Biology subjects

Chapman, M. S.

Publications and source records attributed to Chapman, M. S..

10 recordsLinked to original sources

bioRxiv2026

Cryo-EM Reveals VP1/VP2 N-Terminal Conformations in Adeno-Associated Virus 2 Capsids.

The AAV2 capsid assembles from three viral proteins, VP1, VP2, and VP3, expressed from a single cap gene. In the assembled T=1 60-subunit icosahedral capsid, they have a canonical stoichiometry of 1:1:10. Relative to VP3, VP1 and VP2 have N-terminal extensions that are required for endosomal escape and nuclear trafficking; however, these regions remain poorly resolved due to low stoichiometry, symmetry mismatches, and conformational heterogeneity of their N-termini. Here, we generated empty AAV2 capsids using a baculovirus expression system with time-course harvests, yielding approximately 2:3:5 and 1:2:7. VP1:VP2:VP3 ratios on days 3 and 5 post-infection, respectively. Cryo-EM single-particle and subparticle reconstructions revealed distinct N-terminal configurations associated with these different VP stoichiometries. Day 3 capsids contained an internal density protruding from the twofold axis and lacked the ordered {beta}A preceding residue 237, whereas day 5 capsids lacked the twofold protrusion and instead showed density associated with the fivefold axis, with {beta}A ordered to approximately residues 219-221. Fivefold subparticle reconstruction of day 5 capsids further resolved a density plug occluding the pore and an underlying density ring. These findings link VP stoichiometry to distinct conformations of the VP1/VP2 N-termini and provide structural evidence for alternative organization of these regions within the AAV2 capsid, offering new insights into the conformational landscape of AAV capsid heterogeneity.

biophysics↗

bioRxiv2025

Segregating DNA lesions point to high selective advantage of tumor initiating cells

The complications with identifying cells at the origin of cancer and tracking their early divisions impede studies of cancer initiation. Recently, it was shown that some DNA lesions generated by a pulse of damage-inducing mutagen persist over multiple rounds of replication. Segregation of DNA lesions in the early genealogy of an expanding clone leaves a statistically interpretable footprint of cancer initiating events. Specifically, it allows for estimating the number of cell divisions between the initiating DNA lesion and the most recent common ancestor of the tumor. Here, we analyze footprints of segregating lesions from a previously published experimental mouse system, as well as post-chemotherapy human metastatic tumors and the blood of chemotherapy treated patients. In all contexts, clones tend to start early, usually within the span of 4 cell generations from mutagen exposure. Using a branching process model, we show that fitness advantage of early cancer drivers exceeds 30%, with each early division leading to at least 1.3 self-renewing cells. We highlight an example of a blood-derived single cell phylogeny with major subclones separated by just two cell divisions. Broadly, our approach allows inference of tumor initiation and growth parameters based on events preceding the most recent common ancestor of the initiating clone as opposed to characteristics of fully grown tumors.

cancer biology↗

bioRxiv2025

JAK2 signalling to HNRNPA1 represses retrotransposon activity in haematopoietic stem cells

Retrotransposon expression must be tightly controlled, particularly in long-lived multipotent stem cells, to prevent deleterious consequences including insertional mutations. Several mechanisms are known to repress retrotransposon transcription during development which are generally thought to persist thereafter. However, integration of retrotransposable elements into host genomes has also provided a major source of genetic variation across evolution. This has generated many sequences that have acquired advantageous host functions, but little is known about retrotransposon expression and mobility in adult stem cells or about how these processes might be dynamically regulated. Here we describe the landscape of somatic retrotransposition in haematopoietic stem cells and identify a previously unrecognised pathway which links cytokine signalling, RNA-modulating HNRNP complexes and repression of retrotransposon activity. Activation of JAK2, by thrombopoietin or gain-of-function JAK2 mutations, triggers tyrosine phosphorylation of HNRNPA1 that represses expression of ERVs, LINEs and SINEs and reduces insertional mutagenesis. This pathway coordinates retrotransposon activity with cellular context and state and provides a mechanism for protecting the haematopoietic stem cell genome.

cell biology↗

bioRxiv2024

Artifacts in single-cell mitochondrial DNA mutation analyses misinform phylogenetic inference

Sequencing mitochondrial DNA (mtDNA) variants from single cells has resolved clonality and lineage in native human samples and clinical specimens. Prior work established that heteroplasmic mtDNA variants can be used to delineate clonality in hematopoiesis, but they have limited ability to reconstruct cellular phylogenies. However, a recent report by Weng et al. challenges the current paradigm by describing an unprecedented number of shared mtDNA variants between cells that reportedly resolve high-resolution phylogenetic trees. We re-examined the claims of Weng et al., and identified two major points of concern regarding this unprecedented connectedness. First, shared variants between cells are disproportionately detected in a single molecule per cell, and second, these variants are enriched 10-20-fold at the edges of mtDNA molecules, reminiscent of artifacts reported in other sequencing approaches. Further, our analyses show that pruning low support and likely artificial mtDNA variants removes nearly all of the reported phylogenetic structure. Thus, we strongly caution against using mtDNA variant calling workflows that rely on minimal evidence, including the computational pipeline introduced in Weng et al., as variants with high connectedness and low evidence are likely artifacts that lead to the construction of false phylogenies.

genomics↗

bioRxiv2024

Clonal evolution of hematopoietic stem cells after cancer chemotherapy

Normal hematopoietic stem and progenitor cells (HSPCs) inherently accumulate somatic mutations and lose clonal diversity with age, processes implicated in the development of myeloid malignancies1. The impact of exogenous stressors, such as cancer chemotherapies, on the genomic integrity and clonal dynamics of normal HSPCs is not well defined. We conducted whole-genome sequencing on 1,032 single-cell-derived HSPC colonies from 10 patients with multiple myeloma (MM), who had undergone various chemotherapy regimens. Our findings reveal that melphalan treatment distinctly increases mutational burden with a unique mutation signature, whereas other MM chemotherapies do not significantly affect the normal mutation rate of HSPCs. Among these therapy-induced mutations were several oncogenic drivers such as TET2 and PPM1D. Phylogenetic analysis showed a clonal architecture in post-treatment HSPCs characterized by extensive convergent evolution of mutations in genes such as TP53 and PPM1D. Consequently, the clonal diversity and structure of post-treatment HSPCs mirror those observed in normal elderly individuals, suggesting an accelerated clonal aging due to chemotherapy. Furthermore, analysis of matched therapy-related myeloid neoplasm (t-MN) samples, which occurred 1-8 years later, enabled us to trace the clonal origin of t-MNs to a single HSPC clone among a group of clones with competing malignant potential, indicating the critical role of secondary mutations in dictating clonal dominance and malignant transformation. Our findings suggest that cancer chemotherapy promotes an oligoclonal architecture with multiple HSPC clones possessing competing leukemic potentials, setting the stage for the selective emergence of a singular clone that evolves into t-MNs after acquiring secondary mutations. These results underscore the importance of further systematic research to elucidate the long-term hematological consequences of cancer chemotherapy.

cancer biology↗

bioRxiv2022

Cross-species permissivity: structure of a goat adeno-associated virus and its complex with the human receptor, AAVR

Adeno-associated virus (AAV) is a small ssDNA satellite virus of high interest (in recombinant form) as a safe and effective gene therapy vector. AAVs human cell entry receptor (AAVR) contains Polycystic Kidney Disease (PKD) domains bound by AAV. Seeking understanding of the spectrum of interactions, goat AAVGo.1 is investigated, because its host is the species most distant from human with reciprocal cross-species cell susceptibility. The structure of AAVGo.1, solved by cryo-EM to 2.9 [A] resolution, is most similar to AAV5. Through ELISA studies, it is shown that AAVGo.1 binds to human AAVR (huAAVR) more strongly than do AAV2 or AAV5, and that it joins AAV5 in a class that binds exclusively to PKD domain 1 (PKD1), in contrast to other AAVs that interact primarily with PKD2. The AAVGo.1 cryo-EM structure of a complex with a PKD12 fragment of huAAVR at 2.4 [A] resolution shows PKD1 bound with minimal change in virus structure, except for disordering of a neighboring surface loop. Only 4 of the 42 capsid protein sequence differences between AAVGo.1 and AAV5 occur at the PKD1 binding interface. These result in only minor conformational changes in AAVR, including a near rigid domain rotation with maximal displacement of the receptor by ~1 [A]. A picture emerges of two classes of AAV with completely different modes of binding to the same AAVR receptor, but within each class atomic interactions are mostly conserved. IMPORTANCEAdeno-Associated Virus (AAV) is a small ssDNA satellite parvovirus. As a recombinant vector with a protein shell encapsidating a transgene, recombinant AAV (rAAV) is a leading delivery vehicle for gene therapy with two FDA-approved treatments and 150 clinical trials for 30 diseases. The human entry receptor huAAVR has five PKD domains. To date, all serotypes, except AAV5, have interacted primarily with the second PKD domain, PKD2. Goat is the AAV host most distant from human with cross-species cell infectivity. AAVGo.1 is similar in structure to AAV5, the two forming a class with a distinct mode of receptor-binding. Within the two classes, binding interactions are mostly conserved, giving an indication of the latitude available in modulating delivery vectors.

microbiology↗

bioRxiv2022

Cryo-EM Structure of Adeno-associated virus-4 at 2.2 A resolution

Adeno-associated virus (AAV) is the vector of choice for several approved gene therapy treatments and is the basis for many ongoing clinical trials. Various strains of AAV exist (referred to as serotypes), each with their own transfection characteristics. Here, we present a high-resolution cryo-electron microscopy structure (2.2 [A]) for AAV serotype 4 (AAV4). The receptor responsible for transduction of the AAV4 clade of AAV viruses (including AAV11, 12 and rh32.33) is unknown. Other AAVs interact with the same cell receptor, Adeno-associated virus receptor (AAVR), in one of two different ways. AAV5-like viruses interact exclusively with the polycystic kidney disease-like [PKD]-1 domain of AAVR while most other AAVs interact primarily with the PKD2 domain. A comparison of the present AAV4 structure with prior corresponding structures of AAV5, AAV2 and AAV1 in complex with AAVR, provides a foundation for understanding why the AAV4-like clade is unable to interact with either PKD1 or PKD2. The conformation of the AAV4 capsid in variable regions I, III, IV and V on the viral surface appears to be sufficiently different from AAV2 to ablate binding with PKD2. Differences between AAV4 and AAV5 in variable region VII appear sufficient to exclude binding with PKD1.

microbiology↗

bioRxiv2022

Adeno-associated Virus Receptor-binding: Flexible Domains and Alternative Conformations through cryo-Electron Tomography of AAV2 and AAV5 complexes

Recombinant forms of adeno-associated virus (rAAV) are vectors of choice in the development of treatments for a number of genetic dispositions. Greater understanding of AAVs molecular virology is needed to underpin needed improvements in efficiency and specificity. Recent advances have included identification of a near universal entry receptor, AAVR, and structures by cryo-electron microscopy (EM) single particle analysis (SPA) that revealed, at high resolution, only the domains of AAVR most tightly bound to AAV. Here, cryogenic electron tomography (cryo-ET) is applied to reveal the neighboring domains of the flexible receptor. For AAV5, where the PKD1 domain is bound strongly, PKD2 is seen in three configurations extending away from the virus. AAV2 binds tightly to the PKD2 domain at a distinct site, and cryo-ET now reveals four configurations of PKD1, all different from that seen in AAV5. The AAV2 receptor complex also shows unmodeled features on the inner surface that appear to be an equilibrium alternate configuration. Other AAV structures start near the 5-fold axis, but now {beta}-strand A is the minor conformer and, for the major conformer, partially ordered N-termini near the 2-fold axis join the canonical capsid jellyroll fold at the {beta}A-{beta}B turn. The addition of cryo-ET is revealing unappreciated complexity that is likely relevant to viral entry and to the development of improved gene therapy vectors. IMPORTANCEWith 150 clinical trials for 30 diseases underway, AAV is a leading gene therapy vector. Immunotoxicity at high doses used to overcome inefficient transduction, has occasionally proven fatal and highlighted gaps in fundamental virology. AAV enters cells, interacting through distinct sites with different domains of the AAVR receptor, according to AAV clade. Single domains are resolved in structures by cryogenic electron microscopy. Here, the adjoining domains are revealed by cryo-electron tomography of AAV2 and AAV5 complexes. They are in flexible configurations interacting minimally with AAV, despite measurable dependence of AAV2 transduction on both domains.

microbiology↗

bioRxiv2021

The longitudinal dynamics and natural history of clonal haematopoiesis

Human cells acquire somatic mutations throughout life, some of which can drive clonal expansion. Such expansions are frequent in the haematopoietic system of healthy individuals and have been termed clonal haematopoiesis (CH). While CH predisposes to myeloid neoplasia and other diseases, we have limited understanding of how and when CH develops, what factors govern its behaviour, how it interacts with ageing and how these variables relate to malignant progression. Here, we track 697 CH clones from 385 individuals aged 55 or older over a median of 13 years. We find that 92.4% of clones expanded at a stable exponential rate over the study period, with different mutations driving substantially different growth rates, ranging from 5% (DNMT3A, TP53) to over 50%/yr (SRSF2-P95H). Growth rates of clones with the same mutation differed by approximately +/-5%/yr, proportionately impacting "slow" drivers more substantially. By combining our time-series data with phylogenetic analysis of 1,731 whole genome-sequenced haematopoietic colonies from 7 older individuals, we reveal distinct patterns of lifelong clonal behaviour. DNMT3A-mutant clones preferentially expanded early in life and displayed slower growth in old age, in the context of an increasingly competitive oligoclonal landscape. By contrast, splicing gene mutations only drove expansion later in life, while growth of TET2-mutant clones showed minimal age-dependency. Finally, we show that mutations driving faster clonal growth carry a higher risk of malignant progression. Our findings characterise the lifelong natural history of CH and give fundamental insights into the interactions between somatic mutation, ageing and clonal selection.

cancer biology↗

bioRxiv2020

Patterns of within-host genetic diversity in SARS-CoV-2

Monitoring the spread of SARS-CoV-2 and reconstructing transmission chains has become a major public health focus for many governments around the world. The modest mutation rate and rapid transmission of SARS-CoV-2 prevents the reconstruction of transmission chains from consensus genome sequences, but within-host genetic diversity could theoretically help identify close contacts. Here we describe the patterns of within-host diversity in 1,181 SARS-CoV-2 samples sequenced to high depth in duplicate. 95% of samples show within-host mutations at detectable allele frequencies. Analyses of the mutational spectra revealed strong strand asymmetries suggestive of damage or RNA editing of the plus strand, rather than replication errors, dominating the accumulation of mutations during the SARS-CoV-2 pandemic. Within and between host diversity show strong purifying selection, particularly against nonsense mutations. Recurrent within-host mutations, many of which coincide with known phylogenetic homoplasies, display a spectrum and patterns of purifying selection more suggestive of mutational hotspots than recombination or convergent evolution. While allele frequencies suggest that most samples result from infection by a single lineage, we identify multiple putative examples of co-infection. Integrating these results into an epidemiological inference framework, we find that while sharing of within-host variants between samples could help the reconstruction of transmission chains, mutational hotspots and rare cases of superinfection can confound these analyses.

genomics↗