The rockefeller university

Hepatitis C virus (HCV) infection is characterized by persistent replication of a complex mixture of viruses termed a "quasispecies." Transmission is generally associated with a stringent population bottleneck characterized by infection by limited numbers of "transmitted/founder" (T/F) viruses. Characterization of T/F genomes of human immunodeficiency virus type 1 (HIV-1) has been integral to studies of transmission, immunopathogenesis, and vaccine development. Here, we describe the identification of complete T/F genomes of HCV by single-genome sequencing of plasma viral RNA from acutely infected subjects. A total of 2,739 single-genome-derived amplicons comprising 10,966,507 bp from 18 acute-phase and 11 chronically infected subjects were analyzed. Acute-phase sequences diversified essentially randomly, except for the poly(U/UC) tract, which was subject to polymerase slippage. Fourteen acute-phase subjects were productively infected by more than one genetically distinct virus, permitting assessment of recombination between replicating genomes. No evidence of recombination was found among 1,589 sequences analyzed. Envelope sequences of T/F genomes lacked transmission signatures that could distinguish them from chronic infection viruses. Among chronically infected subjects, higher nucleotide substitution rates were observed in the poly(U/UC) tract than in envelope hypervariable region 1. Fourteen full-length molecular clones with variable poly(U/UC) sequences corresponding to seven genotype 1a, 1b, 3a, and 4a T/F viruses were generated. Like most unadapted HCV clones, T/F genomes did not replicate efficiently in Huh 7.5 cells, indicating that additional cellular factors or viral adaptations are necessary for in vitro replication. Full-length T/F HCV genomes and their progeny provide unique insights into virus transmission, virus evolution, and virus-host interactions associated with immunopathogenesis. IMPORTANCE Hepatitis C virus (HCV) infects 2% to 3% of the world's population and exhibits extraordinary genetic diversity. This diversity is mirrored by HIV-1, where characterization of transmitted/founder (T/F) genomes has been instrumental in studies of virus transmission, immunopathogenesis, and vaccine development. Here, we show that despite major differences in genome organization, replication strategy, and natural history, HCV (like HIV-1) diversifies essentially randomly early in infection, and as a consequence, sequences of actual T/F viruses can be identified. This allowed us to capture by molecular cloning the full-length HCV genomes that are responsible for infecting the first hepatocytes and eliciting the initial immune responses, weeks before these events could be directly analyzed in human subjects. These findings represent an enabling experimental strategy, not only for HCV and HIV-1 research, but also for other RNA viruses of medical importance, including West Nile, chikungunya, dengue, Venezuelan encephalitis, and Ebola viruses.

Hepatitis C virus (HCV) uniquely requires the liver-specific microRNA-122 for replication, yet global effects on endogenous miRNA targets during infection are unexplored. Here, high-throughput sequencing and crosslinking immunoprecipitation (HITS-CLIP) experiments of human Argonaute (AGO) during HCV infection showed robust AGO binding on the HCV 5'UTR at known and predicted miR-122 sites. On the human transcriptome, we observed reduced AGO binding and functional mRNA de-repression of miR-122 targets during virus infection. This miR-122 "sponge'' effect was relieved and redirected to miR-15 targets by swapping the miRNA tropism of the virus. Single-cell expression data from reporters containing miR-122 sites showed significant derepression during HCV infection depending on expression level and site number. We describe a quantitative mathematical model of HCV-induced miR-122 sequestration and propose that such miR-122 inhibition by HCV RNA may result in global derepression of host miR-122 targets, providing an environment fertile for the long-term oncogenic potential of HCV.

Human-infecting microbial pathogens all face a serious problem of elimination by the host immune response. Antigenic variation is an effective immune evasion mechanism where the pathogen regularly switches its major surface antigen. In many cases, the major surface antigen is encoded by genes from the same gene family, and its expression is strictly monoallelic. Among pathogens that undergo antigenic variation, Trypanosoma brucei (a kinetoplastid), which causes human African trypanosomiasis, Plasmodium falciparum (an apicomplexan), which causes malaria, Pneumocystis jirovecii (a fungus), which causes pneumonia, and Borrelia burgdorferi (a bacterium), which causes Lyme disease, also express their major surface antigens from loci next to the telomere. Except for Plasmodium, DNA recombination-mediated gene conversion is a major pathway for surface antigen switching in these pathogens. In the last decade, more sophisticated molecular and genetic tools have been developed in T. brucei, and our knowledge of functions of DNA recombination in antigenic variation has been greatly advanced. VSG is the major surface antigen in T. brucei. In subtelomeric VSG expression sites (ESs), VSG genes invariably are flanked by a long stretch of upstream 70-bp repeats. Recent studies have shown that DNA double-strand breaks (DSBs), particularly those in 70-bp repeats in the active ES, are a natural potent trigger for antigenic variation in T. brucei. In addition, telomere proteins can influence VSG switching by reducing the DSB amount at subtelomeric regions. These findings will be summarized and their implications will be discussed in this review.

Interleukin-1 receptor-associated kinase 4 (IRAK-4) deficiency is a primary immunodeficiency of innate immunity. This is the case of a previous healthy toddler and his sibling, who both died of fulminant sepsis due to Pseudomonas aeruginosa. Subsequent genetic analysis demonstrated IRAK-4 deficiency with compound heterozygous splice mutations. Fulminant fatal P. aeruginosa sepsis may be the first manifestation of IRAK-4 deficiency.

Over half a century has passed since interferons (IFNs) were discovered and shown to inhibit virus infection in cultured cells. Since then, researchers have steadily brought to light the molecular details of IFN signaling, catalogued their pleiotropic effects on cells, and harnessed their therapeutic potential for a variety of maladies. While advances have been plentiful, several fundamental questions have yet to be answered and much complexity remains to be unraveled. We explore the current knowledge surrounding four main questions: are type I IFN subtypes differentially produced in response to distinct pathogens? How are IFN subtypes distinguished by cells? What are the mechanisms and consequences of viral antagonism? Lastly, how can the IFN response be harnessed to improve vaccine efficacy?

Nonlinear systems are capable of displaying complex behavior even if this is the result of a small number of interacting time scales. A widely studied case is when complex dynamics emerges out of a nonlinear system being forced by a simple harmonic function. In order to identify if a recorded time series is the result of a nonlinear system responding to a simpler forcing, we develop a discrete nonlinear transformation for time series based on synchronization techniques. This allows a parameter estimation procedure which simultaneously searches for a good fit of the recorded data, and small complexity of a fluctuating driving parameter. We illustrate this procedure using data from respiratory patterns during birdsong production. (C) 2015 AIP Publishing LLC.

A search for resonances and quantum black holes is performed using the dijet mass spectra measured in proton-proton collisions at root s = 8 TeV with the CMS detector at the LHC. The data set corresponds to an integrated luminosity of 19.7 fb(-1). In a search for narrow resonances that couple to quark-quark, quark-gluon, or gluon-gluon pairs, model-independent upper limits, at 95% confidence level, are obtained on the production cross section of resonances, with masses above 1.2 TeV. When interpreted in the context of specific models the limits exclude string resonances with masses below 5.0 TeV; excited quarks below 3.5 TeV; scalar diquarks below 4.7 TeV; W' bosons below 1.9 TeV or between 2.0 and 2.2 TeV; Z' bosons below 1.7 TeV; and Randall-Sundrum gravitons below 1.6 TeV. A separate search is conducted for narrow resonances that decay to final states including b quarks. The first exclusion limit is set for excited b quarks, with a lower mass limit between 1.2 and 1.6 TeV depending on their decay properties. Searches are also carried out for wide resonances, assuming for the first time width-to-mass ratios up to 30%, and for quantum black holes with a range of model parameters. The wide resonance search excludes axigluons and colorons with mass below 3.6 TeV, and color-octet scalars with mass below 2.5 TeV. Lower bounds between 5.0 and 6.3 TeV are set on the masses of quantum black holes.

Dissecting and studying cellular systems requires the ability to specifically isolate distinct proteins along with the co-assembled constituents of their associated complexes. Affinity capture techniques leverage high affinity, high specificity reagents to target and capture proteins of interest along with specifically associated proteins from cell extracts. Affinity capture coupled to mass spectrometry (MS)-based proteomic analyses has enabled the isolation and characterization of a wide range of endogenous protein complexes. Here, we outline effective procedures for the affinity capture of protein complexes, highlighting best practices and common pitfalls.

Following their synthesis in the endoplasmic reticulum (ER), voltage-gated sodium channels (Na-V) are transported to the membranes of excitable cells, where they often cluster, such as at the axon initial segment of neurons. Although the mechanisms by which Na-V channels form and maintain clusters have been extensively examined, the processes that govern their transport and degradation have received less attention. Our entry into the study of these processes began with the isolation of a new allele of the zebrafish mutant alligator, which we found to be caused by mutations in the gene encoding really interesting new gene (RING) finger protein 121 (RNF121), an E3-ubiquitin ligase present in the ER and cis-Golgi compartments. Here we demonstrate that RNF121 facilitates two opposing fates of Na-V channels: (i) ubiquitin-mediated proteasome degradation and (ii) membrane localization when coexpressed with auxiliary Na-V beta subunits. Collectively, these results indicate that RNF121 participates in the quality control of NaV channels during their synthesis and subsequent transport to the membrane.