Genome-wide sequencing-based identification of methylation quantitative trait loci and their role in schizophrenia risk.

DNA methylation (DNAm) is an epigenetic regulator of gene expression and a hallmark of gene-environment interaction. Using whole-genome bisulfite sequencing, we have surveyed DNAm in 344 samples of human postmortem brain tissue from neurotypical subjects and individuals with schizophrenia. We identify genetic influence on local methylation levels throughout the genome, both at CpG sites and CpH sites, with 86% of SNPs and 55% of CpGs being part of methylation quantitative trait loci (meQTLs). These associations can further be clustered into regions that are differentially methylated by a given SNP, highlighting the genes and regions with which these loci are epigenetically associated. These findings can be used to better characterize schizophrenia GWAS-identified variants as epigenetic risk variants. Regions differentially methylated by schizophrenia risk-SNPs explain much of the heritability associated with risk loci, despite covering only a fraction of the genomic space. We provide a comprehensive, single base resolution view of association between genetic variation and genomic methylation, and implicate schizophrenia GWAS-associated variants as influencing the epigenetic plasticity of the brain.

Cortical cellular diversity and development in schizophrenia.

While a definitive understanding of schizophrenia etiology is far from current reality, an increasing body of evidence implicates perturbations in early development that alter the trajectory of brain maturation in this disorder, leading to abnormal function in early childhood and adulthood. This atypical development likely arises from an interaction of many brain cell types that follow distinct developmental paths. Because both cellular identity and development are governed by the transcriptome and epigenome, two levels of gene regulation that have the potential to reflect both genetic and environmental influences, mapping "omic" changes over development in diverse cells is a fruitful avenue for schizophrenia research. In this review, we provide a survey of human brain cellular composition and development, levels of genomic regulation that determine cellular identity and developmental trajectories, and what is known about how genomic regulation is dysregulated in specific cell types in schizophrenia. We also outline technical challenges and solutions to conducting cell type-specific functional genomic studies in human postmortem brain.

Characterizing the dynamic and functional DNA methylation landscape in the developing human cortex.

DNA methylation (DNAm) is a key epigenetic regulator of gene expression across development. The developing prenatal brain is a highly dynamic tissue, but our understanding of key drivers of epigenetic variability across development is limited. We, therefore, assessed genomic methylation at over 39 million sites in the prenatal cortex using whole-genome bisulfite sequencing and found loci and regions in which methylation levels are dynamic across development. We saw that DNAm at these loci was associated with nearby gene expression and enriched for enhancer chromatin states in prenatal brain tissue. Additionally, these loci were enriched for genes associated with neuropsychiatric disorders and genes involved with neurogenesis. We also found autosomal differences in DNAm between the sexes during prenatal development, though these have less clear functional consequences. We lastly confirmed that the dynamic methylation at this critical period is specifically CpG methylation, with generally low levels of CpH methylation. Our findings provide detailed insight into prenatal brain development as well as clues to the pathogenesis of psychiatric traits seen later in life.

Dissecting transcriptomic signatures of neuronal differentiation and maturation using iPSCs.

Human induced pluripotent stem cells (hiPSCs) are a powerful model of neural differentiation and maturation. We present a hiPSC transcriptomics resource on corticogenesis from 5 iPSC donor and 13 subclonal lines across 9 time points over 5 broad conditions: self-renewal, early neuronal differentiation, neural precursor cells (NPCs), assembled rosettes, and differentiated neuronal cells. We identify widespread changes in the expression of both individual features and global patterns of transcription. We next demonstrate that co-culturing human NPCs with rodent astrocytes results in mutually synergistic maturation, and that cell type-specific expression data can be extracted using only sequencing read alignments without cell sorting. We lastly adapt a previously generated RNA deconvolution approach to single-cell expression data to estimate the relative neuronal maturity of iPSC-derived neuronal cultures and human brain tissue. Using many public datasets, we demonstrate neuronal cultures are maturationally heterogeneous but contain subsets of neurons more mature than previously observed.

Schizophrenia risk variants influence multiple classes of transcripts of sorting nexin 19 (SNX19).

Genome-wide association studies (GWAS) have identified many genomic loci associated with risk for schizophrenia, but unambiguous identification of the relationship between disease-associated variants and specific genes, and in particular their effect on risk conferring transcripts, has proven difficult. To better understand the specific molecular mechanism(s) at the schizophrenia locus in 11q25, we undertook cis expression quantitative trait loci (cis-eQTL) mapping for this 2 megabase genomic region using postmortem human brain samples. To comprehensively assess the effects of genetic risk upon local expression, we evaluated multiple transcript features: genes, exons, and exon-exon junctions in multiple brain regions-dorsolateral prefrontal cortex (DLPFC), hippocampus, and caudate. Genetic risk variants strongly associated with expression of SNX19 transcript features that tag multiple rare classes of SNX19 transcripts, whereas they only weakly affected expression of an exon-exon junction that tags the majority of abundant transcripts. The most prominent class of SNX19 risk-associated transcripts is predicted to be overexpressed, defined by an exon-exon splice junction between exons 8 and 10 (junc8.10) and that is predicted to encode proteins that lack the characteristic nexin C terminal domain. Risk alleles were also associated with either increased or decreased expression of multiple additional classes of transcripts. With RACE, molecular cloning, and long read sequencing, we found a number of novel SNX19 transcripts that further define the set of potential etiological transcripts. We explored epigenetic regulation of SNX19 expression and found that DNA methylation at CpG sites near the primary transcription start site and within exon 2 partially mediate the effects of risk variants on risk-associated expression. ATAC sequencing revealed that some of the most strongly risk-associated SNPs are located within a region of open chromatin, suggesting a nearby regulatory element is involved. These findings indicate a potentially complex molecular etiology, in which risk alleles for schizophrenia generate epigenetic alterations and dysregulation of multiple classes of SNX19 transcripts.

Characterizing the nuclear and cytoplasmic transcriptomes in developing and mature human cortex uncovers new insight into psychiatric disease gene regulation.

Transcriptome compartmentalization by the nuclear membrane provides both stochastic and functional buffering of transcript activity in the cytoplasm, and has recently been implicated in neurodegenerative disease processes. Although many mechanisms regulating transcript compartmentalization are also prevalent in brain development, the extent to which subcellular localization differs as the brain matures has yet to be addressed. To characterize the nuclear and cytoplasmic transcriptomes during brain development, we sequenced both RNA fractions from homogenate prenatal and adult human postmortem cortex using poly(A)+ and Ribo-Zero library preparation methods. We find that while many genes are differentially expressed by fraction and developmental expression changes are similarly detectable in nuclear and cytoplasmic RNA, the compartmented transcriptomes become more distinct as the brain matures, perhaps reflecting increased utilization of nuclear retention as a regulatory strategy in adult brain. We examined potential mechanisms of this developmental divergence including alternative splicing, RNA editing, nuclear pore composition, RNA-binding protein motif enrichment, and RNA secondary structure. Intron retention is associated with greater nuclear abundance in a subset of transcripts, as is enrichment for several splicing factor binding motifs. Finally, we examined disease association with fraction-regulated gene sets and found nuclear-enriched genes were also preferentially enriched in gene sets associated with neurodevelopmental psychiatric disorders. These results suggest that although gene-level expression is globally comparable between fractions, nuclear retention of transcripts may play an underappreciated role in developmental regulation of gene expression in brain, particularly in genes whose dysregulation is related to neuropsychiatric disorders.

Divergent neuronal DNA methylation patterns across human cortical development reveal critical periods and a unique role of CpH methylation.

BACKGROUND: DNA methylation (DNAm) is a critical regulator of both development and cellular identity and shows unique patterns in neurons. To better characterize maturational changes in DNAm patterns in these cells, we profile the DNAm landscape at single-base resolution across the first two decades of human neocortical development in NeuN+ neurons using whole-genome bisulfite sequencing and compare them to non-neurons (primarily glia) and prenatal homogenate cortex. RESULTS: We show that DNAm changes more dramatically during the first 5 years of postnatal life than during the entire remaining period. We further refine global patterns of increasingly divergent neuronal CpG and CpH methylation (mCpG and mCpH) into six developmental trajectories and find that in contrast to genome-wide patterns, neighboring mCpG and mCpH levels within these regions are highly correlated. We integrate paired RNA-seq data and identify putative regulation of hundreds of transcripts and their splicing events exclusively by mCpH levels, independently from mCpG levels, across this period. We finally explore the relationship between DNAm patterns and development of brain-related phenotypes and find enriched heritability for many phenotypes within identified DNAm features. CONCLUSIONS: By profiling DNAm changes in NeuN-sorted neurons over the span of human cortical development, we identify novel, dynamic regions of DNAm that would be masked in homogenate DNAm data; expand on the relationship between CpG methylation, CpH methylation, and gene expression; and find enrichment particularly for neuropsychiatric diseases in genomic regions with cell type-specific, developmentally dynamic DNAm patterns.

Regional Heterogeneity in Gene Expression, Regulation, and Coherence in the Frontal Cortex and Hippocampus across Development and Schizophrenia.

The hippocampus formation, although prominently implicated in schizophrenia pathogenesis, has been overlooked in large-scale genomics efforts in the schizophrenic brain. We performed RNA-seq in hippocampi and dorsolateral prefrontal cortices (DLPFCs) from 551 individuals (286 with schizophrenia). We identified substantial regional differences in gene expression and found widespread developmental differences that were independent of cellular composition. We identified 48 and 245 differentially expressed genes (DEGs) associated with schizophrenia within the hippocampus and DLPFC, with little overlap between the brain regions. 124 of 163 (76.6%) of schizophrenia GWAS risk loci contained eQTLs in any region. Transcriptome-wide association studies in each region identified many novel schizophrenia risk features that were brain region-specific. Last, we identified potential molecular correlates of in vivo evidence of altered prefrontal-hippocampal functional coherence in schizophrenia. These results underscore the complexity and regional heterogeneity of the transcriptional correlates of schizophrenia and offer new insights into potentially causative biology.

Fragment-based drug discovery and its application to challenging drug targets.

Fragment-based drug discovery (FBDD) is a technique for identifying low molecular weight chemical starting points for drug discovery. Since its inception 20 years ago, FBDD has grown in popularity to the point where it is now an established technique in industry and academia. The approach involves the biophysical screening of proteins against collections of low molecular weight compounds (fragments). Although fragments bind to proteins with relatively low affinity, they form efficient, high quality binding interactions with the protein architecture as they have to overcome a significant entropy barrier to bind. Of the biophysical methods available for fragment screening, X-ray protein crystallography is one of the most sensitive and least prone to false positives. It also provides detailed structural information of the protein-fragment complex at the atomic level. Fragment-based screening using X-ray crystallography is therefore an efficient method for identifying binding hotspots on proteins, which can then be exploited by chemists and biologists for the discovery of new drugs. The use of FBDD is illustrated here with a recently published case study of a drug discovery programme targeting the challenging protein-protein interaction Kelch-like ECH-associated protein 1:nuclear factor erythroid 2-related factor 2.

HIV-1 uses dynamic capsid pores to import nucleotides and fuel encapsidated DNA synthesis.

During the early stages of infection, the HIV-1 capsid protects viral components from cytosolic sensors and nucleases such as cGAS and TREX, respectively, while allowing access to nucleotides for efficient reverse transcription. Here we show that each capsid hexamer has a size-selective pore bound by a ring of six arginine residues and a 'molecular iris' formed by the amino-terminal ß-hairpin. The arginine ring creates a strongly positively charged channel that recruits the four nucleotides with on-rates that approach diffusion limits. Progressive removal of pore arginines results in a dose-dependent and concomitant decrease in nucleotide affinity, reverse transcription and infectivity. This positively charged channel is universally conserved in lentiviral capsids despite the fact that it is strongly destabilizing without nucleotides to counteract charge repulsion. We also describe a channel inhibitor, hexacarboxybenzene, which competes for nucleotide binding and efficiently blocks encapsidated reverse transcription, demonstrating the tractability of the pore as a novel drug target.

HIV-1 Resistance to the Capsid-Targeting Inhibitor PF74 Results in Altered Dependence on Host Factors Required for Virus Nuclear Entry.

UNLABELLED: During HIV-1 infection of cells, the viral capsid plays critical roles in reverse transcription and nuclear entry of the virus. The capsid-targeting small molecule PF74 inhibits HIV-1 at early stages of infection. HIV-1 resistance to PF74 is complex, requiring multiple amino acid substitutions in the viral CA protein. Here we report the identification and analysis of a novel PF74-resistant mutant encoding amino acid changes in both domains of CA, three of which are near the pocket where PF74 binds. Interestingly, the mutant virus retained partial PF74 binding, and its replication was stimulated by the compound. The mutant capsid structure was not significantly perturbed by binding of PF74; rather, the mutations inhibited capsid interactions with CPSF6 and Nup153 and altered HIV-1 dependence on these host factors and on TNPO3. Moreover, the replication of the mutant virus was markedly impaired in activated primary CD4(+) T cells and macrophages. Our results suggest that HIV-1 escapes a capsid-targeting small molecule inhibitor by altering the virus's dependence on host factors normally required for entry into the nucleus. They further imply that clinical resistance to inhibitors targeting the PF74 binding pocket is likely to be strongly limited by functional constraints on HIV-1 evolution. IMPORTANCE: The HIV-1 capsid plays critical roles in early steps of infection and is an attractive target for therapy. Here we show that selection for resistance to a capsid-targeting small molecule inhibitor can result in viral dependence on the compound. The mutant virus was debilitated in primary T cells and macrophages--cellular targets of infection in vivo. The mutations also altered the virus's dependence on cellular factors that are normally required for HIV-1 entry into the nucleus. This work provides new information regarding mechanisms of HIV-1 resistance that should be useful in efforts to develop clinically useful drugs targeting the HIV-1 capsid.

Engineering targeted chromosomal amplifications in human breast epithelial cells.

Chromosomal amplifications are among the most common genetic alterations found in human cancers. However, experimental systems to study the processes that lead to specific, recurrent amplification events in human cancers are lacking. Moreover, some common amplifications, such as that at 8p11-12 in breast cancer, harbor multiple driver oncogenes, which are poorly modeled by conventional overexpression approaches. We sought to develop an experimental system to model recurrent chromosomal amplification events in human cell lines. Our strategy is to use homologous-recombination-mediated gene targeting to deliver a dominantly selectable, amplifiable marker to a specified chromosomal location. We used adeno-associated virus vectors to target human MCF-7 breast cancer cells at the ZNF703 locus, in the recurrent 8p11-12 amplicon, using the E. coli inosine monophosphate dehydrogenase (IMPDH) enzyme as a marker. We applied selective pressure using IMPDH inhibitors. Surviving clones were found to have increased copy number of ZNF703 (average 2.5-fold increase) by droplet digital PCR and FISH. Genome-wide array comparative genomic hybridization confirmed that amplifications had occurred on the short arm of chromosome 8, without changes on 8q or other chromosomes. Patterns of amplification were variable and similar to those seen in primary human breast cancers, including "sawtooth" patterns, distal copy number loss, and large continuous regions of copy number gain. This system will allow study of the cis- and trans-acting factors that are permissive for chromosomal amplification and provide a model to analyze oncogene cooperativity in amplifications harboring multiple candidate driver genes.

Activation of human γδ T cells by cytosolic interactions of BTN3A1 with soluble phosphoantigens and the cytoskeletal adaptor periplakin.

The three butyrophilin BTN3A molecules, BTN3A1, BTN3A2, and BTN3A3, are members of the B7/butyrophilin-like group of Ig superfamily receptors, which modulate the function of T cells. BTN3A1 controls activation of human Vγ9/Vδ2 T cells by direct or indirect presentation of self and nonself phosphoantigens (pAg). We show that the microbial metabolite (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate binds to the intracellular B30.2 domain of BTN3A1 with an affinity of 1.1 µM, whereas the endogenous pAg isopentenyl pyrophosphate binds with an affinity of 627 µM. Coculture experiments using knockdown cell lines showed that in addition to BTN3A1, BTN3A2 and BTN3A3 transmit activation signals to human γδ T cells in response to (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate and the aminobisphosphonate drug zoledronate that causes intracellular accumulation of isopentenyl pyrophosphate. The plakin family member periplakin, identified in yeast two-hybrid assays, interacted with a membrane-proximal di-leucine motif, located proximal to the B30.2 domain in the BTN3A1 cytoplasmic tail. Periplakin did not interact with BTN3A2 or BTN3A3, which do not contain the di-leucine motif. Re-expression into a BTN3A1 knockdown line of wild-type BTN3A1, but not of a variant lacking the periplakin binding motif, BTN3A1Δexon5, restored γδ T cell responses, demonstrating a functional role for periplakin interaction. These data, together with the widespread expression in epithelial cells, tumor tissues, and macrophages detected using BTN3A antiserum, are consistent with complex functions for BTN3A molecules in tissue immune surveillance and infection, linking the cell cytoskeleton to γδ T cell activation by indirectly presenting pAg to the Vγ9/Vδ2 TCR.

Host cofactors and pharmacologic ligands share an essential interface in HIV-1 capsid that is lost upon disassembly.

The HIV-1 capsid is involved in all infectious steps from reverse transcription to integration site selection, and is the target of multiple host cell and pharmacologic ligands. However, structural studies have been limited to capsid monomers (CA), and the mechanistic basis for how these ligands influence infection is not well understood. Here we show that a multi-subunit interface formed exclusively within CA hexamers mediates binding to linear epitopes within cellular cofactors NUP153 and CPSF6, and is competed for by the antiretroviral compounds PF74 and BI-2. Each ligand is anchored via a shared phenylalanine-glycine (FG) motif to a pocket within the N-terminal domain of one monomer, and all but BI-2 also make essential interactions across the N-terminal domain: C-terminal domain (NTD:CTD) interface to a second monomer. Dissociation of hexamer into CA monomers prevents high affinity interaction with CPSF6 and PF74, and abolishes binding to NUP153. The second interface is conformationally dynamic, but binding of NUP153 or CPSF6 peptides is accommodated by only one conformation. NUP153 and CPSF6 have overlapping binding sites, but each makes unique CA interactions that, when mutated selectively, perturb cofactor dependency. These results reveal that multiple ligands share an overlapping interface in HIV-1 capsid that is lost upon viral disassembly.

HIV-1 evades innate immune recognition through specific cofactor recruitment.

Human immunodeficiency virus (HIV)-1 is able to replicate in primary human macrophages without stimulating innate immunity despite reverse transcription of genomic RNA into double-stranded DNA, an activity that might be expected to trigger innate pattern recognition receptors. We reasoned that if correctly orchestrated HIV-1 uncoating and nuclear entry is important for evasion of innate sensors then manipulation of specific interactions between HIV-1 capsid and host factors that putatively regulate these processes should trigger pattern recognition receptors and stimulate type 1 interferon (IFN) secretion. Here we show that HIV-1 capsid mutants N74D and P90A, which are impaired for interaction with cofactors cleavage and polyadenylation specificity factor subunit 6 (CPSF6) and cyclophilins (Nup358 and CypA), respectively, cannot replicate in primary human monocyte-derived macrophages because they trigger innate sensors leading to nuclear translocation of NF-κB and IRF3, the production of soluble type 1 IFN and induction of an antiviral state. Depletion of CPSF6 with short hairpin RNA expression allows wild-type virus to trigger innate sensors and IFN production. In each case, suppressed replication is rescued by IFN-receptor blockade, demonstrating a role for IFN in restriction. IFN production is dependent on viral reverse transcription but not integration, indicating that a viral reverse transcription product comprises the HIV-1 pathogen-associated molecular pattern. Finally, we show that we can pharmacologically induce wild-type HIV-1 infection to stimulate IFN secretion and an antiviral state using a non-immunosuppressive cyclosporine analogue. We conclude that HIV-1 has evolved to use CPSF6 and cyclophilins to cloak its replication, allowing evasion of innate immune sensors and induction of a cell-autonomous innate immune response in primary human macrophages.

HIV-1 capsid undergoes coupled binding and isomerization by the nuclear pore protein NUP358.

BACKGROUND: Lentiviruses such as HIV-1 can be distinguished from other retroviruses by the cyclophilin A-binding loop in their capsid and their ability to infect non-dividing cells. Infection of non-dividing cells requires transport through the nuclear pore but how this is mediated is unknown. RESULTS: Here we present the crystal structure of the N-terminal capsid domain of HIV-1 in complex with the cyclophilin domain of nuclear pore protein NUP358. The structure reveals that HIV-1 is positioned to allow single-bond resonance stabilisation of exposed capsid residue P90. NMR exchange experiments demonstrate that NUP358 is an active isomerase, which efficiently catalyzes cis-trans isomerization of the HIV-1 capsid. In contrast, the distantly related feline lentivirus FIV can bind NUP358 but is neither isomerized by it nor requires it for infection. CONCLUSION: Isomerization by NUP358 may be preserved by HIV-1 to target the nuclear pore and synchronize nuclear entry with capsid uncoating.

CPSF6 defines a conserved capsid interface that modulates HIV-1 replication.

The HIV-1 genome enters cells inside a shell comprised of capsid (CA) protein. Variation in CA sequence alters HIV-1 infectivity and escape from host restriction factors. However, apart from the Cyclophilin A-binding loop, CA has no known interfaces with which to interact with cellular cofactors. Here we describe a novel protein-protein interface in the N-terminal domain of HIV-1 CA, determined by X-ray crystallography, which mediates both viral restriction and host cofactor dependence. The interface is highly conserved across lentiviruses and is accessible in the context of a hexameric lattice. Mutation of the interface prevents binding to and restriction by CPSF6-358, a truncated cytosolic form of the RNA processing factor, cleavage and polyadenylation specific factor 6 (CPSF6). Furthermore, mutations that prevent CPSF6 binding also relieve dependence on nuclear entry cofactors TNPO3 and RanBP2. These results suggest that the HIV-1 capsid mediates direct host cofactor interactions to facilitate viral infection.

Diverse HIV viruses are targeted by a conformationally dynamic antiviral.

Rhesus macaque TRIMCyp (RhTC) is a potent primate antiviral host protein that inhibits the replication of diverse HIV viruses. Here we show that it has acquired the ability to target multiple viruses by evolving an active site that interconverts between multiple conformations. Mutations that have relieved active site constraints allow RhTC to dynamically sample conformational space, including radically different conformers that target both HIV-1 and HIV-2 viruses. Introduction of a reversible constraint into RhTC allows specificity to be switched between a single conformation specific for HIV-1 and a dynamic ensemble that targets multiple viruses. These results show that conformational diversity can be used to expand the target diversity of innate immune receptors by supplementing their limited genetic variability with variability in protein structure.

HIV-1 capsid-cyclophilin interactions determine nuclear import pathway, integration targeting and replication efficiency.

Lentiviruses such as HIV-1 traverse nuclear pore complexes (NPC) and infect terminally differentiated non-dividing cells, but how they do this is unclear. The cytoplasmic NPC protein Nup358/RanBP2 was identified as an HIV-1 co-factor in previous studies. Here we report that HIV-1 capsid (CA) binds directly to the cyclophilin domain of Nup358/RanBP2. Fusion of the Nup358/RanBP2 cyclophilin (Cyp) domain to the tripartite motif of TRIM5 created a novel inhibitor of HIV-1 replication, consistent with an interaction in vivo. In contrast to CypA binding to HIV-1 CA, Nup358 binding is insensitive to inhibition with cyclosporine, allowing contributions from CypA and Nup358 to be distinguished. Inhibition of CypA reduced dependence on Nup358 and the nuclear basket protein Nup153, suggesting that CypA regulates the choice of the nuclear import machinery that is engaged by the virus. HIV-1 cyclophilin-binding mutants CA G89V and P90A favored integration in genomic regions with a higher density of transcription units and associated features than wild type virus. Integration preference of wild type virus in the presence of cyclosporine was similarly altered to regions of higher transcription density. In contrast, HIV-1 CA alterations in another patch on the capsid surface that render the virus less sensitive to Nup358 or TRN-SR2 depletion (CA N74D, N57A) resulted in integration in genomic regions sparse in transcription units. Both groups of CA mutants are impaired in replication in HeLa cells and human monocyte derived macrophages. Our findings link HIV-1 engagement of cyclophilins with both integration targeting and replication efficiency and provide insight into the conservation of viral cyclophilin recruitment.

Conformational adaptation of Asian macaque TRIMCyp directs lineage specific antiviral activity.

TRIMCyps are anti-retroviral proteins that have arisen independently in New World and Old World primates. All TRIMCyps comprise a CypA domain fused to the tripartite domains of TRIM5alpha but they have distinct lentiviral specificities, conferring HIV-1 restriction in New World owl monkeys and HIV-2 restriction in Old World rhesus macaques. Here we provide evidence that Asian macaque TRIMCyps have acquired changes that switch restriction specificity between different lentiviral lineages, resulting in species-specific alleles that target different viruses. Structural, thermodynamic and viral restriction analysis suggests that a single mutation in the Cyp domain, R69H, occurred early in macaque TRIMCyp evolution, expanding restriction specificity to the lentiviral lineages found in African green monkeys, sooty mangabeys and chimpanzees. Subsequent mutations have enhanced restriction to particular viruses but at the cost of broad specificity. We reveal how specificity is altered by a scaffold mutation, E143K, that modifies surface electrostatics and propagates conformational changes into the active site. Our results suggest that lentiviruses may have been important pathogens in Asian macaques despite the fact that there are no reported lentiviral infections in current macaque populations.