Non-Coding RNA Features Critical to the Replication of HIV-1

The HIV-1 genome contains RNA sequences and structures that control many aspects of viral replication including, but not limited to transcription, splicing, nuclear export, translation, packaging and reverse transcription. Despite this extensive existing catalogue of RNA sequences that are critical to its replication, chemical probing and targeting mutagenesis studies suggest that the HIV-1 genome may contain many more RNA elements of unknown important function. To determine whether there are additional, undiscovered cis-acting RNA elements in the HIV-1 genome that are important for viral replication, we conducted a global synonymous mutagenesis experiment. Sixteen mutant proviruses containing clusters of ~50 to ~200 synonymous mutations covering nearly the entire HIV-1 protein coding sequence were designed and synthesized. Analyses of these mutant viruses resulted in their division into three phenotypic groups. Group 1 mutants exhibited near wild-type replication, Group 2 mutants exhibited replication defects accompanied by perturbed RNA splicing, and Group 3 mutants had replication defects in the absence of obvious splicing perturbation. The three phenotypes were caused by mutations that exhibited a clear regional bias in their distribution along the viral genome, and those that caused replication defects all caused reductions in the level of unspliced RNA. We characterized in detail the underlying defects for Group 2 mutants. Second-site revertants that enabled viral replication could be derived for Group 2 mutants, and generally contained point mutations that reduced the utilization of proximal splice sites. Mapping of the changes responsible for splicing perturbations in Group 2 viruses revealed the presence of several RNA sequences that apparently suppressed the use of cryptic or canonical splice sites. Some sequences that affected splicing were diffusely distributed, while others could be mapped to discrete elements, proximal or distal to the affected splice sites. This data from the Group 2 mutants indicates complex negative regulation of HIV-1 splicing by RNA elements in various regions of the HIV-1 genome that enable balanced splicing and viral replication. In silico analysis of the Group 3 mutants revealed that our mutagenesis had significantly increased the frequency of CG dinucleotides in sections of the viral genome to that of random sequence. This is important due to the remarkable CG suppression in both the HIV-1 and human genomes, and we had therefore disrupted the dinucleotide congruence that exists between HIV-1 and the genome of its host. We recoded these mutants to selectively remove either only the CG dinucleotides or only remove the mutations that did not encode a CG dinucleotide. Analysis of these mutants clearly demonstrated that the addition of CG dinucleotides were the causative mutations entirely responsible for the observed replication defects. qPCR analysis and smFISH microscopy revealed that the addition of CG dinucleotides to HIV-1 resulted in a depletion of the cytoplasmic mRNA molecules where the CG-dinucleotides were encoded as exons. A targeted siRNA screen for proteins that destabilize cytoplasmic RNA identified the Zinc-finger Antiviral Protein (ZAP) as responsible for the restriction of the CG-high HIV-1, specifically by targeting CGhigh viral RNA. CLIP-Seq experiments demonstrate that ZAP binds directly to CG dinucleotides in both cellular and viral RNA. Collectively these studies implicate ZAP as a cellular protein that can recognize CG-high viral RNA and is possibly a cellular mechanism for determining self from non-self RNA based on the CG composition. TRIM25 has previously been identified as a cofactor for two cytosolic RNA binding proteins that have antiviral functions, RIG-I where it is an essential cofactor, and ZAP where it functions as an enhancing cofactor. The mechanism by which TRIM25 enhances the antiviral activity of ZAP currently remains unclear. Through CLIP-Seq experiments in cells knocked out for TRIM25, we determined that ZAP does not require TRIM25 to recognize CG-high RNA. Using full length mutants of TRIM25 that are deficient for either RNA binding, E3 ligase activity, or formation of higher order multimers, our data suggest that the key biological activity required for TRIM25 to enhance ZAP is the formation of higher order multimers. Analyzing the replication of CG-high HIV-1 in different cell lines indicates that ZAP is not equally potent across all cell lines. The degree of potency ZAP possess against CG-high HIV-1 does not correlate with TRIM25 expression, suggesting the possibility of an additional ZAP cofactor that is heterogeneously expressed in varying cell lines. siRNA screens have been used in an attempt to identify a yet undiscovered cofactor, but so far these experiments have not yielded any such factor

The Identification of the X-ray Counterpart to PSR J2021+4026

We report the probable identification of the X-ray counterpart to the gamma-ray pulsar PSR J2021+4026 using imaging with the Chandra X-ray Observatory ACIS and timing analysis with the Fermi satellite. Given the statistical and systematic errors, the positions determined by both satellites are coincident. The X-ray source position is R.A. 20h21m30.733s, Decl. +40 deg 26 min 46.04sec (J2000) with an estimated uncertainty of 1.3 arsec combined statistical and systematic error. Moreover, both the X-ray to gamma-ray and the X-ray to optical flux ratios are sensible assuming a neutron star origin for the X-ray flux. The X-ray source has no cataloged infrared-to-visible counterpart and, through new observations, we set upper limits to its optical emission of i' >23.0 mag and r' > 25.2mag. The source exhibits an X-ray spectrum with most likely both a powerlaw and a thermal component. We also report on the X-ray and visible light properties of the 43 other sources detected in our Chandra observation.Comment: Accepted for publication in the Astrophysical Journa

[Avian cytogenetics goes functional] Third report on chicken genes and chromosomes 2015

High-density gridded libraries of large-insert clones using bacterial artificial chromosome (BAC) and other vectors are essential tools for genetic and genomic research in chicken and other avian species... Taken together, these studies demonstrate that applications of large-insert clones and BAC libraries derived from birds are, and will continue to be, effective tools to aid high-throughput and state-of-the-art genomic efforts and the important biological insight that arises from them

Expression quantitative trait loci in the developing human brain and their enrichment in neuropsychiatric disorders

BACKGROUND: Genetic influences on gene expression in the human fetal brain plausibly impact upon a variety of postnatal brain-related traits, including susceptibility to neuropsychiatric disorders. However, to date, there have been no studies that have mapped genome-wide expression quantitative trait loci (eQTL) specifically in the human prenatal brain. RESULTS: We performed deep RNA sequencing and genome-wide genotyping on a unique collection of 120 human brains from the second trimester of gestation to provide the first eQTL dataset derived exclusively from the human fetal brain. We identify high confidence cis-acting eQTL at the individual transcript as well as whole gene level, including many mapping to a common inversion polymorphism on chromosome 17q21. Fetal brain eQTL are enriched among risk variants for postnatal conditions including attention deficit hyperactivity disorder, schizophrenia, and bipolar disorder. We further identify changes in gene expression within the prenatal brain that potentially mediate risk for neuropsychiatric traits, including increased expression of C4A in association with genetic risk for schizophrenia, increased expression of LRRC57 in association with genetic risk for bipolar disorder, and altered expression of multiple genes within the chromosome 17q21 inversion in association with variants influencing the personality trait of neuroticism. CONCLUSIONS: We have mapped eQTL operating in the human fetal brain, providing evidence that these confer risk to certain neuropsychiatric disorders, and identifying gene expression changes that potentially mediate susceptibility to these conditions

Omecamtiv mecarbil in chronic heart failure with reduced ejection fraction, GALACTIC‐HF: baseline characteristics and comparison with contemporary clinical trials

Aims: The safety and efficacy of the novel selective cardiac myosin activator, omecamtiv mecarbil, in patients with heart failure with reduced ejection fraction (HFrEF) is tested in the Global Approach to Lowering Adverse Cardiac outcomes Through Improving Contractility in Heart Failure (GALACTIC‐HF) trial. Here we describe the baseline characteristics of participants in GALACTIC‐HF and how these compare with other contemporary trials. Methods and Results: Adults with established HFrEF, New York Heart Association functional class (NYHA) ≥ II, EF ≤35%, elevated natriuretic peptides and either current hospitalization for HF or history of hospitalization/ emergency department visit for HF within a year were randomized to either placebo or omecamtiv mecarbil (pharmacokinetic‐guided dosing: 25, 37.5 or 50 mg bid). 8256 patients [male (79%), non‐white (22%), mean age 65 years] were enrolled with a mean EF 27%, ischemic etiology in 54%, NYHA II 53% and III/IV 47%, and median NT‐proBNP 1971 pg/mL. HF therapies at baseline were among the most effectively employed in contemporary HF trials. GALACTIC‐HF randomized patients representative of recent HF registries and trials with substantial numbers of patients also having characteristics understudied in previous trials including more from North America (n = 1386), enrolled as inpatients (n = 2084), systolic blood pressure < 100 mmHg (n = 1127), estimated glomerular filtration rate < 30 mL/min/1.73 m2 (n = 528), and treated with sacubitril‐valsartan at baseline (n = 1594). Conclusions: GALACTIC‐HF enrolled a well‐treated, high‐risk population from both inpatient and outpatient settings, which will provide a definitive evaluation of the efficacy and safety of this novel therapy, as well as informing its potential future implementation

Global synonymous mutagenesis identifies cis-acting RNA elements that regulate HIV-1 splicing and replication.

The ~9.5 kilobase HIV-1 genome contains RNA sequences and structures that control many aspects of viral replication, including transcription, splicing, nuclear export, translation, packaging and reverse transcription. Nonetheless, chemical probing and other approaches suggest that the HIV-1 genome may contain many more RNA secondary structures of unknown importance and function. To determine whether there are additional, undiscovered cis-acting RNA elements in the HIV-1 genome that are important for viral replication, we undertook a global silent mutagenesis experiment. Sixteen mutant proviruses containing clusters of ~50 to ~200 synonymous mutations covering nearly the entire HIV-1 protein coding sequence were designed and synthesized. Analyses of these mutant viruses resulted in their division into three phenotypic groups. Group 1 mutants exhibited near wild-type replication, Group 2 mutants exhibited replication defects accompanied by perturbed RNA splicing, and Group 3 mutants had replication defects in the absence of obvious splicing perturbation. The three phenotypes were caused by mutations that exhibited a clear regional bias in their distribution along the viral genome, and those that caused replication defects all caused reductions in the level of unspliced RNA. We characterized in detail the underlying defects for Group 2 mutants. Second-site revertants that enabled viral replication could be derived for Group 2 mutants, and generally contained point mutations that reduced the utilization of proximal splice sites. Mapping of the changes responsible for splicing perturbations in Group 2 viruses revealed the presence of several RNA sequences that apparently suppressed the use of cryptic or canonical splice sites. Some sequences that affected splicing were diffusely distributed, while others could be mapped to discrete elements, proximal or distal to the affected splice site(s). Overall, our data indicate complex negative regulation of HIV-1 splicing by RNA elements in various regions of the HIV-1 genome that enable balanced splicing and viral replication

Phenotypes of synonymously mutated HIV-1 viruses.

<p>(A) Summary of the properties of HIV-1 viruses carrying blocks of nucleotides that were synonymously mutated (A-P). The frequency of splice site utilization was assessed in transfected 293T cells (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006824#ppat.1006824.g003" target="_blank">Fig 3</a>), Single-cycle replication assays were used to assess unspliced RNA levels and infectious virus yield (see panels B and C below). Replication competence was determined using spreading replication assays (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006824#ppat.1006824.g002" target="_blank">Fig 2</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006824#ppat.1006824.s006" target="_blank">S1 Fig</a>). (B) Infectious virion yield measured in the supernatant of MT4 cells, infected with each of the mutant viruses at an MOI of 1.0, and harvested 2 days post infection. Values are the mean ±sd n = 3 or n = 2 experiments, *p<0.05, **p<0.005 by students t-test calculated with relative values compared to wildtype virus (*** Values were below the limit of quantitation). (C) Levels of unspliced HIV-1 genomes in RNA extracted from MT4 cells, infected with each of the mutant viruses at an MOI of 1.0, and harvested 2 days post infection, mean ±sd n = 3 or n = 2 experiments, *p<0.05 by students t-test compared to wildtype virus. (*** Values were below the limit of quantitation).</p

Spreading replication properties of mutant viruses.

<p>(A-P) MT4 cells were infected with the indicated virus (harvested from the supernatant of 293T cells transfected with each of the WT(HIV-1_NHG) or mutant (A-P) proviral plasmids at an MOI of 0.002. Aliquots of infected cells were withdrawn each day, fixed in 4% PFA and the proportion of infected cells determined by FACS analysis of GFP expression. a Representative replication curve for WT(HIV-1_NHG) is plotted in each chart as grey symbols and line, while each mutant is plotted using red symbols and lines.</p