Protein composition of the hepatitis A virus quasi-envelope

The nonlytic cellular egress of picornaviruses in extracellular vesicles is likely to be important in disease pathogenesis, but the mechanism(s) underlying this process and the origins of the membranes surrounding virions exiting the cell are poorly understood. We describe a quantitative proteomics analysis of quasi-enveloped hepatitis A virus (eHAV) virions that shows capsids are selected as cargo for vesicular export via a highly specific process, and that infectious eHAV virions possess a host protein complement similar to that of exosomes with CD9 and DPP4 displayed on their surface. eHAV-associated proteins are highly enriched for endolysosomal components and lack markers of autophagy, suggesting an exosome-like mechanism of endosomal sorting complex required for transport-mediated eHAV biogenesis involving endosomal budding that is distinct from the autophagosome-mediated release proposed previously for enteroviruses

Human pDCs preferentially sense enveloped hepatitis A virions

Unlike other picornaviruses, hepatitis A virus (HAV) is cloaked in host membranes when released from cells, providing protection from neutralizing antibodies and facilitating spread in the liver. Acute HAV infection is typified by minimal type I IFN responses; therefore, we questioned whether plasmacytoid dendritic cells (pDCs), which produce IFN when activated, are capable of sensing enveloped virions (eHAV). Although concentrated nonenveloped virus failed to activate freshly isolated human pDCs, these cells produced substantial amounts of IFN-α via TLR7 signaling when cocultured with infected cells. pDCs required either close contact with infected cells or exposure to concentrated culture supernatants for IFN-α production. In isopycnic and rate-zonal gradients, pDC-activating material cosedimented with eHAV but not membrane-bound acetylcholinesterase, suggesting that eHAV, and not viral RNA exosomes, is responsible for IFN-α induction. pDC activation did not require virus replication and was associated with efficient eHAV uptake, which was facilitated by phosphatidylserine receptors on pDCs. In chimpanzees, pDCs were transiently recruited to the liver early in infection, during or shortly before maximal intrahepatic IFN-stimulated gene expression, but disappeared prior to inflammation onset. Our data reveal that, while membrane envelopment protects HAV against neutralizing antibody, it also facilitates an early but limited detection of HAV infection by pDCs

A pathogenic picornavirus acquires an envelope by hijacking cellular membranes

Animal viruses are broadly categorized structurally by the presence or absence of an envelope composed of a lipid-bilayer membrane1, attributes that profoundly affect stability, transmission, and immune recognition. Among those lacking an envelope, the Picornaviridae are a large and diverse family of positive-strand RNA viruses that includes hepatitis A virus (HAV), an ancient human pathogen that remains a common cause of enterically-transmitted hepatitis2–4. HAV infects in a stealth-like manner and replicates efficiently in the liver5. Virus-specific antibodies appear only after 3–4 weeks of infection, and typically herald its resolution3,4. Although unexplained mechanistically, both anti-HAV antibody and inactivated whole-virus vaccines prevent disease when administered as late as 2 weeks after exposure6, when virus replication is well established in the liver5. Here, we show that HAV released from cells is cloaked in host-derived membranes, thereby protecting the virion from antibody-mediated neutralization. These enveloped viruses (“eHAV”) resemble exosomes7, small vesicles that are increasingly recognized to play important roles in intercellular communications. They are fully infectious, sensitive to chloroform extraction, and circulate in the blood of infected humans. Their biogenesis is dependent upon host proteins associated with endosomal-sorting complexes required for transport (ESCRT)8, VPS4B and ALIX. While the hijacking of membranes by HAV facilitates escape from neutralizing antibodies and likely promotes virus spread within the liver, anti-capsid antibodies restrict replication following infection with eHAV, suggesting a possible explanation for post-exposure prophylaxis. Membrane hijacking by HAV blurs the classic distinction between “enveloped” and “nonenveloped” viruses, and has broad implications for mechanisms of viral egress from infected cells as well as host immune responses

Loss of immune escape mutations during persistent HCV infection in pregnancy enhances replication of vertically transmitted viruses

Globally, about 1% of pregnant women are persistently infected with the hepatitis C virus (HCV)1. Vertical transmission occurs in 3–5% of cases2 and accounts for most new childhood HCV infections1,3. HCV-specific CD8+ cytotoxic T-lymphocytes (CTLs) play a vital role in the clearance of acute infections4–6, but in the 60–80% of infections that persist these cells become functionally exhausted or select mutant viruses that escape T-cell recognition7–9. Increased HCV replication during pregnancy10,11 suggests that maternofetal immune tolerance mechanisms12 may further impair HCV-specific CTLs, limiting their selection pressure on persistent viruses. To assess this possibility, we characterized the circulating viral quasispecies during and after consecutive pregnancies. This revealed a loss of some escape mutations in class I epitopes in pregnancy associated with emergence of more fit viruses13. CTL selection pressure was reimposed after childbirth, when escape mutations in these epitopes again predominated in the quasispecies and viral load dropped sharply14. Importantly, viruses transmitted perinatally were those with enhanced fitness due to reversion of escape mutations. Our findings indicate that immunoregulatory changes of pregnancy reduce CTL selection pressure on HCV class I epitopes, thereby facilitating vertical transmission of viruses with optimized replicative fitness

Deduced consensus sequence of Sindbis virus strain AR339: mutations contained in laboratory strains which affect cell culture and in vivo phenotypes.

The consensus sequence of the Sindbis virus AR339 isolate, the prototype alphavirus, has been deduced. THe results presented here suggest (i) that a substantial proportion of the sequence divergence evident between the consensus sequence and sequences of laboratory strains of AR339 has resulted from selection for efficient growth in cell culture, (ii) that many of these changes affect the virulence of the virus in animal models, and (iii) that such modified genetic backgrounds present in laboratory strains can exert a significant influence on genetic studies of virus pathogenesis and host range. A laboratory strain of Sindbis virus AR339 was sequenced and cloned as a cDNA (pTRSB) from which infectious virus (TRSB) could be derived. The consensus sequence was deduced from the complete sequences of pTRSB and HRsp (E. G. Strauss, C. M. Rice, and J. H. Strauss, Virology 133:92-110, 1984), from partial sequences of the glycoprotein genes of three other AR339 laboratory strains, and by comparison with the sequences of the glycoprotein genes of three other AR339 sequence. HRsp differed form the consensus sequence by eight coding changes, and TRSB differed by three coding changes. In the 5' untranslated region, HRsp differed from the consensus sequence at nucleotide (nt) 5. These differences were likely the result of cell culture passage of the original AR339 isolate. At three of the difference loci (one in TRSB and two in HRsp), selection of cell-culture-adaptive mutations was documented with Sindbis virus or other alphaviruses. Selection in cell culture often results in attenuation of virulence in animals. Considering the TRSB and HRsp sequences together, one noncoding difference from the consensus (an A-for-G substitution in the 5' untranslated region at nt 5) and six coding differences in the glycoprotein genes (at E2 amino acids 1, 3, 70, and 172 and at E1 amino acids 72 and 237) were at loci which, either individually or in combination, significantly affected alphavirus virulence in mice. Although the levels of virulence of isogenic strains containing either nt 5 A or nt 5 G did not differ significantly in neonatal mice, the presence of nt 5 A greatly enhanced the effect of a second attenuating mutation in the E2 gene. These results suggest that minimal differences in the "wild type" genetic background into which an additional mutation is introduced can have a dramatic effect on apparent virulence and pathogenesis phenotypes. A cDNA clone of the consensus AR339 sequence, a sequence devoid of occult attenuating mutations introduced by cell culture passage, will allow the molecular genetic examination of cell culture and in vivo phenotypes of a virus which may best reflect the sequence of Sindbis virus AR339 at the time of its isolation

Spectrum of KV2.1 Dysfunction in KCNB1‐Associated Neurodevelopmental Disorders

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/152486/1/ana25607.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/152486/2/ana25607_am.pd

Hundreds of variants clustered in genomic loci and biological pathways affect human height

Most common human traits and diseases have a polygenic pattern of inheritance: DNA sequence variants at many genetic loci influence the phenotype. Genome-wide association (GWA) studies have identified more than 600 variants associated with human traits, but these typically explain small fractions of phenotypic variation, raising questions about the use of further studies. Here, using 183,727 individuals, we show that hundreds of genetic variants, in at least 180 loci, influence adult height, a highly heritable and classic polygenic trait. The large number of loci reveals patterns with important implications for genetic studies of common human diseases and traits. First, the 180 loci are not random, but instead are enriched for genes that are connected in biological pathways (P = 0.016) and that underlie skeletal growth defects (P < 0.001). Second, the likely causal gene is often located near the most strongly associated variant: in 13 of 21 loci containing a known skeletal growth gene, that gene was closest to the associated variant. Third, at least 19 loci have multiple independently associated variants, suggesting that allelic heterogeneity is a frequent feature of polygenic traits, that comprehensive explorations of already-discovered loci should discover additional variants and that an appreciable fraction of associated loci may have been identified. Fourth, associated variants are enriched for likely functional effects on genes, being over-represented among variants that alter amino-acid structure of proteins and expression levels of nearby genes. Our data explain approximately 10% of the phenotypic variation in height, and we estimate that unidentified common variants of similar effect sizes would increase this figure to approximately 16% of phenotypic variation (approximately 20% of heritable variation). Although additional approaches are needed to dissect the genetic architecture of polygenic human traits fully, our findings indicate that GWA studies can identify large numbers of loci that implicate biologically relevant genes and pathways.

Nonlytic cellular release of hepatitis A virus requires dual capsid recruitment of the ESCRT-associated Bro1 domain proteins HD-PTP and ALIX

Although picornaviruses are conventionally considered ‘nonenveloped’, members of multiple picornaviral genera are released nonlytically from infected cells in extracellular vesicles. The mechanisms underlying this process are poorly understood. Here, we describe interactions of the hepatitis A virus (HAV) capsid with components of host endosomal sorting complexes required for transport (ESCRT) that play an essential role in release. We show release of quasi-enveloped virus (eHAV) in exosome-like vesicles requires a conserved export signal located within the 8 kDa C-terminal VP1 pX extension that functions in a manner analogous to late domains of canonical enveloped viruses. Fusing pX to a self-assembling engineered protein nanocage (EPN-pX) resulted in its ESCRT-dependent release in extracellular vesicles. Mutational analysis identified a 24 amino acid peptide sequence located within the center of pX that was both necessary and sufficient for nanocage release. Deleting a YxxL motif within this sequence ablated eHAV release, resulting in virus accumulating intracellularly. The pX export signal is conserved in non-human hepatoviruses from a wide range of mammalian species, and functional in pX sequences from bat hepatoviruses when fused to the nanocage protein, suggesting these viruses are released as quasi-enveloped virions. Quantitative proteomics identified multiple ESCRT-related proteins associating with EPN-pX, including ALG2-interacting protein X (ALIX), and its paralog, tyrosine-protein phosphatase non-receptor type 23 (HD-PTP), a second Bro1 domain protein linked to sorting of ubiquitylated cargo into multivesicular endosomes. RNAi-mediated depletion of either Bro1 domain protein impeded eHAV release. Super-resolution fluorescence microscopy demonstrated colocalization of viral capsids with endogenous ALIX and HD-PTP. Co-immunoprecipitation assays using biotin-tagged peptides and recombinant proteins revealed pX interacts directly through the export signal with N-terminal Bro1 domains of both HD-PTP and ALIX. Our study identifies an exceptionally potent viral export signal mediating extracellular release of virus-sized protein assemblies and shows release requires non-redundant activities of both HD-PTP and ALIX

Genome-wide association identifies nine common variants associated with fasting proinsulin levels and provides new insights into the pathophysiology of type 2 diabetes.

OBJECTIVE: Proinsulin is a precursor of mature insulin and C-peptide. Higher circulating proinsulin levels are associated with impaired β-cell function, raised glucose levels, insulin resistance, and type 2 diabetes (T2D). Studies of the insulin processing pathway could provide new insights about T2D pathophysiology. RESEARCH DESIGN AND METHODS: We have conducted a meta-analysis of genome-wide association tests of ∼2.5 million genotyped or imputed single nucleotide polymorphisms (SNPs) and fasting proinsulin levels in 10,701 nondiabetic adults of European ancestry, with follow-up of 23 loci in up to 16,378 individuals, using additive genetic models adjusted for age, sex, fasting insulin, and study-specific covariates. RESULTS: Nine SNPs at eight loci were associated with proinsulin levels (P < 5 × 10(-8)). Two loci (LARP6 and SGSM2) have not been previously related to metabolic traits, one (MADD) has been associated with fasting glucose, one (PCSK1) has been implicated in obesity, and four (TCF7L2, SLC30A8, VPS13C/C2CD4A/B, and ARAP1, formerly CENTD2) increase T2D risk. The proinsulin-raising allele of ARAP1 was associated with a lower fasting glucose (P = 1.7 × 10(-4)), improved β-cell function (P = 1.1 × 10(-5)), and lower risk of T2D (odds ratio 0.88; P = 7.8 × 10(-6)). Notably, PCSK1 encodes the protein prohormone convertase 1/3, the first enzyme in the insulin processing pathway. A genotype score composed of the nine proinsulin-raising alleles was not associated with coronary disease in two large case-control datasets. CONCLUSIONS: We have identified nine genetic variants associated with fasting proinsulin. Our findings illuminate the biology underlying glucose homeostasis and T2D development in humans and argue against a direct role of proinsulin in coronary artery disease pathogenesis

Regulatory T Cell Responses in Participants with Type 1 Diabetes after a Single Dose of Interleukin-2: A Non-Randomised, Open Label, Adaptive Dose-Finding Trial

BACKGROUND: Interleukin-2 (IL-2) has an essential role in the expansion and function of CD4+ regulatory T cells (Tregs). Tregs reduce tissue damage by limiting the immune response following infection and regulate autoreactive CD4+ effector T cells (Teffs) to prevent autoimmune diseases, such as type 1 diabetes (T1D). Genetic susceptibility to T1D causes alterations in the IL-2 pathway, a finding that supports Tregs as a cellular therapeutic target. Aldesleukin (Proleukin; recombinant human IL-2), which is administered at high doses to activate the immune system in cancer immunotherapy, is now being repositioned to treat inflammatory and autoimmune disorders at lower doses by targeting Tregs. METHODS AND FINDINGS: To define the aldesleukin dose response for Tregs and to find doses that increase Tregs physiologically for treatment of T1D, a statistical and systematic approach was taken by analysing the pharmacokinetics and pharmacodynamics of single doses of subcutaneous aldesleukin in the Adaptive Study of IL-2 Dose on Regulatory T Cells in Type 1 Diabetes (DILT1D), a single centre, non-randomised, open label, adaptive dose-finding trial with 40 adult participants with recently diagnosed T1D. The primary endpoint was the maximum percentage increase in Tregs (defined as CD3+CD4+CD25highCD127low) from the baseline frequency in each participant measured over the 7 d following treatment. There was an initial learning phase with five pairs of participants, each pair receiving one of five pre-assigned single doses from 0.04 × 106 to 1.5 × 106 IU/m2, in order to model the dose-response curve. Results from each participant were then incorporated into interim statistical modelling to target the two doses most likely to induce 10% and 20% increases in Treg frequencies. Primary analysis of the evaluable population (n = 39) found that the optimal doses of aldesleukin to induce 10% and 20% increases in Tregs were 0.101 × 106 IU/m2 (standard error [SE] = 0.078, 95% CI = -0.052, 0.254) and 0.497 × 106 IU/m2 (SE = 0.092, 95% CI = 0.316, 0.678), respectively. On analysis of secondary outcomes, using a highly sensitive IL-2 assay, the observed plasma concentrations of the drug at 90 min exceeded the hypothetical Treg-specific therapeutic window determined in vitro (0.015-0.24 IU/ml), even at the lowest doses (0.040 × 106 and 0.045 × 106 IU/m2) administered. A rapid decrease in Treg frequency in the circulation was observed at 90 min and at day 1, which was dose dependent (mean decrease 11.6%, SE = 2.3%, range 10.0%-48.2%, n = 37), rebounding at day 2 and increasing to frequencies above baseline over 7 d. Teffs, natural killer cells, and eosinophils also responded, with their frequencies rapidly and dose-dependently decreased in the blood, then returning to, or exceeding, pretreatment levels. Furthermore, there was a dose-dependent down modulation of one of the two signalling subunits of the IL-2 receptor, the β chain (CD122) (mean decrease = 58.0%, SE = 2.8%, range 9.8%-85.5%, n = 33), on Tregs and a reduction in their sensitivity to aldesleukin at 90 min and day 1 and 2 post-treatment. Due to blood volume requirements as well as ethical and practical considerations, the study was limited to adults and to analysis of peripheral blood only. CONCLUSIONS: The DILT1D trial results, most notably the early altered trafficking and desensitisation of Tregs induced by a single ultra-low dose of aldesleukin that resolves within 2-3 d, inform the design of the next trial to determine a repeat dosing regimen aimed at establishing a steady-state Treg frequency increase of 20%-50%, with the eventual goal of preventing T1D. TRIAL REGISTRATION: ISRCTN Registry ISRCTN27852285; ClinicalTrials.gov NCT01827735.This is the final version of the article. It first appeared from the Public Library of Science via http://dx.doi.org/10.1371/journal.pmed.100213