Retroviral Integration Research Articles

Long Terminal Repeats of Gammaretroviruses Retain Stable Expression after Integration Retargeting.

Retroviruses integrate into the genomes of infected host cells to form proviruses, a genetic platform for stable viral gene expression. Epigenetic silencing can, however, hamper proviral transcriptional activity. As gammaretroviruses (γRVs) preferentially integrate into active promoter and enhancer sites, the high transcriptional activity of γRVs can be attributed to this integration preference. In addition, long terminal repeats (LTRs) of some γRVs were shown to act as potent promoters by themselves. Here, we investigate the capacity of different γRV LTRs to drive stable expression within a non-preferred epigenomic environment in the context of diverse retroviral vectors. We demonstrate that different γRV LTRs are either rapidly silenced or remain active for long periods of time with a predominantly active proviral population under normal and retargeted integration. As an alternative to the established γRV systems, the feline leukemia virus and koala retrovirus LTRs are able to drive stable, albeit intensity-diverse, transgene expression. Overall, we show that despite the occurrence of rapid silencing events, most γRV LTRs can drive stable expression outside of their preferred chromatin landscape after retrovirus integrations.

The primate-specific presence of interferon regulatory factor-5 pseudogene 1.

Interferon regulatory factor 5 (IRF5) is a key transcription factor in inflammatory and immune responses, with its dysregulation linked to autoimmune diseases. Using bioinformatic approaches, including Basic Local Alignment Search Tool (BLAST) for sequence similarity searches, BLAST-Like Alignment Tool (BLAT) for genome-wide alignments, and several phylogenetics software, such as Multiple Alignment using Fast Fourier Transform (MAFFT), for phylogenetic analyses, we characterized the structure, origin, and evolutionary history of the human IRF5 pseudogene 1 (IRF5P1). Our analyses reveal that IRF5P1 is a chimeric processed pseudogene containing sequences derived from multiple sources, including IRF5-like sequences from disparate organisms. We find that IRF5P1 is specific to higher primates, likely originating through an ancient retroviral integration event approximately 60 million years ago. Interestingly, IRF5P1 resides within the triple QxxK/R motif-containing (TRIQK) gene, and its antisense strand is predominantly expressed as part of the TRIQK pre-messenger RNA (mRNA). Analysis of publicly available RNA-seq data suggests potential expression of antisense IRF5P1 RNA. We hypothesize that this antisense RNA may regulate IRF5 expression through complementary binding to IRF5 mRNA, with human genetic variants potentially modulating this interaction. The conservation of IRF5P1 in the primate lineage suggests its positive effects on primate evolution and innate immunity. This study highlights the importance of investigating pseudogenes and their potential regulatory roles in shaping lineage-specific immune adaptations.

HIV-1 Intasomes Assembled with Excess Integrase C-Terminal Domain Protein Facilitate Structural Studies by Cryo-EM and Reveal the Role of the Integrase C-Terminal Tail in HIV-1 Integration.

Retroviral integration is mediated by intasome nucleoprotein complexes wherein a pair of viral DNA ends are bridged together by a multimer of integrase (IN). Atomic-resolution structures of HIV-1 intasomes provide detailed insights into the mechanism of integration and inhibition by clinical IN inhibitors. However, previously described HIV-1 intasomes are highly heterogeneous and have the tendency to form stacks, which is a limiting factor in determining high-resolution cryo-EM maps. We have assembled HIV-1 intasomes in the presence of excess IN C-terminal domain protein, which was readily incorporated into the intasomes. The purified intasomes were largely homogeneous and exhibited minimal stacking tendencies. The cryo-EM map resolution was further improved to 2.01 Å, which will greatly facilitate structural studies of IN inhibitor action and drug resistance mechanisms. The C-terminal 18 residues of HIV-1 IN, which are critical for virus replication and integration in vitro, have not been well resolved in previous intasome structures, and its function remains unclear. We show that the C-terminal tail participates in intasome assembly, resides within the intasome core, and forms a small alpha helix (residues 271-276). Mutations that disrupt alpha helix integrity impede IN activity in vitro and disrupt HIV-1 infection at the step of viral DNA integration.

Limitations of current high-throughput sequencing technologies lead to biased expression estimates of endogenous retroviral elements.

Human endogenous retroviruses (HERVs), the remnants of ancient germline retroviral integrations, comprise almost 8% of the human genome. The elucidation of their biological roles is hampered by our inability to link HERV mRNA and protein production with specific HERV loci. To solve the riddle of the integration-specific RNA expression of HERVs, several bioinformatics approaches have been proposed; however, no single process seems to yield optimal results due to the repetitiveness of HERV integrations. The performance of existing data-bioinformatics pipelines has been evaluated against real world datasets whose true expression profile is unknown, thus the accuracy of widely-used approaches remains unclear. Here, we simulated mRNA production from specific HERV integrations to evaluate second and third generation sequencing technologies along with widely used bioinformatic approaches to estimate the accuracy in describing integration-specific expression. We demonstrate that, while a HERV-family approach offers accurate results, per-integration analyses of HERV expression suffer from substantial expression bias, which is only partially mitigated by algorithms developed for calculating the per-integration HERV expression, and is more pronounced in recent integrations. Hence, this bias could erroneously result into biologically meaningful inferences. Finally, we demonstrate the merits of accurate long-read high-throughput sequencing technologies in the resolution of per-locus HERV expression.

Cyclophilin A Facilitates HIV-1 DNA Integration.

HIV-1 capsid interaction with host cellular factors is essential for establishing a productive infection. However, the molecular details of such virus-host interactions are not fully understood. Cyclophilin A (CypA) is the first host protein identified to specifically bind to the HIV-1 capsid. Now it is established that CypA promotes reverse transcription and nuclear entry steps of HIV-1 infection. In this report, we show that CypA promotes HIV-1 integration by binding to the viral capsid. Specifically, our results demonstrate that CypA promotes HIV-1 integration by stimulating the activity of the viral preintegration complex and identifies a novel role of CypA during HIV-1 infection. This new knowledge is important because recent reports suggest that an operationally intact HIV-1 capsid enters the nucleus of an infected cell.

Risk of Second Tumors and T-Cell Lymphoma after CAR T-Cell Therapy

BackgroundThe risk of second tumors after chimeric antigen receptor (CAR) T-cell therapy, especially the risk of T-cell neoplasms related to viral vector integration, is an emerging concern.MethodsWe reviewed our clinical experience with adoptive cellular CAR T-cell therapy at our institution since 2016 and ascertained the occurrence of second tumors. In one case of secondary T-cell lymphoma, a broad array of molecular, genetic, and cellular techniques were used to interrogate the tumor, the CAR T cells, and the normal hematopoietic cells in the patient.ResultsA total of 724 patients who had received T-cell therapies at our center were included in the study. A lethal T-cell lymphoma was identified in a patient who had received axicabtagene ciloleucel therapy for diffuse large B-cell lymphoma, and both lymphomas were deeply profiled. Each lymphoma had molecularly distinct immunophenotypes and genomic profiles, but both were positive for Epstein–Barr virus and were associated with DNMT3A and TET2 mutant clonal hematopoiesis. No evidence of oncogenic retroviral integration was found with the use of multiple techniques.ConclusionsOur results highlight the rarity of second tumors and provide a framework for defining clonal relationships and viral vector monitoring. (Funded by the National Cancer Institute and others.)

Comprehensive genome assembly reveals genetic diversity and carcass consumption insights in critically endangered Asian king vultures

The Asian king vulture (AKV), a vital forest scavenger, is facing globally critical endangerment. This study aimed to construct a reference genome to unveil the mechanisms underlying its scavenger abilities and to assess the genetic relatedness of the captive population in Thailand. A reference genome of a female AKV was assembled from sequencing reads obtained from both PacBio long-read and MGI short-read sequencing platforms. Comparative genomics with New World vultures (NWVs) and other birds in the Family Accipitridae revealed unique gene families in AKV associated with retroviral genome integration and feather keratin, contrasting with NWVs’ genes related to olfactory reception. Expanded gene families in AKV were linked to inflammatory response, iron regulation and spermatogenesis. Positively selected genes included those associated with anti-apoptosis, immune response and muscle cell development, shedding light on adaptations for carcass consumption and high-altitude soaring. Using restriction site-associated DNA sequencing (RADseq)-based genome-wide single nucleotide polymorphisms (SNPs), genetic relatedness and inbreeding status of five captive AKVs were determined, revealing high genomic inbreeding in two females. In conclusion, the AKV reference genome was established, providing insights into its unique characteristics. Additionally, the potential of RADseq-based genome-wide SNPs for selecting AKV breeders was demonstrated.

Brief Histories of Retroviral Integration Research and Associated International Conferences.

The field of retroviral integration research has a long history that started with the provirus hypothesis and subsequent discoveries of the retroviral reverse transcriptase and integrase enzymes. Because both enzymes are essential for retroviral replication, they became valued targets in the effort to discover effective compounds to inhibit HIV-1 replication. In 2007, the first integrase strand transfer inhibitor was licensed for clinical use, and subsequently approved second-generation integrase inhibitors are now commonly co-formulated with reverse transcriptase inhibitors to treat people living with HIV. International meetings specifically focused on integrase and retroviral integration research first convened in 1995, and this paper is part of the Viruses Special Issue on the 7th International Conference on Retroviral Integration, which was held in Boulder Colorado in the summer of 2023. Herein, we overview key historical developments in the field, especially as they pertain to the development of the strand transfer inhibitor drug class. Starting from the mid-1990s, research advancements are presented through the lens of the international conferences. Our overview highlights the impact that regularly scheduled, subject-specific international meetings can have on community-building and, as a result, on field-specific collaborations and scientific advancements.

HIV-1 Integrase Assembles Multiple Species of Stable Synaptic Complex Intasomes That Are Active for Concerted DNA Integration In vitro

Retroviral DNA integration is mediated by nucleoprotein complexes (intasomes) in which a pair of viral DNA ends are bridged by a multimer of integrase (IN). Most of the high-resolution structures of HIV-1 intasomes are based on an HIV-1 IN with an Sso7d protein domain fused to the N-terminus. Sso7d-IN aggregates much less than wild-type IN and has been critical for structural studies of HIV-1 intasomes. Unexpectedly, these structures revealed that the common core architecture that mediates catalysis could be assembled in various ways, giving rise to both tetrameric and dodecameric intasomes, together with other less well-characterized species. This differs from related retroviruses that assemble unique multimeric intasomes, although the number of protomers in the intasome varies between viruses. The question of whether the additional Sso7d domain contributes to the heterogeneity of HIV-1 intasomes is therefore raised. We have addressed this by biochemical and structural studies of intasomes assembled with wild-type HIV-1 IN. Negative stain and cryo-EM reveal a similar range of multimeric intasome species as with Sso7d-IN with the same common core architecture. Stacks of intasomes resulting from domain swapping are also seen with both wild-type and Sso7d-IN intasomes. The propensity to assemble multimeric intasome species is, therefore, an intrinsic property of HIV-1 IN and is not conferred by the presence of the Sso7d domain. The recently solved intasome structures of different retroviral species, which have been reported to be tetrameric, octameric, dodecameric, and hexadecameric, highlight how a common intasome core architecture can be assembled in different ways for catalysis.

Endogenous retroviral solo-LTRs in human genome.

Human endogenous retroviruses (HERVs) are derived from the infection and integration of exogenetic retroviruses. HERVs account for 8% of human genome, and the majority of HERVs are solitary LTRs (solo-LTRs) due to homologous recombination. Multiple findings have showed that solo-LTRs could provide an enormous reservoir of transcriptional regulatory sequences involved in diverse biological processes, especially carcinogenesis and cancer development. The link between solo-LTRs and human diseases still remains poorly understood. This review focuses on the regulatory modules of solo-LTRs, which contribute greatly to the diversification and evolution of human genes. More importantly, although inactivating mutations, insertions and deletions have been identified in solo-LTRs, the inherited regulatory elements of solo-LTRs initiate the expression of chimeric lncRNA transcripts, which have been reported to play crucial roles in human health and disease. These findings provide valuable insights into the evolutionary and functional mechanisms underlying the presence of HERVs in human genome. Taken together, in this review, we will present evidences showing the regulatory and encoding capacity of solo-LTRs as well as the significant impact on various aspects of human biology.

Virus specificity and nucleoporin requirements for MX2 activity are affected by GTPase function and capsid-CypA interactions.

Human myxovirus resistance 2 (MX2/MXB) is an interferon-induced GTPase that inhibits human immunodeficiency virus-1 (HIV-1) infection by preventing nuclear import of the viral preintegration complex. The HIV-1 capsid (CA) is the major viral determinant for sensitivity to MX2, and complex interactions between MX2, CA, nucleoporins (Nups), cyclophilin A (CypA), and other cellular proteins influence the outcome of viral infection. To explore the interactions between MX2, the viral CA, and CypA, we utilized a CRISPR-Cas9/AAV approach to generate CypA knock-out cell lines as well as cells that express CypA from its endogenous locus, but with specific point mutations that would abrogate CA binding but should not affect enzymatic activity or cellular function. We found that infection of CypA knock-out and point mutant cell lines with wild-type HIV-1 and CA mutants recapitulated the phenotypes observed upon cyclosporine A (CsA) addition, indicating that effects of CsA treatment are the direct result of blocking CA-CypA interactions and are therefore independent from potential interactions between CypA and MX2 or other cellular proteins. Notably, abrogation of GTP hydrolysis by MX2 conferred enhanced antiviral activity when CA-CypA interactions were abolished, and this effect was not mediated by the CA-binding residues in the GTPase domain, or by phosphorylation of MX2 at position T151. We additionally found that elimination of GTPase activity also altered the Nup requirements for MX2 activity. Our data demonstrate that the antiviral activity of MX2 is affected by CypA-CA interactions in a virus-specific and GTPase activity-dependent manner. These findings further highlight the importance of the GTPase domain of MX2 in regulation of substrate specificity and interaction with nucleocytoplasmic trafficking pathways.

Murine leukemia virus infection of non-dividing dendritic cells is dependent on nucleoporins.

Retroviral reverse transcription starts within the capsid and uncoating and reverse transcription are mutually dependent. There is still debate regarding the timing and cellular location of HIV's uncoating and reverse transcription and whether it occurs solely in the cytoplasm, nucleus or both. HIV can infect non-dividing cells because there is active transport of the preintegration complex (PIC) across the nuclear membrane, but Murine Leukemia Virus (MLV) is thought to depend on cell division for replication and whether MLV uncoating and reverse transcription is solely cytoplasmic has not been studied. Here, we used NIH3T3 and primary mouse dendritic cells to determine where the different stages of reverse transcription occur and whether cell division is needed for nuclear entry. Our data strongly suggest that in both NIH3T3 cells and dendritic cells (DCs), the initial step of reverse transcription occurs in the cytoplasm. However, we detected MLV RNA/DNA hybrid intermediates in the nucleus of dividing NIH3T3 cells and non-dividing DCs, suggesting that reverse transcription can continue after nuclear entry. We also confirmed that the MLV PIC requires cell division to enter the nucleus of NIH3T3 cells. In contrast, we show that MLV can infect non-dividing primary DCs, although integration of MLV DNA in DCs still required the viral p12 protein. Knockdown of several nuclear pore proteins dramatically reduced the appearance of integrated MLV DNA in DCs but not NIH3T3 cells. Additionally, MLV capsid associated with the nuclear pore proteins NUP358 and NUP62 during infection. These findings suggest that simple retroviruses, like the complex retrovirus HIV, gain nuclear entry by traversing the nuclear pore complex in non-mitotic cells.

Biophysical model for DNA mutations induced by retroviral genome insertion based on the probability density function of mutation distribution

Research into the biophysical properties of deoxyribonucleic acid (DNA) and the mechanisms underlying genetic mutations has undergone marked advancements. The intriguing nature of mutations resulting from retroviral DNA insertion has garnered considerable attention. Whether these mutations are random or region-specific, the distribution patterns of mutation sites have been the focus of numerous research endeavours. This mutation mechanism originates from interactions between host DNA and the pre-integration complex (PIC), comprising retroviral DNA and an integrase enzyme that facilitates its incorporation into the host DNA. Our study focused on the Zfp521 gene locus, recognised for its pronounced susceptibility to insertional mutations, particularly around unique palindromic sequences. We employed two biophysical models to predict mutation distribution within a range of 50 base pairs centred on these sequences. The first is a probabilistic collision model emphasising PIC and target DNA interactions. The second model is a DNA diffraction lattice, where the PIC behaves according to probability density. Although both models adeptly illuminated the probability distributions of target sites, the second model was more successful in predicting the PIC integration sites based on DNA biophysical properties. This highlights the pivotal role of intricate interactions between the PIC and target DNA, suggesting that mutations can be predicted in a stochastic manner.

DNA strand breaks and gaps target retroviral intasome binding and integration

Retrovirus integration into a host genome is essential for productive infections. The integration strand transfer reaction is catalyzed by a nucleoprotein complex (Intasome) containing the viral integrase (IN) and the reverse transcribed (RT) copy DNA (cDNA). Previous studies suggested that DNA target-site recognition limits intasome integration. Using single molecule Förster resonance energy transfer (smFRET), we show prototype foamy virus (PFV) intasomes specifically bind to DNA strand breaks and gaps. These break and gap DNA discontinuities mimic oxidative base excision repair (BER) lesion-processing intermediates that have been shown to affect retrovirus integration in vivo. The increased DNA binding events targeted strand transfer to the break/gap site without inducing substantial intasome conformational changes. The major oxidative BER substrate 8-oxo-guanine as well as a G/T mismatch or +T nucleotide insertion that typically introduce a bend or localized flexibility into the DNA, did not increase intasome binding or targeted integration. These results identify DNA breaks or gaps as modulators of dynamic intasome-target DNA interactions that encourage site-directed integration.

HIV Preintegration Transcription and Host Antagonism.

Retrovirus integration is an obligatory step for the viral life cycle, but large amounts of unintegrated DNA (uDNA) accumulate during retroviral infection. For simple retroviruses, in the absence of integration, viral genomes are epigenetically silenced in host cells. For complex retroviruses such as HIV, preintegration transcription has been found to occur at low levels from a large population of uDNA even in the presence of host epigenetic silencing mechanisms. HIV preintegration transcription has been suggested to be a normal early process of HIV infection that leads to the syntheses of all three classes of viral transcripts: multiply-spliced, singly-spliced, and unspliced genomic RNA; only viral early proteins such as Nef are selectively translated at low levels in blood CD4 T cells and macrophages, the primary targets of HIV. The initiation and persistence of HIV preintegration transcription have been suggested to rely on viral accessory proteins, particularly virion Vpr and de novo Tat generated from uDNA; both proteins have been shown to antagonize host epigenetic silencing of uDNA. In addition, stimulation of latently infected resting T cells and macrophages with cytokines, PKC activator, or histone deacetylase inhibitors has been found to greatly upregulate preintegration transcription, leading to low-level viral production or even replication from uDNA. Functionally, Nef synthesized from preintegration transcription is biologically active in modulating host immune functions, lowering the threshold of T cell activation, and downregulating surface CD4, CXCR4/CCR5, and HMC receptors. The early Tat activity from preintegration transcription antagonizes repressive minichromatin assembled onto uDNA. The study of HIV preintegration transcription is important to understanding virus-host interaction and antagonism, viral persistence, and the mechanism of integrase drug resistance. The application of unintegrated lentiviral vectors for gene therapy also offers a safety advantage for minimizing retroviral vector-mediated insertional mutagenesis.

Genealogical Diversity of Endogenous Retrovirus in the Jawless Fish Genome.

Retroviral integration into ancient vertebrate genomes left traces that can shed light on the early history of viruses. In this study, we explored the early evolution of retroviruses by isolating nine Spuma endogenous retroviruses (ERVs) and one Epsilon ERV from the genomes of Agnatha and Chondrichthyes. Phylogenetic analysis of protein sequences revealed a striking pattern of co-evolution between jawless fish ERV and their host, while shark ERV underwent ancient cross-class viral transmission with jawless fish, ray-finned fish, and amphibians. Nucleotide sequence analysis showed that jawless fish ERV emerged in the Palaeozoic period, relatively later than ray-finned fish ERV. Moreover, codon analysis suggested that the jawless fish ERV employed an infection strategy that mimics the host codon. The genealogical diversity of ERVs in the jawless fish genome highlights the importance of studying different viral species. Overall, our findings provide valuable insights into the evolution of retroviruses and their interactions with their hosts.

Unraveling the palindromic and nonpalindromic motifs of retroviral integration site sequences by statistical mixture models.

A weak palindromic nucleotide motif is the hallmark of retroviral integration site alignments. Given that the majority of target sequences are not palindromic, the current model explains the symmetry by an overlap of the nonpalindromic motif present on one of the half-sites of the sequences. Here, we show that the implementation of multicomponent mixture models allows for different interpretations consistent with the existence of both palindromic and nonpalindromic submotifs in the sets of integration site sequences. We further show that the weak palindromic motifs result from freely combined site-specific submotifs restricted to only a few positions proximal to the site of integration. The submotifs are formed by either palindrome-forming nucleotide preference or nucleotide exclusion. Using the mixture models, we also identify HIV-1-favored palindromic sequences in Alu repeats serving as local hotspots for integration. The application of the novel statistical approach provides deeper insight into the selection of retroviral integration sites and may prove to be a valuable tool in the analysis of any type of DNA motifs.

GRL-142 binds to and impairs HIV-1 integrase nuclear localization signal and potently suppresses highly INSTI-resistant HIV-1 variants.

Nuclear localization signal (NLS) of HIV-1 integrase (IN) is implicated in nuclear import of HIV-1 preintegration complex (PIC). Here, we established a multiclass drug-resistant HIV-1 variant (HIVKGD) by consecutively exposing an HIV-1 variant to various antiretroviral agents including IN strand transfer inhibitors (INSTIs). HIVKGD was extremely susceptible to a previously reported HIV-1 protease inhibitor, GRL-142, with IC50 of 130 femtomolar. When cells were exposed to HIVKGD IN-containing recombinant HIV in the presence of GRL-142, significant decrease of unintegrated 2-LTR circular cDNA was observed, suggesting that nuclear import of PIC was severely compromised by GRL-142. X-ray crystallographic analyses revealed that GRL-142 interacts with NLS's putative sequence (DQAEHLK) and sterically blocks the nuclear transport of GRL-142-bound HIVKGD's PIC. Highly INSTI-resistant HIV-1 variants isolated from heavily INSTI-experienced patients proved to be susceptible to GRL-142, suggesting that NLS-targeting agents would serve as salvage therapy agents for highly INSTI-resistant variant-harboring individuals. The data should offer a new modality to block HIV-1 infectivity and replication and shed light on developing NLS inhibitors for AIDS therapy.

The HIV-1 Capsid-Targeted Inhibitor GSK878 Alters Selection of Target Sites for HIV DNA Integration.

Decades of effort have yielded highly effective antiviral agents to treat HIV, but viral strains have evolved resistance to each inhibitor type, focusing attention on the importance of developing new inhibitor classes. A particularly promising new target is the HIV capsid, the function of which can be disrupted by highly potent inhibitors that persist long term in treated subjects. Studies with such inhibitors have contributed to an evolving picture of the role of capsid itself-the inhibitors, like certain capsid protein (CA) amino acid substitutions, can disrupt intracellular trafficking to alter the selection of target sites for HIV DNA integration in cellular chromosomes. In this study, we compare effects on HIV integration targeting for two potent inhibitors-a new molecule targeting CA, GSK878, and the previously studied lenacapavir (LEN, formerly known as GS-6207). We find that both inhibitors reduce integration in active transcription units and near epigenetic marks associated with active transcription. A careful study of integration near repeated sequences indicated frequencies were also altered for integration within multiple repeat classes. One notable finding was increased integration in centromeric satellite repeats in the presence of LEN and GSK878, which is of interest because proviruses integrated in centromeric repeats have been associated with transcriptional repression, inducibility, and latency. These data add to the picture that CA protein remains associated with preintegration complexes through the point in infection during which target sites for integration are selected, and specify new aspects of the consequences of disrupting this mechanism.

Wild sheep on St Kilda.

Veterinary RecordVolume 192, Issue 11 p. 450-452 Animal Welfare Wild sheep on St Kilda First published: 02 June 2023 https://doi.org/10.1002/vetr.3145Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL No abstract is available for this article. References 1Jewell PA, Milner C, Morton Boyd J. Island Survivors: The Ecology of the Soay Sheep on St Kilda. London, Athlone, 1974 2Clutton-Brock J, Dennis-Bryan K, Armitage P, et al. Osteology of the Soay sheep. https://bit.ly/42X1hMc (accessed 16 May 2023) 3Kijas JW, Lenstra JA, Hayes B, et al. Genomic analysis of the world's sheep breeds reveals high levels of historic mixture and strong recent selection. PLoS Biol 2012; 10:e1001258 4Chessa B, Pereira F, Arnaud F, et al. Revealing the history of sheep domestication using retrovirus integrations. Science 2009; 324: 532– 6 5 Home Office. Animals (Scientific Procedures) Act 1986. Working with animals taken from the wild. https://tinyurl.com/4pkyd2f4 (accessed 24 May 2023) 6Fleming A. Soay sheep: the back story. Cam Archaeol J 2021; 31: 419– 36 7Harman M. An Isle called Hirte: History and Culture of the St Kildans to 1930. Maclean Press, 1997 8Clutton-Brock TH. The causes and consequences of instability. In: TH Clutton-Brock, JM Pemberton eds. Soay Sheep: Dynamics and Selection in an Island Population. Cambridge University Press, 2004: 304, 307 9Buckland D, Charlesworth G. PE2021: Ensure the definition of protected animals in the Animal Health and Welfare (Scotland) Act 2006 applies to the sheep on St Kilda. https://tinyurl.com/276skns9 (accessed 24 May 2023) Volume192, Issue113/10 June 2023Pages 450-452 ReferencesRelatedInformation