Mutations, substitutions, and selection: Linking mutagenic processes to cancer using evolutionary theory.

Cancers are the product of evolutionary events, where molecular variation occurs and accumulates in tissues and tumors. Sequencing of this molecular variation informs not only which variants are driving tumorigenesis, but also the mechanisms behind what is fueling mutagenesis. Both of these details are crucial for preventing premature deaths due to cancer, whether it is by targeting the variants driving the cancer phenotype or by measures to prevent exogenous mutations from contributing to somatic evolution. Here, we review tools to determine both molecular signatures and cancer drivers, and avenues by which these metrics may be linked.

Variation in mesenchymal KITL/SCF and IGF1 expression in middle age underlies steady-state hematopoietic stem cell aging.

ABSTRACT: Intrinsic molecular programs and extrinsic factors including proinflammatory molecules are understood to regulate hematopoietic aging. This is based on foundational studies using genetic perturbation to evaluate causality. However, individual organisms exhibit natural variation in the hematopoietic aging phenotypes and the molecular basis of this heterogeneity is poorly understood. Here, we generated individual single-cell transcriptomic profiles of hematopoietic and nonhematopoietic cell types in 5 young adult and 9 middle-aged C57BL/6J female mice, providing a web-accessible transcriptomic resource for the field. Among all assessed cell types, hematopoietic stem cells (HSCs) exhibited the greatest phenotypic variation in expansion among individual middle-aged mice. We computationally pooled samples to define modules representing the molecular signatures of middle-aged HSCs and interrogated, which extrinsic regulatory cell types and factors would predict the variance in these signatures between individual middle-aged mice. Decline in signaling mediated by adiponectin, kit ligand (KITL) and insulin-like growth factor 1 (IGF1) from mesenchymal stromal cells (MSCs) was predicted to have the greatest transcriptional impact on middle-aged HSCs, as opposed to signaling mediated by endothelial cells or mature hematopoietic cell types. In individual middle-aged mice, lower expression of Kitl and Igf1 in MSCs was highly correlated with reduced lymphoid lineage commitment of HSCs and increased signatures of differentiation-inactive HSCs. These signatures were independent of expression of aging-associated proinflammatory cytokines including interleukin-1ß (IL-1ß), IL-6, tumor necrosis factor α and RANTES. In sum, we find that Kitl and Igf1 expression are coregulated and variable between individual mice at the middle age and expression of these factors is predictive of HSC activation and lymphoid commitment independently of inflammation.

Mesenchymal Stromal Cell Senescence Induced by Dnmt3a -Mutant Hematopoietic Cells is a Targetable Mechanism Driving Clonal Hematopoiesis and Initiation of Hematologic Malignancy.

Clonal hematopoiesis (CH) can predispose to blood cancers due to enhanced fitness of mutant hematopoietic stem and progenitor cells (HSPCs), but the mechanisms driving this progression are not understood. We hypothesized that malignant progression is related to microenvironment-remodelling properties of CH-mutant HSPCs. Single-cell transcriptomic profiling of the bone marrow microenvironment in Dnmt3a R878H/+ mice revealed signatures of cellular senescence in mesenchymal stromal cells (MSCs). Dnmt3a R878H/+ HSPCs caused MSCs to upregulate the senescence markers SA-ß-gal, BCL-2, BCL-xL, Cdkn1a (p21) and Cdkn2a (p16), ex vivo and in vivo . This effect was cell contact-independent and can be replicated by IL-6 or TNFα, which are produced by Dnmt3a R878H/+ HSPCs. Depletion of senescent MSCs in vivo reduced the fitness of Dnmt3a R878H/+ hematopoietic cells and the progression of CH to myeloid neoplasms using a sequentially inducible Dnmt3a ; Npm1 -mutant model. Thus, Dnmt3a -mutant HSPCs reprogram their microenvironment via senescence induction, creating a self-reinforcing niche favoring fitness and malignant progression. Statement of Significance: Mesenchymal stromal cell senescence induced by Dnmt3a -mutant hematopoietic stem and progenitor cells drives clonal hematopoiesis and initiation of hematologic malignancy.

Transcriptional and functional consequences of Oncostatin M signaling on young Dnmt3a-mutant hematopoietic stem cells.

Age-associated clonal hematopoiesis (CH) occurs due to somatic mutations accrued in hematopoietic stem cells (HSCs) that confer a selective growth advantage in the context of aging. The mechanisms by which CH-mutant HSCs gain this advantage with aging are not comprehensively understood. Using unbiased transcriptomic approaches, we identified Oncostatin M (OSM) signaling as a candidate contributor to age-related Dnmt3a-mutant CH. We found that Dnmt3a-mutant HSCs from young adult mice (3-6 months old) subjected to acute OSM stimulation do not demonstrate altered proliferation, apoptosis, hematopoietic engraftment, or myeloid differentiation. Dnmt3a-mutant HSCs from young mice do transcriptionally upregulate an inflammatory cytokine network in response to acute in vitro OSM stimulation as evidenced by significant upregulation of the genes encoding IL-6, IL-1ß, and TNFα. OSM-stimulated Dnmt3a-mutant HSCs also demonstrate upregulation of the anti-inflammatory genes Socs3, Atf3, and Nr4a1. In the context of an aged bone marrow (BM) microenvironment, Dnmt3a-mutant HSCs upregulate proinflammatory genes but not the anti-inflammatory genes Socs3, Atf3, and Nr4a1. The results from our studies suggest that aging may exhaust the regulatory mechanisms that HSCs employ to resolve inflammatory states in response to factors such as OSM.

Oncostatin M is a Master Regulator of an Inflammatory Network in Dnmt3a -Mutant Hematopoietic Stem Cells.

Age-associated clonal hematopoiesis (CH) occurs due to somatic mutations accrued in hematopoietic stem cells (HSCs) that confer a selective advantage in the context of aging. The mechanisms by which CH-mutant HSCs gain this advantage with aging are not comprehensively understood. Using unbiased transcriptomic approaches, we identify Oncostatin M (OSM) signaling as a candidate contributor to aging-driven Dnmt3a -mutant CH. We find that Dnmt3a -mutant HSCs from young mice do not functionally respond to acute OSM stimulation with respect to proliferation, apoptosis, hematopoietic engraftment, or myeloid differentiation. However, young Dnmt3a -mutant HSCs transcriptionally upregulate an inflammatory cytokine network in response to acute OSM stimulation including genes encoding IL-6, IL-1ß and TNFα. In addition, OSM-stimulated Dnmt3a -mutant HSCs upregulate the anti-inflammatory genes Socs3, Atf3 and Nr4a1 , creating a negative feedback loop limiting sustained activation of the inflammatory network. In the context of an aged bone marrow (BM) microenvironment with chronically elevated levels of OSM, Dnmt3a -mutant HSCs upregulate pro-inflammatory genes but do not upregulate Socs3, Atf3 and Nr4a1 . Together, our work suggests that chronic inflammation with aging exhausts the regulatory mechanisms in young CH-mutant HSCs that resolve inflammatory states, and that OSM is a master regulator of an inflammatory network that contributes to age-associated CH.

Expression Microdissection for the Analysis of miRNA in a Single-Cell Type.

Cell-specific microRNA (miRNA) expression estimates are important in characterizing the localization of miRNA signaling within tissues. Much of these data are obtained from cultured cells, a process known to significantly alter miRNA expression levels. Thus, our knowledge of in vivo cell miRNA expression estimates is poor. We previously demonstrated expression microdissection-miRNA-sequencing (xMD-miRNA-seq) to acquire in vivo estimates, directly from formalin-fixed tissues, albeit with a limited yield. In this study, we optimized each step of the xMD process, including tissue retrieval, tissue transfer, film preparation, and RNA isolation, to increase RNA yields and ultimately show strong enrichment for in vivo miRNA expression by qPCR array. These method improvements, such as the development of a noncrosslinked ethylene vinyl acetate membrane, resulted in a 23- to 45-fold increase in miRNA yield, depending on the cell type. By qPCR, miR-200a increased by 14-fold in xMD-derived small intestine epithelial cells, with a concurrent 336-fold reduction in miR-143 relative to the matched nondissected duodenal tissue. xMD is now an optimized method to obtain robust in vivo miRNA expression estimates from cells. xMD will allow formalin-fixed tissues from surgical pathology archives to make theragnostic biomarker discoveries.

An Educational Needs Assessment for Outpatient Palliative Care Clinicians.

Introduction: As the field of palliative medicine continues to grow in community-based settings, outpatient palliative care clinics have become an important site for providing upstream palliative care to patients and families. It is unclear whether current training models, focused predominantly on the inpatient setting, adequately prepare clinicians for outpatient palliative care practice. Methods: We performed an online educational needs assessment survey of physicians and advanced practice providers working in outpatient palliative care clinics. Survey questions focused on the importance of specific palliative care knowledge, skills, and attitudes in outpatient practice using the Accreditation Council of Graduate Medical Education Hospice and Palliative Medicine (HPM) curricular milestones to guide survey development. We also explored clinician perception of training adequacy and current educational needs relevant to outpatient practice. Results: One hundred sixty-four clinicians, including 122 (74.4%) physicians, 32 (19.5%) nurse practitioners, and 8 (4.9%) physician assistants, completed our survey. Clinicians had a median of 10 years of HPM experience and 6 years of outpatient experience. We identified two main areas of perceived knowledge or skill deficit: navigating insurance and prior authorizations and co-management of pain and opioid use disorder. Conclusion: Addressing gaps in education and preparedness for outpatient practice is essential to improve clinician competence and efficiency as well as patient care, safety, and care coordination. This study identifies practice management and opioid stewardship as potential targets for educational interventions. The development of curricula related to these outpatient skills may improve clinicians' ability to provide safe, patient-centered care with confidence.

Distinct Tumor Necrosis Factor Alpha Receptors Dictate Stem Cell Fitness versus Lineage Output in Dnmt3a-Mutant Clonal Hematopoiesis.

Clonal hematopoiesis resulting from the enhanced fitness of mutant hematopoietic stem cells (HSC) associates with both favorable and unfavorable health outcomes related to the types of mature mutant blood cells produced, but how this lineage output is regulated is unclear. Using a mouse model of a clonal hematopoiesis-associated mutation, DNMT3AR882/+ (Dnmt3aR878H/+), we found that aging-induced TNFα signaling promoted the selective advantage of mutant HSCs and stimulated the production of mutant B lymphoid cells. The genetic loss of the TNFα receptor TNFR1 ablated the selective advantage of mutant HSCs without altering their lineage output, whereas the loss of TNFR2 resulted in the overproduction of mutant myeloid cells without altering HSC fitness. These results nominate TNFR1 as a target to reduce clonal hematopoiesis and the risk of associated diseases and support a model in which clone size and mature blood lineage production can be independently controlled to modulate favorable and unfavorable clonal hematopoiesis outcomes. SIGNIFICANCE: Through the identification and dissection of TNFα signaling as a key driver of murine Dnmt3a-mutant hematopoiesis, we report the discovery that clone size and production of specific mature blood cell types can be independently regulated. See related commentary by Niño and Pietras, p. 2724. This article is highlighted in the In This Issue feature, p. 2711.

The "true" acetabular anteversion angle (AV angle): 2D CT versus 3D model.

INTRODUCTION: Different factors can lead to inconsistencies in measurement for the acetabular version using 2D axial CT-cuts. We have defined a "true" anteversion angle (AV angle) in the physiological position of the pelvis in 3D with the largest European population measured to our knowledge. MATERIAL AND METHODS: We analyzed 258 hemipelvises and created 3D models. We compared the results of our AV angle 3D method with the cross-sectional cuts of the same acetabula. We included factors like side, sex, body mass index, and patient positioning. RESULTS: Overall, the mean (SD) AV angle was 16.1 (5.9)° as measured with the 3D method and 22.0 (6.0)° as measured with the 2D method (p < 0.0001). Measured with both the 3D and the 2D method, the AV angle was significantly larger in female than in male individuals (p < 0.0001). In the 2D method, the AV angle estimation was influenced by the pelvic tilt. CONCLUSION: We propose a more accurate method for the measurement of the AV angle of the acetabulum in a 3D model that is not influenced by patient positioning or pelvic tilt. We provide a computational model that will facilitate operative decisions and improve preoperative planning. We confirm that 3D measurement should be the gold standard in measuring the acetabular anteversion.

A randomized controlled trial of everolimus for neurocognitive symptoms in PTEN hamartoma tumor syndrome.

PTEN hamartoma tumor syndrome (PHTS) is a complex neurodevelopmental disorder characterized by mechanistic target of rapamycin (mTOR) overactivity. Limited data suggest that mTOR inhibitors may be therapeutic. No placebo-controlled studies have examined mTOR inhibition on cognition and behavior in humans with PHTS with/without autism. We conducted a 6-month phase II, randomized, double-blinded, placebo-controlled trial to examine the safety profile and efficacy of everolimus (4.5 mg/m2) in individuals (5-45 years) with PHTS. We measured several cognitive and behavioral outcomes, and electroencephalography (EEG) biomarkers. The primary endpoint was a neurocognitive composite derived from Stanford Binet-5 (SB-5) nonverbal working memory score, SB-5 verbal working memory, Conners' Continuous Performance Test hit reaction time and Purdue Pegboard Test score. Forty-six participants underwent 1:1 randomization: n = 24 (everolimus) and n = 22 (placebo). Gastrointestinal adverse events were more common in the everolimus group (P < 0.001). Changes in the primary endpoint between groups from baseline to Month 6 were not apparent (Cohen's d = -0.10, P = 0.518). However, several measures were associated with modest effect sizes (≥0.2) in the direction of improvement, including measures of nonverbal IQ, verbal learning, autism symptoms, motor skills, adaptive behavior and global improvement. There was a significant difference in EEG central alpha power (P = 0.049) and central beta power (P = 0.039) 6 months after everolimus treatment. Everolimus is well tolerated in PHTS; adverse events were similar to previous reports. The primary efficacy endpoint did not reveal improvement. Several secondary efficacy endpoints moved in the direction of improvement. EEG measurements indicate target engagement following 6 months of daily oral everolimus. Trial Registration Information: ClinicalTrials.gov NCT02991807 Classification of Evidence: I.

Physical Activity and Food Environments in and around Schools: A Case Study in Regional North-West Tasmania.

A better understanding of the physical activity (PA) infrastructure in schools, the walkability of neighborhoods close to schools, and the food environments around schools, particularly in rural, socioeconomically challenged areas such as the North-West (NW) of Tasmania, could be important in the wider effort to improve the health of school-age children. Accordingly, this research aimed to assess PA resources, walkability, and food environments in and around schools in three socioeconomically disadvantaged, regional/rural Local Government Areas (LGAs) of Tasmania, Australia. A census of schools (including assessment of the PA infrastructure quality within school grounds), a walkability assessment, and a census of food outlets surrounding schools (through geospatial mapping) were executed. Most of the schools in the study region had access to an oval, basketball/volleyball/netball court, and free-standing exercise equipment. In all instances (i.e., regardless of school type), the quality of the available infrastructure was substantially higher than the number of incivilities observed. Most schools also had good (i.e., within the first four deciles) walkability. Numerous food outlets were within the walking zones of all schools in the study region, with an abundance of food outlets that predominantly sold processed unhealthy food.

Validation of a computational phenotype for finding patients eligible for genetic testing for pathogenic PTEN variants across three centers.

BACKGROUND: Computational phenotypes are most often combinations of patient billing codes that are highly predictive of disease using electronic health records (EHR). In the case of rare diseases that can only be diagnosed by genetic testing, computational phenotypes identify patient cohorts for genetic testing and possible diagnosis. This article details the validation of a computational phenotype for PTEN hamartoma tumor syndrome (PHTS) against the EHR of patients at three collaborating clinical research centers: Boston Children's Hospital, Children's National Hospital, and the University of Washington. METHODS: A combination of billing codes from the International Classification of Diseases versions 9 and 10 (ICD-9 and ICD-10) for diagnostic criteria postulated by a research team at Cleveland Clinic was used to identify patient cohorts for genetic testing from the clinical data warehouses at the three research centers. Subsequently, the EHR-including billing codes, clinical notes, and genetic reports-of these patients were reviewed by clinical experts to identify patients with PHTS. RESULTS: The PTEN genetic testing yield of the computational phenotype, the number of patients who needed to be genetically tested for incidence of pathogenic PTEN gene variants, ranged from 82 to 94% at the three centers. CONCLUSIONS: Computational phenotypes have the potential to enable the timely and accurate diagnosis of rare genetic diseases such as PHTS by identifying patient cohorts for genetic sequencing and testing.

A Spatial Analysis of Access to Physical Activity Infrastructure and Healthy Food in Regional Tasmania.

Prevalence of physical inactivity and obesity continues to increase in regional areas such as North-West (NW) Tasmania and show no signs of abating. It is possible that limited access to physical activity infrastructure (PAI) and healthier food options are exacerbating the low levels of habitual physical activity and obesity prevalence in these communities. Despite a burgeoning research base, concomitant exploration of both physical activity and food environments in rural and regional areas remain scarce. This research evaluated access (i.e., coverage, variety, density, and proximity) to physical activity resources and food outlets in relation to socioeconomic status (SES) in three NW Tasmanian communities. In all three study areas, the PAI and food outlets were largely concentrated in the main urban areas with most recreational tracks and natural amenities located along the coastline or river areas. Circular Head had the lowest total number of PAI (n = 43) but a greater proportion (30%) of free-to-access outdoor amenities. There was marked variation in accessibility to infrastructure across different areas of disadvantage within and between sites. For a considerable proportion of the population, free-to-access natural amenities/green spaces and recreational tracks (73 and 57%, respectively) were beyond 800 m from their households. In relation to food accessibility, only a small proportion of the food outlets across the region sells predominantly healthy (i.e., Tier 1) foods (~6, 13, and 10% in Burnie, Circular Head and Devonport, respectively). Similarly, only a small proportion of the residents are within a reasonable walking distance (i.e., 5-10 min walk) from outlets. In contrast, a much larger proportion of residents lived close to food outlets selling predominantly energy-dense, highly processed food (i.e., Tier 2 outlets). Circular Head had at least twice as many Tier 1 food stores per capita than Devonport and Burnie (0.23 vs. 0.10 and 0.06; respectively) despite recording the highest average distance (4.35 and 5.66 km to Tier 2/Tier 1 stores) to a food outlet. As such, it is possible that both food and physical activity environment layouts in each site are contributing to the obesogenic nature of each community.

Structural understanding of non-nucleoside inhibition in an elongating herpesvirus polymerase.

All herpesviruses encode a conserved DNA polymerase that is required for viral genome replication and serves as an important therapeutic target. Currently available herpesvirus therapies include nucleoside and non-nucleoside inhibitors (NNI) that target the DNA-bound state of herpesvirus polymerase and block replication. Here we report the ternary complex crystal structure of Herpes Simplex Virus 1 DNA polymerase bound to DNA and a 4-oxo-dihydroquinoline NNI, PNU-183792 (PNU), at 3.5 Å resolution. PNU bound at the polymerase active site, displacing the template strand and inducing a conformational shift of the fingers domain into an open state. These results demonstrate that PNU inhibits replication by blocking association of dNTP and stalling the enzyme in a catalytically incompetent conformation, ultimately acting as a nucleotide competing inhibitor (NCI). Sequence conservation of the NCI binding pocket further explains broad-spectrum activity while a direct interaction between PNU and residue V823 rationalizes why mutations at this position result in loss of inhibition.

A randomized double-blind controlled trial of everolimus in individuals with PTEN mutations: Study design and statistical considerations.

This randomized, double-blind controlled trial of everolimus in individuals with germline phosphatase and tensin homolog mutations (PTEN) was designed to evaluate the safety of everolimus compared with placebo and to evaluate the efficacy of everolimus on neurocognition and behavior compared to placebo as measured by standardized neurocognitive and motor measures as well as behavioral questionnaires. The safety profile of everolimus is characterized by manageable adverse events that are generally reversible and non-cumulative. The primary safety endpoint of this study was drop-out rate due to side effects, comparing everolimus versus placebo. We also sought to determine the frequency of adverse events by type and severity. The main efficacy endpoint was a neurocognitive composite computed in two ways: 1) an average for working memory, processing speed, and fine motor subtests; and 2) the same average as above except weighted 2/3, and an additional average based on all other available neurocognitive testing measures assessing the additional domains of nonverbal ability, visuomotor skills, verbal learning, and receptive and expressive language, weighted 1/3. Secondary efficacy endpoints examined the effect of everolimus on overall global clinical improvement, autism symptoms, behavioral problems, and adaptive abilities as measured by validated, standardized instruments. We predicted that the rate of adverse events would be no more than 10% higher in the everolimus group compared to placebo, and overall severity of side effects would be minimal. We also expected that individuals receiving everolimus would show more improvement, relative to those taking placebo, on the composite neurocognitive index.

Structures of active-state orexin receptor 2 rationalize peptide and small-molecule agonist recognition and receptor activation.

Narcolepsy type 1 (NT1) is a chronic neurological disorder that impairs the brain's ability to control sleep-wake cycles. Current therapies are limited to the management of symptoms with modest effectiveness and substantial adverse effects. Agonists of the orexin receptor 2 (OX2R) have shown promise as novel therapeutics that directly target the pathophysiology of the disease. However, identification of drug-like OX2R agonists has proven difficult. Here we report cryo-electron microscopy structures of active-state OX2R bound to an endogenous peptide agonist and a small-molecule agonist. The extended carboxy-terminal segment of the peptide reaches into the core of OX2R to stabilize an active conformation, while the small-molecule agonist binds deep inside the orthosteric pocket, making similar key interactions. Comparison with antagonist-bound OX2R suggests a molecular mechanism that rationalizes both receptor activation and inhibition. Our results enable structure-based discovery of therapeutic orexin agonists for the treatment of NT1 and other hypersomnia disorders.

Long-term trans-inhibition of the hepatitis B and D virus receptor NTCP by taurolithocholic acid.

Human hepatic bile acid transporter Na+/taurocholate cotransporting polypeptide (NTCP) represents the liver-specific entry receptor for the hepatitis B and D viruses (HBV/HDV). Chronic hepatitis B and D affect several million people worldwide, but treatment options are limited. Recently, HBV/HDV entry inhibitors targeting NTCP have emerged as promising novel drug candidates. Nevertheless, the exact molecular mechanism that NTCP uses to mediate virus binding and entry into hepatocytes is still not completely understood. It is already known that human NTCP mRNA expression is downregulated under cholestasis. Furthermore, incubation of rat hepatocytes with the secondary bile acid taurolithocholic acid (TLC) triggers internalization of the rat Ntcp protein from the plasma membrane. In the present study, the long-term inhibitory effect of TLC on transport function, HBV/HDV receptor function, and membrane expression of human NTCP were analyzed in HepG2 and human embryonic kidney (HEK293) cells stably overexpressing NTCP. Even after short-pulse preincubation, TLC had a significant long-lasting inhibitory effect on the transport function of NTCP, but the NTCP protein was still present at the plasma membrane. Furthermore, binding of the HBV/HDV myr-preS1 peptide and susceptibility for in vitro HDV infection were significantly reduced by TLC preincubation. We hypothesize that TLC rapidly accumulates in hepatocytes and mediates long-lasting trans-inhibition of the transport and receptor function of NTCP via a particular TLC-binding site at an intracellularly accessible domain of NTCP. Physiologically, this trans-inhibition might protect hepatocytes from toxic overload of bile acids. Pharmacologically, it provides an interesting novel NTCP target site for potential long-acting HBV/HDV entry inhibitors.NEW & NOTEWORTHY The hepatic bile acid transporter NTCP is a high-affinity receptor for hepatitis B and D viruses. This study shows that TLC rapidly accumulates in NTCP-expressing hepatoma cells and mediates long-lasting trans-inhibition of NTCP's transporter and receptor function via an intracellularly accessible domain, without substantially affecting its membrane expression. This domain is a promising novel NTCP target site for pharmacological long-acting HBV/HDV entry inhibitors.

The current global state of movement and physical activity - the health and economic costs of the inactive phenotype.

Physical inactivity is one of the major contributing factors to the global pandemic of non-communicable diseases. Unfortunately, low levels of habitual movement and physical activity (PA) are seen in an increasing proportion of populations across low- and middle-income countries and high-income countries alike. This new normal - the inactive phenotype - is a significant contributor to multiple health and economic costs. Here we provide a brief historical overview of societal declines in PA, roughly consistent with major transitions in PA and nutrition in recent decades. This is followed by a synthesis of research evidence linking inactivity with poor health outcomes and prevention approaches needed to impact a perpetuation of poor lifestyle behaviors. A major focus of the paper is on the economic/health costs and the reduction of the inactive phenotype. In summary, we demonstrate that the consequences of insufficient PA are manifold, and if sustained, impact short and long-term health and quality of life, along with substantial economic costs.

Chikungunya Virus Strains from Each Genetic Clade Bind Sulfated Glycosaminoglycans as Attachment Factors.

Chikungunya virus (CHIKV) is an arthritogenic alphavirus that causes debilitating musculoskeletal disease. CHIKV displays broad cell, tissue, and species tropism, which may correlate with the attachment factors and entry receptors used by the virus. Cell surface glycosaminoglycans (GAGs) have been identified as CHIKV attachment factors. However, the specific types of GAGs and potentially other glycans to which CHIKV binds and whether there are strain-specific differences in GAG binding are not fully understood. To identify the types of glycans bound by CHIKV, we conducted glycan microarray analyses and discovered that CHIKV preferentially binds GAGs. Microarray results also indicate that sulfate groups on GAGs are essential for CHIKV binding and that CHIKV binds most strongly to longer GAG chains of heparin and heparan sulfate. To determine whether GAG binding capacity varies among CHIKV strains, a representative strain from each genetic clade was tested. While all strains directly bound to heparin and chondroitin sulfate in enzyme-linked immunosorbent assays (ELISAs) and depended on heparan sulfate for efficient cell binding and infection, we observed some variation by strain. Enzymatic removal of cell surface GAGs and genetic ablation that diminishes GAG expression reduced CHIKV binding and infectivity of all strains. Collectively, these data demonstrate that GAGs are the preferred glycan bound by CHIKV, enhance our understanding of the specific GAG moieties required for CHIKV binding, define strain differences in GAG engagement, and provide further evidence for a critical function of GAGs in CHIKV cell attachment and infection.IMPORTANCE Alphavirus infections are a global health threat, contributing to outbreaks of disease in many parts of the world. Recent epidemics caused by CHIKV, an arthritogenic alphavirus, resulted in more than 8.5 million cases as the virus has spread into new geographic regions, including the Western Hemisphere. CHIKV causes disease in the majority of people infected, leading to severe and debilitating arthritis. Despite the severity of CHIKV disease, there are no licensed therapeutics. Since attachment factors and receptors are determinants of viral tropism and pathogenesis, understanding these virus-host interactions can enhance our knowledge of CHIKV infection. We analyzed over 670 glycans and identified GAGs as the main glycan bound by CHIKV. We defined specific GAG components required for CHIKV binding and assessed strain-specific differences in GAG binding capacity. These studies provide insight about cell surface molecules that CHIKV binds, which could facilitate the development of antiviral therapeutics targeting the CHIKV attachment step.