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Alphaviruses, like many other arthropod-borne viruses, infect vertebrate species and insect vectors separated by hundreds of millions of years of evolutionary history. Entry into evolutionarily divergent host cells can be accomplished by recognition of different cellular receptors in different species, or by binding to receptors that are highly conserved across species. Although multiple alphavirus receptors have been described1–3, most are not shared among vertebrate and invertebrate hosts. Here we identify the very low-density lipoprotein receptor (VLDLR) as a receptor for the prototypic alphavirus Semliki forest virus. We show that the E2 and E1 glycoproteins (E2–E1) of Semliki forest virus, eastern equine encephalitis virus and Sindbis virus interact with the ligand-binding domains (LBDs) of VLDLR and apolipoprotein E receptor 2 (ApoER2), two closely related receptors. Ectopic expression of either protein facilitates cellular attachment, and internalization of virus-like particles, a VLDLR LBD–Fc fusion protein or a ligand-binding antagonist block Semliki forest virus E2–E1-mediated infection of human and mouse neurons in culture. The administration of a VLDLR LBD–Fc fusion protein has protective activity against rapidly fatal Semliki forest virus infection in mouse neonates. We further show that invertebrate receptor orthologues from mosquitoes and worms can serve as functional alphavirus receptors. We propose that the ability of some alphaviruses to infect a wide range of hosts is a result of their engagement of evolutionarily conserved lipoprotein receptors and contributes to their pathogenesis., Studies using viral coat glycoproteins show that alphaviruses can enter cells via the very low-density lipoprotein receptor (VLDLR) and apolipoprotein E receptor 2 (ApoER2), members of an evolutionarily conserved family of lipoprotein receptors.

Additional file 4: Supplemental Fig. 1. Effects of human brain extracts on spontaneous firing of cultured cortical neurons are heat labile and are strongest in a high molecular weight fraction. (A) Images of primary rat cortical cultures stained for neuronal markers TAU and DAPI the nuclear marker following 4-day treatment with control brain extract (MGH1887) or AD brain extract (ALB01). Scale bars = 100 μm. (B) Titration of AD brain extract (ALB01, red) compared to an LP-NCI brain extract (MGH1887, blue) at 4-days post-treatment in primary rat cortical neurons. (C) Denaturing extracts by boiling rescues effects on spontaneous firing Spontaneous firing activity within rat cortical cultures following 4-day treatment with native (open points) or boiled (shaded points) human brain extracts. Each point represents a single multi-electrode array, bars represent mean ± SEM. Significant differences between conditions indicated by brackets and associated p-value. Supplemental Fig. 2. Generation and testing of cell-derived GM2A in neurite integrity assay. (A) Western blot probed for GM2A measuring GM2A levels in TBS-soluble brain extracts from 4 individuals: (1) MGH1887, (2) MGH1837, (3) RM304, and (4) RM305. Serial diluted recombinant GM2A ranging from 0.005 to 0.05 μg served as standards. GM2A is estimated to be ~ 1 ng/uL in the brain extracts. (B) Neurite integrity of Day 21 human iNs treated with 2 or 5 ng/uL recombinant GM2A was comparable to that of untreated iNs (0 ng/uL recombinant GM2A). (C) Western blot probed for GM2A measuring GM2A levels in TBS-soluble protein lysates from HEK293 transfected with GM2A or control cDNA construct. Lysates from 2 independent transfections of the GM2A construct were measured. Serial diluted recombinant GM2A ranging from 0.0025 to 0.01 μg served as standards. Total GM2A (endogenous and GFP-tagged) is estimated to be ~ 25 ng/uL in the TBS-soluble protein lysates from GM2A-transfected HEK293 cells. (D-E) Neurite integrity of Day 21 human iNs treated with 0.25 or 0.5 ng/uL HEK293-derived, TBS-soluble GM2A started to show significant reduction 4 hr. after the treatment (E) and GM2A-mediated reduction of neurite integrity is dose-dependent (D&E). N = 10 for each condition. Statistical analyses used unpaired t-test at each time point with Holm-Šídák method. *, p < 0.05; **, p < 0.01; ****, p < 0.0001. (F-G) Neurite integrity of Day 21 human iNs treated with TBS-soluble protein lysates derived from HEK293 cells transfected with GM2A or control cDNA construct. Neurite integrity assessed every 4 hrs (F) and 24-hr post-treatment (G) are presented. N = 15 for each condition. Error bars represent mean ± SEM. Statistical analyses used unpaired t-test at each time point with Holm-Šídák method. **, p < 0.01. Supplemental Fig. 3. Expression of synaptic proteins in neurons with elevated GM2A. (A-G) Western blot probed for VGLUT2, HOMER1, GM2A, and GAPDH in Day 21 human iNs overexpressing GM2A or control at MOI = 5 (transduced on Day 5), and in Day 21 human iNs treated with AD brain extract (ALB) or control (treated from Day 17). N = 5 for each condition. (H-L) Images of VGLUT2 and HOMER1 immunostains in Day 21 human iNs overexpressing GM2A or control at MOI = 5 (transduced on Day 5), and in Day 21 human iNs treated with AD brain extract (ALB) or control (treated from Day 17). The insets show the quantified, representative neuritic segments (N = 15 for each condition). Scale bars: 20 μm. Statistical analyses used unpaired t-test. *, p < 0.05; **, p < 0.01; ****, p < 0.0001; ns, not significant.

Mutations in NHE6 (also termed SLC9A6) cause the X-linked neurological disorder Christianson syndrome (CS) in males. The purpose of this study was to examine the phenotypic spectrum of female carriers of NHE6 mutations. Twenty female carriers from 9 pedigrees were enrolled, ranging from approximately age 2 to 65. A subset of female carriers was assessed using standardized neuropsychological measures. Also, the association of NHE6 expression with markers of brain age was evaluated using 740 participants in the Religious Orders Study (ROS) and Rush Memory and Aging Project (MAP). A majority, but not all, female carriers demonstrated a deficit in at least one neurocognitive domain (85%). A recognizable neuropsychological profile emerged, revealing impairments in visuospatial function, attention, and executive function. Common neuropsychiatric diagnoses included: intellectual disability/developmental delay (20%), learning difficulties (31%), speech/language delays (30%), and attention-deficit/hyperactivity disorder (20%). Notable neurological diagnoses in aging CS female carriers include corticobasal degeneration and atypical parkinsonism. In postmortem brains from the ROS/MAP dataset of normal and pathological aging, decreased NHE6 expression was correlated with greater tau deposition. Our study provides an examination of the phenotypic range in female carriers of NHE6 mutations. The findings indicate that NHE6-related disease in females represents a new neurogenetic condition.