· Finkel, Y. et al. Comprehensive annotations of human herpesvirus 6A and 6B genomes reveal novel and conserved genomic features. eLife 9, e (). CAS Article Google Scholar · Online dating can offer significant benefits and has generated new forms of dating and as a result, sexual behaviour (Clemens et al., ; Finkel et al., ).Estimated Reading Time: 9 mins · Speed dating. Within 1–14 d of the presession (M = ), participants attended their first speed-dating event (Finkel et al., ).Each of the six events included 31–40 participants (M = ) with approximately equal numbers of men and blogger.com took place midday in
Study Questions Whether Women Are More Selective at Dating | Live Science
Thank you for visiting nature. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser or turn off compatibility mode in Internet Explorer. In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.
Severe acute respiratory syndrome coronavirus 2 SARS-CoV-2 is the cause of the ongoing coronavirus disease COVID pandemic 1. To understand finkel et al online dating pathogenicity and antigenic potential of SARS-CoV-2 and to develop therapeutic tools, it is essential to profile the full repertoire of its expressed proteins.
The current map of SARS-CoV-2 coding capacity is based on computational predictions and relies on homology with other coronaviruses. As the protein complement varies among coronaviruses, especially in regard to the variety of accessory proteins, it is crucial to characterize the specific range of SARS-CoV-2 proteins in an unbiased and open-ended manner. Here, using a suite of ribosome-profiling techniques 234we present a high-resolution map of coding regions in the SARS-CoV-2 genome, which enables us to accurately quantify the expression of canonical viral open reading frames ORFs and to identify 23 unannotated viral ORFs.
These ORFs include upstream ORFs that are likely to have a regulatory role, several in-frame internal ORFs within existing ORFs, resulting in N-terminally truncated products, as well as internal out-of-frame ORFs, which generate novel polypeptides. We further show that viral mRNAs are not translated more efficiently than host mRNAs; instead, virus translation dominates host translation because of the high levels of finkel et al online dating transcripts.
Our work provides a resource that will form the basis of future functional studies. SARS-CoV-2 is an enveloped virus consisting of a positive-sense, single-stranded RNA genome of around 30 kb. Two overlapping ORFs, ORF1a and ORF1b, are translated from the positive-strand genomic RNA and generate continuous polypeptides, which are cleaved into a total of 16 nonstructural proteins NSPs.
Negative-strand RNA intermediates are produced from the viral genome and serve as templates for the synthesis of positive-strand genomic RNA and subgenomic RNAs 5. The different subgenomic RNAs encode four conserved structural proteins—spike Senvelope Emembrane M and nucleocapsid N —and several accessory proteins. To capture the full coding capacity of SARS-CoV-2, we applied a range of ribosome-profiling approaches to Vero E6 cells infected with SARS-CoV-2 for 5 or 24 h Fig.
At 24 h post-infection hpi the vast majority of cells were infected and cells were still intact Extended Data Fig. For each time point, we prepared three ribosome-profiling libraries Ribo-seqeach one in two biological replicates.
To facilitate mapping of translation initiation sites, we prepared two Ribo-seq libraries by treating cells with lactimidomycin LTM or harringtonine Harrtwo drugs with distinct mechanisms that prevent elongation at 80S ribosomes at translation initiation sites. These treatments lead to excessive accumulation of ribosomes precisely at the sites of translation initiation and depletion of ribosomes over the body of the ORF Fig.
The third Ribo-seq library was prepared from cells treated with the translation elongation inhibitor cycloheximide CHXand provides a snapshot of actively translating ribosomes across the body of the translated ORF Fig.
In parallel, finkel et al online dating, RNA-sequencing RNA-seq was applied to map viral transcripts. Analysis of cellular genes from the different Ribo-seq finkel et al online dating revealed the expected distinct profiles in both replicates. Ribosome footprints displayed a strong peak at the translation initiation site, which, as expected, is more pronounced in the Harr and LTM libraries; the CHX library also exhibited a distribution of ribosomes across the entire coding region, and the mapped ribosome footprints were enriched in fragments that align to the translated frame Fig.
As expected, the RNA-seq reads were uniformly distributed across coding and non-coding regions Fig. The footprint profiles of viral coding sequences at 5 hpi fit the expected profile of translated sequences Fig. Notably, the footprint profile over the viral genome at 24 hpi, did not fit the expected profile of translating ribosomes and was generally not affected by Harr or LTM treatments Extended Data Fig.
To further examine the characteristics of the footprints, we applied a fragment length organization similarity score FLOSS that measures the magnitude of disagreement between the footprint distribution on a given transcript and the footprint distribution on canonical coding sequences CDSs At 5 hpi, protected fragments from SARS-CoV-2 ORFs did not differ from highly expressed cellular transcripts Fig.
However, reads at 24 hpi could be clearly distinguished from cellular CDSs Fig. We conclude that the footprint data from 5 hpi finkel et al online dating robust and reproducible ribosome footprint information but that viral protected fragments at 24 hpi may reflect additional interactions with viral RNA that occur at late time points in infection.
aVero E6 and Calu3 cells infected with SARS-CoV-2 were collected at 5, 24 Vero E6 and 7 Calu3 hpi for RNA-seq, and finkel et al online dating Ribo-seq using LTM, Harr or CHX treatments. bMetagene analysis of read densities relative to the maximal signal of each gene around the start and stop codons of cellular CDSs at 5 hpi.
cMetagene analysis around the start codon, finkel et al online dating, as described in bfor viral ORFs at 5 hpi. deFLOSS score for cellular and SARS-CoV-2 ORFs at 5 hpi d and 24 hpi e. Reduction in footprint density between ORF1a and ORF1b reflects the proportion of ribosomes that terminate at the ORF1a stop codon instead of frameshifting into ORF1b Extended Data Fig. Similar to observations in MHV and avian infectious bronchitis virus IBV 311we did not observe noticeable ribosome pausing before or at the frameshift site, but we identified several potential pausing finkel et al online dating within ORF1a and ORF1b Extended Data Fig, finkel et al online dating.
aRNA-seq green and Ribo-seq CHX red read densities number of reads at 5 hpi along the SARS-CoV-2 genome. SARS-CoV-2 canonical ORFs are labelled.
bTranscript abundance relative to ribosome densities of each SARS-CoV-2 canonical ORF at 5 hpi. cScatter plot of the abundance of reads that span canonical leader-dependent junctions rednon-canonical leader-dependent junctions greennon-canonical leader-independent finkel et al online dating purple or genomic deletions cyan at 5 and 24 hpi.
Besides ORF1a and ORF1b, all other canonical viral ORFs are translated from subgenomic RNAs. As raw RNA-seq densities represent the cumulative sum of genomic and subgenomic RNAs, we calculated transcript abundance using two approaches: deconvolution of RNA densities, in which RNA expression of each ORF is calculated by subtracting the RNA-read density of cumulative densities upstream of the ORF region; and relative abundances of RNA reads spanning leader—body junctions of each of the canonical subgenomic RNAs.
We next compared footprint densities to RNA abundance. For the majority of viral ORFs, transcript abundance correlated almost perfectly with footprint densities Fig. The translation efficiencies of ORF1a and ORF1b were considerably lower. The third outlier is ORF7b, for which we identified very few body—leader junctions; nevertheless, it exhibited relatively high translation, probably owing to ribosome leaky scanning of the ORF7a transcript, as was suggested for SARS-CoV Many transcripts derived from non-canonical junctions have been identified for SARS-CoV-2 9 We estimated the frequency of junction-spanning reads finkel et al online dating our RNA libraries and obtained excellent agreement between our replicates Extended Data Fig.
We also identified five abundant leader-independent junctions that, to our knowledge, were unique to our data Supplementary Table 2. We noted that three of these junctions represent short in-frame deletions in the spike protein that overlap deletions in the furin-like cleavage site that were recently described 9 Extended Data Fig. The recurrence of the same genomic deletion supports the conclusion that this deletion is being selected for during passage in Vero E6 cells.
To examine whether additional non-canonical junctions are derived from genomic deletions, we sequenced the genomic RNA of the virus we used in our infections, finkel et al online dating.
In addition to the deletions at the furin-like cleavage site, we identified an 8 amino acid aa deletion in ORF-E in 2. When we compared the frequency of junctions between 5 h and 24 h time points, the leader-dependent junctions and the genomic deletions showed good correlation, but the leader-independent junctions were specifically increased at 24 hpi Fig.
These data show that a small number of the leader-independent junctions represent genomic deletions and a larger subset increases at late stages of infection when finkel et al online dating replication is dominant and therefore probably do not substantially affect viral transcripts and translated ORFs.
Examination of SARS-CoV-2 translation as reflected by the diverse Ribo-seq libraries, revealed unannotated translated ORFs. We detected in-frame internal ORFs iORFs within existing ORFs, resulting in N-terminally truncated products. These include relatively long truncated versions of canonical ORFs, such as the one found in ORF6 Fig. We also detected internal out-of-frame translations, finkel et al online dating, which would yield novel polypeptides, such as ORFs within ORF3a 41 aa and 33 aa in size, respectively; Fig.
Additionally, we observed a aa extended ORF-M, in addition to the canonical ORF-M, which is predicted to start at the near-cognate codon AUA Fig. a finkel et al online dating iRibosome density profiles number of reads of CHX, Harr and LTM samples at 5 hpi. Filled and open rectangles indicate the canonical and novel ORFs, respectively. ORFs starting in a near cognate codon are labelled with stripes.
abIn-frame iORFs within ORF6 a and within ORF7a b. c — eOut-of-frame internal initiations within ORF-3a, within ORF-S d and within ORF-M e. ffinkel et al online dating, An extended version of ORF-M. gAn uORF that overlaps ORF10 initiation and an in-frame internal initiation generating truncated ORF ifinkel et al online dating, Non-canonical CUG initiation upstream of the TRS leader, finkel et al online dating.
Reads that were cut to fit the scale are indicated with horizontal lines. The presence of the annotated ORF10 was recently called into question, as almost no subgenomic reads were found for its corresponding transcript 12 Although we also did not detect subgenomic RNA designated for ORF10 translation Supplementary Table 1the ribosome footprint densities indicate the presence of a translation initiation signal in ORF10 Fig.
Of note, we additionally detected two putative ORFs in this region, an upstream out-of-frame ORF that overlaps ORF10 initiation and an in-frame internal initiation that leads to a truncated ORF10 product.
Further research is needed to delineate how ORFs in this region are translated and whether they have any functional roles. Three of these encode for uORFs that are located just upstream of ORF1a; the first initiating at an AUG uORF1 and the other two initiating at near-cognate codons uORF2 and extended uORF2; Fig.
These uORFs are in line with findings in other coronaviruses 3 The fourth site is the most prominent peak in the ribosome footprint densities on the SARS-CoV-2 genome and is located on a CUG finkel et al online dating at position 59, just 10 nucleotides upstream of the TRS leader Fig.
The reads mapped to this site have the tight length-distribution characteristic of ribosome-protected fragments Extended Data Fig. The occupancy at the CUG is higher than the downstream translation signal Fig. Notably, potenial ribosome pauses located just upstream of the TRS leader were also identified in MHV and IBV genomes 3 Owing to its location upstream of the TRS leader, footprints mapping to this site could potentially derive from any of the subgenomic or genomic RNAs.
Therefore, to view this initiation in its context, we aligned the footprints to the genomic RNA or to the most abundant subgenomic N transcript. On the genome and on ORF-N transcript, this initiation results in translation of uORFs, which, on the genome would generate an extension of uORF1 Extended Data Fig. To assess the distribution of footprints at this initiation on the different viral transcripts, viral transcripts were divided into three groups on the basis of their sequence similarity downstream of the leader—junction site to enable unique alignment Extended Data Fig.
Of note, substantially more footprints were mapped to the group that includes the genomic RNA and the subgenomic E and M transcripts, than would be expected from their relative RNA abundance Extended Data Fig.
When only footprints that allow unique mapping to genomic RNA or subgenomic M and E transcripts are used sizes 31—33 base pairs bp to discriminate M from genome or E transcript, and sizes finkel et al online dating bp to discriminate E from the genome a strong enrichment of footprints that originate from the genome is observed Extended Data Fig. This footprint enrichment on genomic RNA suggests that ribosome pausing might be more prominent on the genome or that ribosomes engage with genomic RNA differently than with subgenomic transcripts.
The proximity of this pause to the leader TRS, which seems to be conserved in MHV and IBV 311together with the relative enrichment on the viral genome, raises the possibility that a ribosome at this position might affect discontinuous transcription either by sterically blocking the TRS-L site or by affecting RNA secondary structure, finkel et al online dating.
In addition, ribosomes initiating at the CUG have the potential to generate uORFs or ORF extensions in the different subgenomic transcripts Supplementary Table 3. To systematically define the SARS-CoV-2 translated ORFs we used PRICE and ORF-RATER, two computational methods that rely on a combination of translation features to predict novel translated ORFs from ribosome-profiling measurements 16 After application of a minimal expression cut-off and manual curation on the predictions, these classifiers identified 25 ORFs, these included 10 out of the 11 canonical translation initiations and 15 novel viral ORFs.
In addition, ORF-RATER identified three putative ORFs that originate from the CUG initiation and extend to the subgenomic transcripts of S, M and ORF6 Supplementary Table 3. Visual inspection of the ribosome-profiling data suggested eight additional putative novel ORFs, some of which are presented above Fig.
Overall, we identified 23 putative ORFs, in addition to the 12 canonical viral ORFs that are currently annotated in the NCBI database and 3 additional potential ORFs that stem from the CUG initiation upstream of the leader. To confirm the robustness of these annotations, we extended these experiments to human cells.
We first examined the infection efficiency of several human cell lines that were used to study SARS-CoV-2 infection: Calu3, A and Caco Infection of Calu3 was most efficient and the presence of trypsin increased infection efficiency by at least twofold Extended Data Fig.
We infected Calu3 with a different SARS-CoV-2 isolate, which was sequenced to confirm its integrity.
· Online dating can offer significant benefits and has generated new forms of dating and as a result, sexual behaviour (Clemens et al., ; Finkel et al., ).Estimated Reading Time: 9 mins Friends and collaborators can also make it easier to engage in joint goal pursuit (Finkel et al., ; Fitzsimons & Finkel, ) and increase motivation to engage in shared goal pursuits · From disappointing election results (Gilbert et al., ) to lost football games (Wilson, Wheatley, Meyers, Gilbert, & Axsom, ) to unpleasant medical procedures (Riis et al., ), individuals tend to predict that such events will cause greater levels of distress and negative affect than is actually the case
Keine Kommentare:
Kommentar veröffentlichen