , HIV Infections/metabolism , HIV-1/physiology , Tripartite Motif Proteins/metabolism , Ubiquitin-Protein Ligases/metabolism , Antiviral Restriction Factors , Capsid/metabolism , Capsid Proteins/genetics , Clustered Regularly Interspaced Short Palindromic Repeats , HEK293 Cells , HIV Infections/virology , HIV Seropositivity , HIV-1/metabolism , HeLa Cells , Humans , Reverse Transcription , Virus Integration/physiologySubject(s)
Disease Outbreaks , Respiratory Syncytial Virus Infections , Respiratory Syncytial Viruses , Humans , Infant , Respiratory Syncytial Virus Infections/epidemiology , Respiratory Syncytial Virus Infections/genetics , Respiratory Syncytial Virus Infections/virology , Seasons , United States/epidemiology , Respiratory Syncytial Viruses/geneticsABSTRACT
Broadly neutralizing, anti-HIV-1 gp120 mAbs have been isolated from infected individuals, and there is considerable interest in developing these reagents for Ab-based immunoprophylaxis and treatment. As a means to identify potentially new anti-HIV Abs, we exploited humanized NOD-scid IL2rγnull mice systemically infected with HIV-1 to generate a wide variety of Ag-specific human mAbs. The Abs were encoded by a diverse range of variable gene families and Ig classes, including IgA, and several showed significant levels of somatic mutation. Moreover, the isolated Abs not only bound target Ags with similar affinity as broadly neutralizing Abs, they also demonstrated neutralizing ability against multiple HIV-1 clades. The use of humanized mice will allow us to use our knowledge of HIV-1 gp120 structure and function, and the immune response targeting this protein, to generate native human prophylactic Abs to reduce the infection and spread of HIV-1.
Subject(s)
Antibodies, Monoclonal, Humanized/genetics , HIV Antibodies/genetics , HIV Envelope Protein gp120/immunology , HIV-1/immunology , Animals , Animals, Genetically Modified , Antibodies, Monoclonal, Humanized/immunology , Antibodies, Neutralizing/genetics , Antibodies, Neutralizing/immunology , HIV Antibodies/immunology , HIV Infections/immunology , Humans , Mice , Mice, Inbred NOD , Mice, SCID , Neutralization TestsABSTRACT
HIV-1 Nef, a protein important for the development of AIDS, has well-characterized effects on host membrane trafficking and receptor downregulation. By an unidentified mechanism, Nef increases the intrinsic infectivity of HIV-1 virions in a host-cell-dependent manner. Here we identify the host transmembrane protein SERINC5, and to a lesser extent SERINC3, as a potent inhibitor of HIV-1 particle infectivity that is counteracted by Nef. SERINC5 localizes to the plasma membrane, where it is efficiently incorporated into budding HIV-1 virions and impairs subsequent virion penetration of susceptible target cells. Nef redirects SERINC5 to a Rab7-positive endosomal compartment and thereby excludes it from HIV-1 particles. The ability to counteract SERINC5 was conserved in Nef encoded by diverse primate immunodeficiency viruses, as well as in the structurally unrelated glycosylated Gag from murine leukaemia virus. These examples of functional conservation and convergent evolution emphasize the fundamental importance of SERINC5 as a potent anti-retroviral factor.
Subject(s)
HIV-1/physiology , Host-Pathogen Interactions , Membrane Proteins/antagonists & inhibitors , Membrane Proteins/metabolism , Virion/chemistry , Virion/metabolism , nef Gene Products, Human Immunodeficiency Virus/metabolism , Animals , Cell Line , Cell Membrane/metabolism , Cell Membrane/virology , Endosomes/chemistry , Endosomes/metabolism , Evolution, Molecular , Gene Products, gag/metabolism , Gene Products, nef/chemistry , Gene Products, nef/metabolism , HIV-1/chemistry , Host Specificity , Humans , Leukemia Virus, Murine/chemistry , Leukemia Virus, Murine/physiology , Membrane Glycoproteins , Membrane Proteins/analysis , Neoplasm Proteins/metabolism , Primates/virology , Receptors, Cell Surface/metabolism , rab GTP-Binding Proteins/metabolism , rab7 GTP-Binding ProteinsABSTRACT
Type V CRISPR-Cas12a systems provide an alternate nuclease platform to Cas9, with potential advantages for specific genome editing applications. Here we describe improvements to the Cas12a system that facilitate efficient targeted mutagenesis in mammalian cells and zebrafish embryos. We show that engineered variants of Cas12a with two different nuclear localization sequences (NLS) on the C terminus provide increased editing efficiency in mammalian cells. Additionally, we find that pre-crRNAs comprising a full-length direct repeat (full-DR-crRNA) sequence with specific stem-loop G-C base substitutions exhibit increased editing efficiencies compared with the standard mature crRNA framework. Finally, we demonstrate in zebrafish embryos that the improved LbCas12a and FnoCas12a nucleases in combination with these modified crRNAs display high mutagenesis efficiencies and low toxicity when delivered as ribonucleoprotein complexes at high concentration. Together, these results define a set of enhanced Cas12a components with broad utility in vertebrate systems.
Subject(s)
CRISPR-Cas Systems , Endonucleases/genetics , Gene Editing/methods , RNA, Guide, Kinetoplastida/genetics , Ribonucleoproteins/genetics , Animals , Base Sequence , DNA (Cytosine-5-)-Methyltransferase 1/genetics , DNA (Cytosine-5-)-Methyltransferase 1/metabolism , Embryo, Nonmammalian , Endonucleases/metabolism , HEK293 Cells , HeLa Cells , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Humans , Inverted Repeat Sequences , Jurkat Cells , K562 Cells , Nuclear Localization Signals , Nucleic Acid Conformation , Plasmids/chemistry , Plasmids/metabolism , RNA, Guide, Kinetoplastida/metabolism , Ribonucleoproteins/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Transfection , Zebrafish , Zebrafish Proteins/genetics , Zebrafish Proteins/metabolismABSTRACT
The evolution of HIV-1 protein sequences should be governed by a combination of factors including nucleotide mutational probabilities, the genetic code, and fitness. The impact of these factors on protein sequence evolution is interdependent, making it challenging to infer the individual contribution of each factor from phylogenetic analyses alone. We investigated the protein sequence evolution of HIV-1 by determining an experimental fitness landscape of all individual amino acid changes in protease. We compared our experimental results to the frequency of protease variants in a publicly available data set of 32,163 sequenced isolates from drug-naïve individuals. The most common amino acids in sequenced isolates supported robust experimental fitness, indicating that the experimental fitness landscape captured key features of selection acting on protease during viral infections of hosts. Amino acid changes requiring multiple mutations from the likely ancestor were slightly less likely to support robust experimental fitness than single mutations, consistent with the genetic code favoring chemically conservative amino acid changes. Amino acids that were common in sequenced isolates were predominantly accessible by single mutations from the likely protease ancestor. Multiple mutations commonly observed in isolates were accessible by mutational walks with highly fit single mutation intermediates. Our results indicate that the prevalence of multiple-base mutations in HIV-1 protease is strongly influenced by mutational sampling.
Subject(s)
Evolution, Molecular , HIV Protease/genetics , HIV-1/genetics , Point Mutation , Genetic Code , Selection, GeneticABSTRACT
RNA-seq protocols that focus on transcript termini are well suited for applications in which template quantity is limiting. Here we show that, when applied to end-sequencing data, analytical methods designed for global RNA-seq produce computational artifacts. To remedy this, we created the End Sequence Analysis Toolkit (ESAT). As a test, we first compared end-sequencing and bulk RNA-seq using RNA from dendritic cells stimulated with lipopolysaccharide (LPS). As predicted by the telescripting model for transcriptional bursts, ESAT detected an LPS-stimulated shift to shorter 3'-isoforms that was not evident by conventional computational methods. Then, droplet-based microfluidics was used to generate 1000 cDNA libraries, each from an individual pancreatic islet cell. ESAT identified nine distinct cell types, three distinct ß-cell types, and a complex interplay between hormone secretion and vascularization. ESAT, then, offers a much-needed and generally applicable computational pipeline for either bulk or single-cell RNA end-sequencing.
Subject(s)
Islets of Langerhans/cytology , Sequence Analysis, RNA/methods , Single-Cell Analysis/methods , Transcriptome , Animals , Cells, Cultured , Dendritic Cells/cytology , Dendritic Cells/metabolism , Gene Library , Islets of Langerhans/metabolism , Microfluidics/methods , Rats , Sequence Analysis, RNA/standards , Single-Cell Analysis/standardsABSTRACT
Rhesus TRIM5α (rhTRIM5α) potently restricts replication of human immunodeficiency virus type 1 (HIV-1). Restriction is mediated through direct binding of the C-terminal B30.2 domain of TRIM5α to the assembled HIV-1 capsid core. This host-pathogen interaction involves multiple capsid molecules within the hexagonal HIV-1 capsid lattice. However, the molecular details of this interaction and the precise site at which the B30.2 domain binds remain largely unknown. The human orthologue of TRIM5α (hsTRIM5α) fails to block infection by HIV-1 both in vivo and in vitro This is thought to be due to differences in binding to the capsid lattice. To map the species-specific binding surface on the HIV-1 capsid lattice, we used microscale thermophoresis and dual-focus fluorescence correlation spectroscopy to measure binding affinity of rhesus and human TRIM5α B30.2 domains to a series of HIV-1 capsid variants that mimic distinct capsid arrangements at each of the symmetry axes of the HIV-1 capsid lattice. These surrogates include previously characterized capsid oligomers, as well as a novel chemically cross-linked capsid trimer that contains cysteine substitutions near the 3-fold axis of symmetry. The results demonstrate that TRIM5α binding involves multiple capsid molecules along the 2-fold and 3-fold interfaces between hexamers and indicate that the binding interface at the 3-fold axis contributes to the well-established differences in restriction potency between TRIM5α orthologues.IMPORTANCE TRIM5α is a cellular protein that fends off infection by retroviruses through binding to the viruses' protein shell surrounding its genetic material. This shell is composed of several hundred capsid proteins arranged in a honeycomb-like hexagonal pattern that is conserved across retroviruses. By binding to the complex lattice formed by multiple capsid proteins, rather than to a single capsid monomer, TRIM5α restriction activity persists despite the high mutation rate in retroviruses such as HIV-1. In rhesus monkeys, but not in humans, TRIM5α confers resistance to HIV-1. By measuring the binding of human and rhesus TRIM5α to a series of engineered HIV-1 capsid mimics of distinct capsid lattice interfaces, we reveal the HIV-1 capsid surface critical for species-specific binding by TRIM5α.