Crustose coralline algae (CCA) are key producers of carbonate sediment on reefs today. Despite their importance in modern reef ecosystems, the long-term relationship of CCA with reef development has not been quantitatively assessed in the fossil record. This study includes data from 128 Cenozoic coral reefs collected from the Paleobiology Database, the Paleoreefs Database, as well as the original literature and assesses the correlation of CCA abundance with taxonomic diversity  and framework of fossil coral reefs. Chi-squared tests show reef type is significantly correlated with CCA abundance and post-hoc tests indicate higher involvement of CCA is associated with stronger reef structure. Additionally, general linear models show coral reefs with higher amounts of CCA had a higher diversity of reef-dwelling organisms. These data have important implications for paleoecology as they demonstrate that CCA increased building capacity, structural integrity, and diversity of ancient coral reefs. The analyses presented here demonstrate that the function of CCA on modern coral reefs is similar to their function on Cenozoic reefs; thus, studies of ancient coral reef collapse are even more meaningful as modern analogues. Corallinales that secrete Mg-calcite skeletons [Crustose coralline algae (CCA) are non-geniculate, red algae of the order keletons . CCA arekeletons , act as keletons , and arekeletons , 4, 5. Skeletons , CCA havkeletons , 9, 10 ain situ aggregation of densely packed organisms  often encrusted by secondary organisms  [in situ corals to create the primary reef structure [The services that CCA provide to corals and reefs today have been shown to substantially impact reef type and diversity in modern ecosystems. CCA are critical for reef framework particularly in high-energy, intertidal or outer ridge habitats where wave-resistivity is important , 9. A frrpulids) , which frpulids) , 8. In mtructure . While ttructure  and a drtructure . Therefotructure , 9.Given their importance in modern systems, CCA are assumed to play an essential role on fossil reefs, especially in the Cenozoic Era when predominantly coral-algal reefs first appear , 15. Folwww.paleobiodb.org): Sporolithaceae, Hapalidiaceae, Mastophoroideae, Lithophylloideae, Metagoniolithoideae . This angen.de; , from thCCA play many roles on reefs, so simple presence/absence data is insufficient for assessing the interaction between CCA and corals. Therefore, a three-point “Algae Score” scale was developed to describe the role of CCA on coral reefs . This Alwww.r-project.org), specifically RStudio; see the Data compiled from previously published works are not always comparable as different studies have distinct objectives and scopes . For exasensu [in situ structures with syndepositional relief made by skeletal organisms that build a rigid framework [The type of reef  was evaluated following the terminology in the PaleoReefs database (sensu ). Ecologramework , and areramework . Reef moramework  were conReef dweller alpha diversity data was not normally distributed ; therefoThe chi-squared test indicates reef type is dependent upon Algae Score in Late Cretaceous and Cenozoic coral reefs (p = 0.006). When sub-sampled, 557 of 1000 tests were significant (p<0.05); however, the number of significant tests increased to 783/1000 when p<0.1. Because ecological reefs are the only constructions with definite framework, CCA presence is linked with framework and thus structural rigidity of the reef. Post-Hoc standardized residuals indicate that Algae Score 1 is most associated with ecological reefs, while the other reef types are positively associated with an Algae Score of 2 see . TherefoThe association between CCA and framework matches modern observations; reefs with significant CCA involvement in building, encrusting, and binding have more structural integrity than those without , 9. Highsensu stricto, or a biostrome, which does not produce the magnitude of carbonate of an ecological reef. When ecological reefs degrade, they can transition into reef mounds, biostromes, or become overgrown with soft algae or microbes [Reef type can also be a measure of a healthy carbonate factory. Increased carbonate productivity enhances the ability to grow vertically, in a topographically complex way, with robust framework , and tenmicrobes , 35, 36.Reef dweller diversity, i.e. total biodiversity of the reef, including corals, algae, echinoids, foraminifera, etc., is significantly different for each Algae Score . A KruskCCA take an active role in promoting reef biodiversity in several ways. By contributing to framework and binding reef sediments , 8, CCA Coral diversity  showed sThe implications of the results presented here are significant to interpretations of fossil coral reefs and predictions for future reefs. CCA are vital members of the coral reef community and have been since the lower-mid Cretaceous  to assess specifically how this relationship is affected by climate events similar to those predicted for the near future. As can be seen in Given what is known about CCA sensitivity to ocean acidification and heat stress, time periods where these stressors are present will be ideal case studies. One good example is the Paleocene-Eocene Thermal Maximum, a carbon cycle perturbation event 56 million years ago for which there is evidence of rapid global warming , 46 and Ancient analogues for modern coral reef change can be particularly useful for assessing regional and global patterns of migration in response to past environmental change , 50, 51 The role of crustose coralline algae (CCA) on fossil reefs is critical to paleoecological interpretations of coral reefs. The quantitative analysis presented here demonstrates a strong association between CCA and reef diversity as well as reef building capacity in fossil coral reefs. The significance of this association is similar to observations on modern reefs; CCA are key ecosystem engineers on coral reefs, providing protection, structural support, and enhancing biodiversity in the reef ecosystems. Without CCA, coral reefs risk shifts to alternative  stable states or even complete collapse. The results presented herein confirm that Cenozoic coral reefs had similar ecologies to modern coral reefs, and underscores the potential for past reef collapse as analogues for current and future reef stresses. Further studies of the relationship between CCA and coral reefs—and the conditions under which this relationship persists or breaks down—are of paramount importance for the conservation about modern reefs in current and future climate change.S1 Database(XLS)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.S1 Fig(PDF)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.


Complete genomic sequences of a non-redundant set of 70 recombinants between three serotypes of attenuated Sabin polioviruses as well as location  of crossover sites of 28 additional recombinants were determined and compared with the previously published data. It is demonstrated that the genomes of Sabin viruses contain distinct strain-specific segments that are eliminated by recombination. The presumed low fitness of these segments could be linked to mutations acquired upon derivation of the vaccine strains and/or may have been present in wild-type parents of Sabin viruses. These “weak” segments contribute to the propensity of these viruses to recombine with each other and with other enteroviruses as well as determine the choice of crossover sites. The knowledge of location of such segments opens additional possibilities for the design of more genetically stable and/or more attenuated variants, i.e., candidates for new oral polio vaccines. The results also suggest that the genome of wild polioviruses, and, by generalization, of other RNA viruses, may harbor hidden low-fitness segments that can be readily eliminated only by recombination. Genetic recombination was demonstrated to occur in practically all viruses investigated in this respect , althougThese strains were generated by Albert Sabin by selection from three serotypes of wild-type (wt) polioviruses . Upon thAiming to understand these reasons, in this study we have analyzed some aspects of a unique natural mega-experiment when a mixture of three well-studied and related viruses  was given to billions of people and a plethora of progeny isolates were available for investigation. Specifically, we have sequenced, fully or partially, 98 genomes of intertypic poliovirus recombinants and analyzed these data together with the previously published results to infer common patterns of organization of the recombinant genomes. As a result, some important conclusions have emerged relevant not only for the understanding of the nature of Sabin viruses and their penchant for recombination but also of some general issues of biology of RNA viruses.Virus strains derived from Sabin polioviruses were mainly collected in Russia and countries of the former Soviet Union from patients with acute flaccid paralysis (AFP), patients with other diseases as well as from healthy individuals and environment. Isolation, identification and intratypic characterization were performed according to the standard WHO polio surveillance protocol .Nucleotide sequences were determined by full-genome deep sequencing as follows. Cell culture supernatants were treated with micrococcal nuclease to digest free nucleic acids, and RNA was isolated by RNeasy kit . It was then fragmented by using focused ultrasonicator  to produce 200–400 nt-long molecules. cDNA was prepared by reverse transcription using Superscript III . Illumina adapters with index oligonucleotides (barcodes) were added and the resulting DNA library was amplified by PCR. Sequencing was done using Illumina Miseq in multiplex format with 10–12 barcoded samples analyzed in one run. For most samples treated this way, poliovirus-specific sequence reads represented the majority of all reads (50–95%), and the depth of coverage was between 10,000 and 200,000 reads per genomic position. All new nucleotide sequences were deposited in GenBank (accession numbers MG212427-MG212495).Bioinformatic analysis was performed using two in-house software packages: ‘swarm’ program running on MacPro  computers in UNIX (Darwin) environment, and cloud-based HIVE  system . The anaThe location of crossovers in some recombinants investigated years ago and reported for the first time here were determined as follows. Viral RNA was extracted from the cultural medium of virus-infected cells with the Trizol reagent  and reverse transcribed with avian myeloblastosis virus reverse transcriptase (Promega) using random hexamer primers . Approximate positions of the crossover were mapped by using the restriction fragment length polymorphism (RFLP) assay ,21. To tThe set of intertypic recombinants between the Sabin vaccine polioviruses analyzed here, 189 samples, included 98 viruses collected mostly in Russia and countries of the former Soviet Union since the 1980s, whose genomic crossover sites were determined in this study either by full genome deep sequencing  sequences were regularly lost, in some cases together with more or less extended parts of the P3 region and sometimes also distal portions of the P2 region  or poorly compatible with other genome parts (epistasis). Some ground for choosing the more likely explanation can provide a comparison of data of Thus, the presence of homotypic 5′- and 3′-adjacent sequences in the type 1 recombinants A could bThe notion of the existence of presumably low-fit serotype-specific segments of poliovirus genomes is also supported by the structures of recombinants between Sabin strains and non-polio enteroviruses. Thus, for example, such recombinants of type 1, which triggered polio outbreaks in the Dominican Republic and Philippines in 2000–2001, demonstrated a replacement of a significant portion of their P2 region ,26; the A plausible assumption is that the observed regularity of losing/gaining distinct genomic segments is linked to their fitness-modulating capacities. Are they somehow related to the type-specific attenuating mutations acquired upon derivation of the vaccine strains? To approach this point, we used an arbitrary classification of presumably low-fit regions based on the frequency of their removal upon intertypic recombination. Those that were lost in 90% or more recombinants of serotypes 2 and 3 and in at least 75% of serotype 1 were considered as “weak” (heavier shading in ts) phenotype-modulating) mutations are located upstream of the recombination-affected parts of the genomes situated, as demonstrated above, in our set of viruses downstream of the VP1/2A border. Moreover only a single known Sabin 1-specific attenuating mutation  are scattered throughout the entire genome , and thecis-element (oriR), which in poliovirus RNA is composed of two hairpins linked to each other by a tertiary (“kissing”) interaction [oriR. Whether one or both of these features could be linked to fitness loss remains unknown. It may also be noted that the distal part of the 3D protein invariably lost in the serotype 2 recombinants differs from its counterparts in types 1 and 3 at only one position, amino acids 362 and 425, respectively. The loss of this segment could, or could not, be simply due to a kind of “passenger effect” because of its proximity to the 3′UTR.Thus, it is reasonable to look at the intrinsic  peculiarities of the “weak” genomic segments. The regular absence of the type 2-specific 3′UTR in the investigated recombinants could be due to certain peculiarities of the structure of this replicative eraction . Althoug15 in 3A. On the other hand, as noted above, it cannot be ruled out that a nearly universal loss of the relevant short fragment resulted merely from the partial overlap of two low-fitness regions located in P2 and P3, respectively.The presence of Arg at position 15 of 3A protein of Sabin-3 could also be related to the removal of this region from nearly all type 3 recombinants. Indeed, none of the wild polioviruses deposited in the GenBank (except the Sabin-3 parent Leon/37 and its descendants) contain ArgThus, the above results are compatible with the hypothesis that the fitness-related features of the genome segments that are acquired/lost upon intertypic recombination between Sabin polioviruses might be determined by not only vaccine-specific mutations but could also be inherited from their wild type progenitors.To better evaluate the choice of the observed crossover sites, it is appropriate to briefly remind what is known about the mechanisms of RNA recombination . The widAnother, nonreplicative, mechanism of RNA recombination implies end-to-end joining of independently synthesized RNA fragments without participation of viral RNA-dependent RNA polymerase (RdRP) and does not require any similarity between the partners involved ,41. AlthAlthough the choice of the replaced segments appears to be related to fitness, there should be mechanistic circumstances to allow recombination events to occur at certain distinct loci. The available large collection of recombinants originating from known parents may be useful for a search for such mechanistic preferences/limitations, if they exist at all. In our set of recombinants, the crossovers were distributed throughout the entire region encoding nonstructural proteins, though clearly non-randomly with relatively “hot” and “cold” areas . RemarkaThe above pattern of crossover distribution was generated manually using 50 nt-long windows by comparing aligned type-specific sequences. To assess the real level of homology around crossover points in the recombining partners, we looked at the sequences of relevant genomes. It turned out that in the overwhelming majority of cases (81%), this homology indeed spanned for at least 4 nt and in 68% cases it was 7 nt-long or longer . This maThe data reported here revealed previously unknown serotype-specific peculiarities of distinct genomic segments of poliovirus vaccine strains, which are likely related to their effects on viral fitness. Although polio eradication campaign launched by the WHO in 1988  met with a marked success, with only a handful of poliomyelitis cases caused by wild poliovirus reported globally in 2017 , the neeUnfortunately, phenotypic differences between Sabin viruses and their intertypic recombinants have not been systematically investigated. In a small-scale experiment, Savolainen-Kopra et al.  have demTo decrease the ability of vaccine strains to recombine it was suggested to introduce certain mutations into the viral RNA-dependent RNA polymerase  and to uA likely pattern emerging from the results presented here is that naturally circulating (“wild”) RNA viruses may contain lineage-specific more or less extended genomic segments with a relatively low fitness. Indeed, our observations suggest that Sabin viruses (at least of serotype 2 or 3) might have inherited from their wt parents certain “weak” RNA sequences not related to the attenuating (and other) mutations acquired during their selection by A. Sabin. Such low-fitness segments could well arise (and probably indeed are not infrequently arising) upon error-prone RNA replication caused by low fidelity of the RdRP  and The analysis of the pattern of inheritance of distinct genomic segments upon intertypic recombination between the Sabin strains of poliovirus allowed us to draw several conclusions and hypotheses. It suggests that Sabin strains contain, in addition to known attenuating mutations, distinct serotype-specific genomic segments with low-fitness. The genetic determinants responsible for their low fitness could be derived not only during the laboratory attenuation of these strains but also could be inherited directly from their wild-type parents. The presence of these segments underlies the propensity of these viruses to recombine with each other and with other enteroviruses as well as makes a significant contribution to the choice of crossovers sites. Thus, our results clearly demonstrate a role for RNA recombination as an efficient tool of adaptive viral evolution. Also, the knowledge of localization of low-fitness segments in Sabin viruses opens additional possibilities for the design of more genetically stable and/or more attenuated vaccine strains.Our results also provide new insights into the problem of fitness, genetic stability and evolvability of wild polioviruses and, by generalization, of other RNA viruses. We propose that the genomes of such viruses may also harbor hidden low-fitness segments, which cannot be readily rehabilitated and can (nearly) exclusively be removed by recombination.Finally, the absence of sufficiently long homologous sequences around crossover sites in the relatively large pairs of recombining partners may be considered as an indirect evidence for possible involvement, in certain cases, of recombination mechanisms other than the classical copy-choice replicative mode. However, more direct data to support this notion are needed.


The rapid expansion of the available genomic data continues to greatly impact biomedical science and medicine. Fulfilling the clinical potential of genetic discoveries requires the development of therapeutics that can specifically modulate the expression of disease-relevant genes. RNA-based drugs, including short interfering RNAs and antisense oligonucleotides, are particularly promising examples of this newer class of biologics. For over two decades, researchers have been trying to overcome major challenges for utilizing such RNAs in a therapeutic context, including intracellular delivery, stability, and immune response activation. This research is finally beginning to bear fruit as the first RNA drugs gain FDA approval and more advance to the final phases of clinical trials. Furthermore, the recent advent of CRISPR, an RNA-guided gene-editing technology, as well as new strides in the delivery of messenger RNA transcribed in vitro, have triggered a major expansion of the RNA-therapeutics field. In this review, we discuss the challenges for clinical translation of RNA-based therapeutics, with an emphasis on recent advances in delivery technologies, and present an overview of the applications of RNA-based drugs for modulation of gene/protein expression and genome editing that are currently being investigated both in the laboratory as well as in the clinic. Fourteen years after the completion of the human genome project, our understanding of human genomics continues to develop at an unprecedented rate. Thanks to advances in next-generation sequencing technology, scientists have been able to identify the genetic roots of many common diseases . DiseaseIn summary, both protein and small-molecule drugs are limited in that they cannot target every disease-relevant protein or gene. The mRNA and DNA precursors of proteins, however, are promising therapeutically in that they can be specifically targeted via Watson–Crick base pairing and, in the case of gene editing, which aims to permanently change the host’s DNA, represent an avenue to cure a genetic defect as opposed to just treating it. Over the past few decades, RNA drugs have emerged as candidates to address diseases at the gene and RNA levels. Although it has been known since 1990 that nucleic acids can be used to modulate protein production in vivo . As suchKa ~7.4), thought to lead to an ion imbalance that results in endosomal rupture [Broadly speaking, RNA delivery can be mediated by viral and non-viral vectors. For viral RNA delivery, there has been a great deal of interest in engineering adeno-associated viruses to carry nucleic acid cargo —however, rupture , 15, alt rupture . Regardl rupture , polyami rupture , and pol rupture , as well rupture , have al rupture , but hav rupture –24 and m rupture .Table 1CLipids and lipid-like materials represent the second major class of nanoparticle-based delivery vehicles for RNA. As with polymers, cationic lipids are often used to electrostatically bind the nucleic acid. Many laboratories, however, have started utilizing ionizable lipids, which are lipids that are positively charged only at acidic pH. This ionizable behavior is thought to enhance efficacy through helping with endosomal escape  and reduN-acetylgalactosamine . Like siRNA, ASOs are designed to block protein translation through Watson–Crick base-pairing with the target mRNA  and can An emerging, albeit less clinically advanced, RNA-based platform for protein knockdown is microRNA (miRNA). Endogenous microRNAs are non-coding RNAs that act as key regulators for a variety of cellular pathways, and are often downregulated in diseases . Thus, eExpression of disease-relevant proteins can be achieved by intracellular delivery of plasmid DNA (pDNA) or messenger RNA (mRNA). Application of DNA or mRNA as protein intermediate enables expression of virtually any desired protein inside the host cells and tissues. This approach can address formulation and delivery challenges encountered with protein-based drugs, especially those aimed at intracellular targets . mRNA-baThe delivery of mRNA therapeutics has been facilitated by significant progress in maximizing the translation and stability of mRNA, preventing its immune-stimulatory activity and the development of in vivo delivery technologies, some of which are discussed below. The 5′ cap and 3′ poly(A) tail are the main contributors to efficient translation and prolonged half-life of mature eukaryotic mRNAs. Incorporation of cap analogs such as ARCA  and poly(A) tail of 120–150 bp into in vitro transcribed (IVT) mRNAs has markedly improved expression of the encoded proteins and mRNA stability , 90. NewTherapeutic applications of mRNA that are currently being explored are vaccinations against cancer and infectious disease, protein-replacement therapy, and gene editing. A comprehensive list of ongoing clinical trials involving mRNA can be found in Table Another RNA vaccination approach is the use of self-amplifying mRNA replicons that have been developed to extend the duration and magnitude of antigen expression as well as boost the immune response , 105. InBy contrast, most mRNA-based protein replacement therapies are still in the preclinical stages of development and involve supplementation of deficient or aberrant proteins as well as modulation of cell behavior by expression of exogenous proteins. The in vivo efficacy of RNA–protein therapy has been demonstrated for a number of diseases. The majority of the studies preferentially target the liver owing to the well-established and efficient methods for RNA delivery into liver tissue. Therapeutically relevant amounts of human FIX (hFIX) protein were reached and sustained physiological activity for 4–9 days upon a single intravenous dose of hFIX mRNA-loaded LNPs in mice with hemophilia B , 113. SiThe RNA-based technologies discussed above constitute a powerful means to transiently repress or overexpress the expression of genes. By contrast, therapeutic gene editing entails replacement or alteration of gene expression by introducing site-specific modifications into the genome of cells, including correction of deleterious or introduction of protective mutations . While tStreptococcus pyogenes (SpCas9). Direct in vivo delivery of spCas9 to diseased cells has recently been used to correct mutations in genes in animal models of Duchenne muscular dystrophy (mdx) [fah) [oct) [PCSK9 [CCR5 to trigger anti-HIV protection or to deplete PD-1 to boost anti-cancer therapy [To date, the most widely used and well characterized gene-editing technology is the CRISPR–Cas9 system with an effector domain originating from hy (mdx) –129, herx) [fah) , 131, anh) [oct)  and to r) [PCSK9 . Ex vivo therapy . Despite therapy , (2) low therapy , and (3) therapy .Acidaminococcus sp. (AsCpf1), with the capability to edit the genome of mammalian cells have been discovered recently [The DNA-targeting specificity of CRISPR–Cas9 can be improved by combining optimized design and synthesis of guide RNAs. In particular, sgRNAs shorter than 20 nucleotides and containing 5′ mismatches have shown fewer off-target effects , 139, whrecently , 144. Cprecently , 145.Obtaining a better efficiency of genome editing by HDR will be necessary to address genetic diseases demanding a high level of therapeutic product, especially when edited cells do not display a positive change in fitness and outcompete their diseased counterparts over time . The effIntracellular delivery of CRISPR-based agents poses one of the most significant challenges for therapeutic genome editing owing to the number of essential components. CRISPR–Cas9 components can be delivered as DNA, RNA, RNA–protein complex (RNP), or a combination of these macromolecules. These macromolecules are not able to spontaneously enter the cells, relying on the use of delivery vehicles such as viral vectors, nanoparticles, or physical and mechanical delivery methods like nucleofection, cell squeezing, or lipofection that utilize electric field, mechanical force, or cationic lipids for temporary disruption of the cell membrane . The latViral delivery of CRISPR–Cas9 has been explored using lentivirus, adeno-virus, and adeno-associated virus (AAV) . AAVs arFah gene in a hereditary tyrosinemia in adult animals [Complementary to the viral systems, an abundance of nanoparticles comprising various bio-compatible materials are being developed for delivery of CRISPR–Cas9. As with their use in protein modulation, nanoparticles for gene editing have demonstrated high loading capacity for nucleic acid cargos, ability to modify payload bio-distribution and pharmacokinetics through active targeting and formulation, as well as simplicity of manufacturing with a high level of control over their physicochemical parameters, such as size/shape and kinetics of payload release . Nanopar animals . The eff animals . Similar animals . Interes animals . This st animals , 159. HoAfter over two decades of development, RNA therapeutics has become a clinical reality. The design and chemistries used to synthesize siRNAs, ASOs, and mRNAs have advanced to a point where they enable adequate stability and immune evasion, while at the same time allowing the maintenance of efficacy and specificity. The delivery technologies have also greatly progressed thanks to the discovery of potent and bio-compatible materials, aided by high-throughput screening technologies. Despite recent setbacks surrounding withdrawal of Alnylams’s siRNA–GalNac conjugate  and Cure


Finally, we identified two steps that lead to association of assembling Gag with unspliced HIV-1 RNA. Independent of viral-RNA-binding, Gag associates with a broad class of RNA granules that largely lacks unspliced viral RNA (step 1). If a viral-RNA-binding domain is present, Gag further localizes to a subset of these granules that contains unspliced viral RNA (step 2). Thus, our data raise the possibility that HIV-1 packaging is initiated not by soluble Gag, but by Gag targeted to a subset of host RNA granules containing unspliced HIV-1 RNA.During immature capsid assembly, HIV-1 genome packaging is initiated when Gag first associates with unspliced HIV-1 RNA by a poorly understood process. Previously, we defined a pathway of sequential intracellular HIV-1 capsid assembly intermediates; here we sought to identify the intermediate in which HIV-1 Gag first associates with unspliced HIV-1 RNA. In provirus-expressing cells, unspliced HIV-1 RNA was not found in the soluble fraction of the cytosol, but instead was largely in complexes ≥30S. We did not detect unspliced HIV-1 RNA associated with Gag in the first assembly intermediate, which consists of soluble Gag. Instead, the earliest assembly intermediate in which we detected Gag associated with unspliced HIV-1 RNA was the second assembly intermediate (~80S intermediate), which is derived from a host RNA granule containing two cellular facilitators of assembly, ABCE1 and the RNA granule protein DDX6. At steady-state, this RNA-granule-derived ~80S complex was the smallest assembly intermediate that contained Gag associated with unspliced viral RNA, regardless of whether lysates contained intact or disrupted ribosomes, or expressed WT or assembly-defective Gag. A similar complex was identified in HIV-1-infected T cells. RNA-granule-derived assembly intermediates were detected  During HIV-1 immature capsid assembly, packaging of the viral genome is initiated when the HIV-1 capsid protein, Gag, first associates with unspliced HIV-1 RNA. Although the complex in which this association initially occurs is critical for formation of infectious virus, the identity, composition, and the mechanism by which this complex forms remain unknown. To address this question, we utilized a previously described temporal pathway of intermediates in HIV-1 immature capsid assembly. The late intermediates in this pathway are derived from host RNA granules, which are diverse complexes utilized for cellular RNA storage and degradation. Here we sought to identify the intracellular capsid assembly intermediate in which HIV-1 Gag initially associates with unspliced HIV-1 RNA. We failed to detect an association between the first assembly intermediate, which contains soluble Gag, and unspliced HIV-1 RNA. Instead, the association between Gag and unspliced HIV-1 RNA was observed only in complexes corresponding to the RNA-granule-derived assembly intermediates. We also showed that Gag uses two determinants to form RNA-granule-derived intermediates that contain unspliced HIV-1 RNA. Together, these studies support a novel model for HIV-1 genome packaging, in which the first association between HIV-1 Gag and unspliced HIV-1 RNA occurs within a host RNA granule. For released HIV-1 particles to be infectious, they must contain two copies of unspliced (full-length) HIV-1 RNA that are packaged during assembly of the immature HIV-1 capsid. Each immature capsid is composed of ~3000 copies of the HIV-1 structural protein Gag, which initially oligomerize in the cytoplasm and subsequently target to the plasma membrane (PM), where Gag multimerization is completed. Packaging of the viral genome is initiated when Gag first associates with unspliced viral RNA during assembly, and requires the nucleocapsid domain (NC) of Gag as well as specific encapsidation signals in unspliced HIV-1 RNA (reviewed in ). In thetrans packaging is the dominant packaging mechanism )In contrast to the modest VLP packaging defect displayed by ΔΨ viral RNA , the assin trans  is sufficient to restore targeting of Gag Zip to the subset of the granules that contains unspliced viral RNA. Because the V1B genomic RNA contains binding sites for the bacteriophage MS2 (referred to as MS2 stem loops), we reasoned that the MS2 coat protein (MCP), which binds with high affinity to MS2 stem loops (reviewed in ), could in situ double labeling immunoelectron microscopy (IEM) to show colocalization of WT Gag (as well as some assembly-competent Gag mutants) with ABCE1, DDX6, and AGO2 (another RNA granule protein) at PM sites of assembly and budding in intact cells [in situ technique to confirm that Gag associates with RNA granule proteins in intact cells. To do this, we sought to identify sites where Gag colocalizes with DDX6 or ABCE1 using the proximity ligation assay (PLA). PLA produces fluorescent spots at sites where two proteins are within 40 nm of each other in situ. Briefly, this method uses species-specific secondary antibodies conjugated to complementary oligonucleotide probes to detect primary antibodies bound to two proteins of interest; when a linker and other reagents are added, the probes on the secondary antibodies anneal if the two proteins of interest are ≤ 40 nm apart, thereby forming the template for a rolling circle amplification product that is recognized by a fluorophore-conjugated oligonucleotide [Previously, to confirm coIP studies suggesting that assembling Gag associates with a host RNA granule, we had used ct cells ,19,20,22cleotide Fig 8A)..in situ cleotide ,19,20,22DDX6 is known to be a marker of P bodies, as shown by high intensity labeling of P bodies by DDX6 IF ; howeverin situ approaches to reveal DDX6-containing assembly intermediates to be present at WT Gag or Gag Zip PM sites of assembly; however, the DDX6-containing granules formed by WT Gag should be colocalized with unspliced viral RNA, while the DDX6-containing granules formed by Gag Zip should not be colocalized with unspliced viral RNA. Previously, we used quantitative IEM to demonstrate that RNA granule proteins  are recruited to sites of WT Gag and Gag Zip assembly at the PM [in situ  with V1B genomic constructs encoding MS2 binding sites and Gag in cis . These cin cis . Many ofin cis . When alin cis . Notablyin cis . Our mosin cis . As expein cis . Thus, Iin cis  along wiin cis –10, whicPreviously, we identified a pathway of sequential  HIV-1 capsid assembly intermediates and used this temporal pathway to understand intracellular events in the assembly of the immature HIV-1 capsid. Since the process of HIV-1 genome packaging occurs at the same time as capsid assembly, we reasoned that assembly intermediates are likely to be packaging intermediates; thus, identification of the assembly intermediate in which HIV-1 Gag first associates with unspliced HIV-1 RNA could shed light on the nature of the earliest HIV-1 packaging intermediate. If the first assembly intermediate (~10S Gag) is associated with unspliced HIV-1 RNA, that would support a model in which a Gag monomer or dimer alone initates this association in cells. In contrast, if the initial association of Gag with unspliced HIV-1 RNA occurs in any other assembly intermediate besides ~10S Gag, that would support a model in which Gag first associates with HIV-1 RNA in host ribonucleoprotein complexes, since all the intermediates except the first assembly intermediate contain host RNA granule proteins. Here, using a variety of approaches, we were unable to identify a population of soluble ~10S Gag associated with unspliced HIV-1 RNA; indeed, to the limit of our detection, almost no unspliced HIV-1 RNA was detected in the soluble fraction when gradients that fully resolve the soluble region were analyzed. Instead, the first assembly intermediate in which Gag was associated with unspliced HIV-1 RNA was the second assembly intermediate, an RNA-granule-derived ~80S complex. Consistent with these findings, ABCE1 and DDX6, which are associated with Gag in the ~80S intermediate, were also associated with unspliced HIV-1 RNA in ~80S fractions. Based on our findings, we hypothesize that the first association of HIV-1 Gag with unspliced HIV-1 RNA occurs only after Gag localizes to an RNA-granule-derived complex containing unspliced HIV-1 RNA ; lysates expressing WT Gag or two well-studied assembly-defective Gag mutants that are arrested in association with unspliced HIV-1 RNA (one arrested in the cytosol and one arrested at the PM); conditions that largely dissociated ribosomes (through use of PuroHS) versus conditions that preserve ribosomes intact; and cells expressing the HIV-1 provirus or a Gag construct with the viral genome provided ion Figs . When lyion Figs  and closion Figs . Thus, wA second issue worthy of discussion involves our identification of a complex in chronically HIV-infected human T cells that was immunoprecipitated using αABCE1, contains unspliced HIV-1 RNA, and appears to correspond to the ~80S assembly intermediate (with formation of a sharp ~80S peak) . In thesImportantly, a model in which HIV-1 RNA packaging is initiated in a host ribonucleoprotein complex , supportOur additional studies of Gag Zip led us to define two steps that direct Gag proteins to ~80S granules containing unspliced HIV-1 RNA . While GImportantly, we do not know the mechanism by which unspliced HIV-1 RNA associates with host RNA granules. Most likely, ribonucleoprotein complexes containing unspliced HIV-1 RNA are generated during transcription, as is the case for cellular mRNA, and undergo successive rounds of remodeling, both during nuclear export and in the cytoplasm, to form cytoplasmic >30S non-translating RNA granules. Additionally, while our coIP studies demonstrate that Gag is associated with unspliced HIV-1 RNA in the packaging/assembly intermediates, we do not know whether Gag and unspliced HIV-1 RNA make direct contact with each other in these complexes. Based on studies by others , we specin trans allowed us to show that the same results were obtained when we repeated key experiments without PuroHS treatment  has an S value similar to the eukaryotic ribosome, which is a large ribonucleoprotein complex containing four species of RNA and 79 cellular proteins. Interestingly, studies by others are consistent with this finding. Specifically, the reported diffusion coefficient of ~70S bacterial ribosomes  is simiIV-1 RNA  and Gag IV-1 RNA , respectIV-1 RNA , and a GIV-1 RNA  and Vif IV-1 RNA , are preIV-1 RNA ,22. In aIV-1 RNA ). The lain situ studies demonstrate that the RNA-granule-derived putative HIV-1 packaging intermediates that we identified are not simply artifacts formed during cell lysis. Previously, we had used quantitative double-label IEM to show that DDX6 is recruited to PM sites of WT Gag assembly, but not to PM sites containing assembly-incompetent MACA Gag [in situ  in situ by light microscopy using PLA  than newly translated Gag, which is initially found either with translating ribosomes or in the soluble compartment. To solve this problem, Gag may have evolved an oligomerization-dependent mechanism for localizing to a subclass of mRNA-containing host RNA granules; association with RNA granules could allow efficient binding of Gag to unspliced HIV-1 RNA, which would in turn localize Gag to the small subset of these granules that contains unspliced HIV-1 RNA. Future studies will be needed to further test this model and better understand how the virus co-opts this subclass of host RNA granules.in trans expression system in which the genome is provided by V1B, a modified proviral plasmid that expresses an RNA encoding an assembly-incompetent truncated gag gene, all cis-acting packaging signals, full-length tat, rev, and vpu genes, and 24 MS2 stem loops that bind to MCP [in trans, where indicated. V1B and the codon optimized SynGag GFP WT and G2A constructs  were used to generate chronically HIV-infected H9 T cells expressing a eviously . 293T ceeviously .COS-1 and 293T cells were maintained in DMEM (Life Technologies) with 10% FBS. H9 cells were maintained in RPMI (Life Technologies) with 10% FBS under puromycin selection. HeLa-MCP-GFP cells were obtained from P. Bieniasz and were maintained in DMEM with 10% FBS, and periodically subjected to blastocidin selection.+2-containing NP40 buffer  in the presence of freshly prepared protease inhibitor cocktail  and RNaseOUT (Invitrogen). Where indicated, lysates were treated with 1 mM puromycin HCl (Invitrogen) for 10 min at 26°C followed by 0.5M KCl for 10 min at 26°C before further analysis; otherwise cells were harvested in 1X NP40 buffer  in the presence of freshly prepared protease inhibitor cocktail  and RNaseOUT (Invitrogen) and analyzed immediately. Lysates were analyzed by WB or RT-qPCR, or subjected to IP as described below. Alternatively, lysates were analyzed by velocity sedimentation, as described below, and gradient fractions were then analyzed by WB, RT-qPCR, or IP.COS-1, 293T, or HeLa-MCP-GFP cells were transfected with 1–5μg DNA using polyethylenimine . Cell lysates were harvested in 1X Mg1 (Bethyl Laboratories) or anti-mouse-IgG or anti-rabbit secondary antibody . In Except where indicated, lysates or gradient fractions were subjected to IP with affinity purified αABCE1 , HIV imm3 COS-1 cells, 2 x104 293T cells and 8.5 x103 HeLa-MCP-GFP cells were used, and results of quantification were normalized to 1 x103 cells. For VLP analysis, aliquots corresponding to VLPs from 1 x105 293T cells and 1.5 x104 HeLa-MCP-GFP cells were used, and results of quantification were normalized to 1 x103 cells. For gradient analysis, ~4 x105 COS-1 cells, 2 x 106 203T cells, or 6 x 105 H9 cells were analyzed on a single 5 ml gradient. Aliquots of gradient fractions or gradient IP fractions were treated with proteinase K (Sigma) at a final concentration of 150 μg/ml in 0.1% SDS, followed by total RNA extraction by using Trizol (Ambion). RNA was precipitated with isopropanol, extracted with BCP (Molecular Research Center), pelleted at 12,000xg for 15 min at 4°C, and the RNA pellet was subjected to DNase I (Invitrogen) treatment (2 u per 50 μl reaction). The iScript Advanced cDNA synthesis kit (Bio-Rad) was used to generate cDNA from 10% of the RNA using random hexamer primers at 42°C for 30 min, followed by heat inactivation. An aliquot of cDNA (2.9%) was used for qPCR using SYBR Green (Bio-Rad) to determine RNA copy number. For HIV-1 viral RNA qPCR, we used the following oligos that target bp 162 to 269 within the Gag open reading frame in HIV-1 LAI (at the end of MA and start of CA) and result in a 108 bp amplicon: 5’-AGAAGGCTGTAGACAAATACTGGG-3’ (forward); 5’-TGATGCACACAATAGAGGGTTG-3’ (reverse). These oligos detect the full length HIV-1 provirus as well as the V1B genomic construct, but do not recognize the Gag GFP constructs that were transfected in trans, as expected since the Gag GFP constructs were codon-optimized. To determine the copy number of other RNA species, we used the following qPCR oligos: Tat mRNA, 5’-TCT ATC AAA GCA ACC CAC CTC-3’ (forward) and 5’-CGT CCC AGA TAA GTG CTA AGG-3’ (reverse); 28S rRNA, 5’-CCC AGT GCT CTG AAT GTC AA-3’ (forward) and 5’-AGT GGG AAT CTC GTT CAT CC-3’ (reverse); 18S rRNA, 5’-GCA ATT ATT CCC CAT GAA CG-3’ (forward) and 5’-GGC CTC ACT AAA CCA TCC AA-3’ (reverse); GAPDH mRNA, 5’-AGG TCA TCC CTG AGC TGA AC-3’ (forward) and 5’-GCA ATG CCA GCC CCA GCG TC-3’ (reverse); and 7SL RNA 5’-GCT ATG CCG ATC GGG TGT CCG-3’ (forward) and 5’-TGC AGT GGC TAT TCA CAG GCG-3’(reverse). All qPCR samples were analyzed in duplicate using the MyiQ RT-PCR detection system and iQ5 software (Bio-Rad). Amplicons corresponding to regions amplified by qPCR were used to generate standard curves. Duplicate nine-point standard curves were included on every qPCR plate, ranging from 101 copies to 108 copies, with a typical efficiency of ~90% or greater and an R2 of 0.99. Standard curves were able to detect 10 copies per reaction but not 1 copy, thereby setting the detection threshold at 10 copies per reaction, which was equivalent to ~1000 copies per 1000 cells for inputs and total viral RNA from gradient fractions, ~100 copies per 1000 cells for IP from total cell lysates or gradient fractions, and ~50 copies per 1000 cells for VLPs. Minus RNA controls were included in each experiment and were always zero. RT minus controls were also included in each experiment and ranged from 0–100 copies per reaction. Mock transfected VLP controls were used to set the baseline in graphs and ranged from 1–1000 copies per 1000 cells. For IP, nonimmune viral RNA copy number was analyzed in parallel and was typically 1–2 logs lower than in immune IP samples.Transfected cells or VLPs were harvested as described above. Where indicated lysates were analyzed by velocity sedimentation and/or IP, as described above except that IP samples that were prepared for viral RNA quantification were washed four times in detergent buffer and once in non-detergent buffer. For total cell lysate analysis, aliquots corresponding to ~5 x10Quantification of the total cell number used in each experiment allowed us to represent all qPCR data as number of viral RNA copies per 1000 cells, except for Protocols for VLP and gradient RT-qPCR are available at:dx.doi.org/10.17504/protocols.io.k73czqndx.doi.org/10.17504/protocols.io.k74czqwg) for 10 min at 4°C, filtered (0.45 μm) to remove remaining cells, and purified through a 30% sucrose cushion in an SW60Ti rotor at 60,000 rpm  for 30 min at 4°C, as described previously [Supernatants of COS-1 or HeLa-MCP-GFP cells, transfected as described above, were centrifuged at 2000 rpm (910 x eviously .g) for 90 min at 4°C. To resolve from ~10S to ~750S, step gradients were prepared from 10%, 15%, 40%, 50%, 60%, 70%, and 80% sucrose in NP40 buffer without MgCl, and subjected to velocity sedimentation in a 5 ml Beckman MLS50 rotor at 45,000 rpm , for 45 min at 4°C. Gradients were fractioned from top to bottom, and aliquots were analyzed by WB, IP, and/or RT-qPCR as described above. Expected S value migrations were determined using a published equation [Transfected COS-1 cells or 293T cells were harvested at 36 h or 15 h posttransfection, respectively, as described above and diluted into 1X NP40 buffer . For each sample, 120 μl lysate was layered on a step gradient. To resolve complexes of ~10S to ~150S, step gradients were prepared from 5%, 10% 15%, 20%, 25%, and 30% sucrose in NP40 buffer without MgCl  and subjected to velocity sedimentation in a 5 ml Beckman MLS50 rotor at 45,000 rpm 162,500 x 2,500 x g293T cells were plated into 6-well dishes containing coverslips with Grace Biolabs CultureWell silicone chambers (Sigma-Aldrich) attached to create four chambers on each coverslip. Cells were transfected with 3 μg of plasmid per well and 16.5 h later were fixed for 15 min in 4% paraformaldehyde in PBS pH 7.4, permeabilized in 0.5% saponin in PBS, pH 7.4 for 10 min, and blocked in Duolink blocking solution (Sigma-Aldrich) at 37°C for 30 min. Cells were incubated in primary antibody (described under IP methods above), followed by Duolink reagents (Sigma-Aldrich): oligo-linked secondary antibody, ligation mix, and red or green amplification/detection mix, with washes in between, as per the Duolink protocol. For concurrent IF, cells were incubated following the final PLA washes for 15 min at RT with 1:1000 secondary antibody, either Alexafluor 594 conjugated to anti-mouse IgG or Alexafluor 488 conjugated to anti-rabbit IgG. Cover slips were mounted using Duolink In Situ Mounting Media with DAPI, sealed to the glass slides with clear nail polish, allowed to dry for 24 h at RT, and stored at -20°C. Imaging was performed with a Zeiss Axiovert 200M deconvolution microscope using Zeiss Plan-Apochromat 63X/ aperture 1.4 objective with oil immersion, using AxioVision Rel. 4.8 software. For quantification, exposure times were set so that all measured PLA spots and Gag IF signal in test fields fell below saturation. Once a non-saturating exposure time was identified, five fields containing at least three IF-positive cells for Figs  were choBecause spots in fields used for quantitation were difficult to see in figures, the gain was increased proportionally in quantified images solely for the purpose of display. Specifically, after imaging and quantification, the red channel gain in representative images was increased proportionally  were placed on grids, treated with 0.05 M glycine for 20 min at RT, rinsed in PBS, blocked for 45 min with 1% bovine serum albumin (EMS), and washed in PBS with 0.1% bovine serum albumin-C (BSA-C) (EMS). For immunogold double labeling, a previously described peptide-specific antiserum directed against DDX6 was affinity purified, desalted, and concentrated [HeLa-MCP-GFP cells were transfected with the indicated constructs. Cells were harvested at 24 h posttransfection in fixative , pelleted, and subjected to high pressure freezing using the Leica EMPACT2, followed by freeze substitution. Samples were infiltrated overnight with LR White embedding resin  in ethanol, changed to straight LR White, embedded in gelatin capsules (Electron Microscopy Sciences (EMS), Hatfield, PA, USA), and cured overnight in a UV light cryo-chamber at 4entrated . Grids wentrated . DDX6 anentrated .GFP antibody was validated by showing absence of labeling in non-transfected control cells.For quantification, images were acquired for ten cells from each of the three groups, with the goal being to analyze similar total PM lengths in each group. Cells were chosen randomly, but excluded for the WT and Gag Zip groups if they had fewer than ten particles at the PM visible at low power. Images encompassed the area of each cell that contained PM assembly sites, with images obtained for ~250 μm of PM total per group. The number of assembly sites analyzed within this ~250 μm of PM are not equivalent since the number of assembly sites depends on VLP phenotype and kinetics. A total of 760 WT events and 409 Gag Zip events were analyzed, but are shown as number of sites per 25 μm PM per cell in S1 Fig(A) COS-1 cells were co-transfected to express a WT or mutant codon-optimized Gag tagged with GFP (Gag GFP) and the V1B genome (Set II constructs in (B) Equivalent aliquots of cell lysates were analyzed by WB for Gag, as were VLPs harvested from the corresponding cell supernatants. Graph shows the number of unspliced viral RNA copies in VLP pellets from the equivalent of 1000 cells, as determined by RT-qPCR. (C) Lysates of transfected cells were also subjected to IP with αGFP or non-immune (N) antibody followed by Gag WB (left), with IP inputs shown (center). IP eluates were also analyzed for unspliced viral RNA copies by RT-qPCR, with NI values subtracted, as shown in graph. Error bars show SEM from duplicate samples. Data are representative of two independent replicate experiments.(TIF)Click here for additional data file.S2 Fig(A) COS-1 cells transfected with the indicated plasmids (Set II constructs in (B) Lysates from A were analyzed by velocity sedimentation, and the number of unspliced viral RNA copies per 1000 cells in each fraction was determined and normalized to inputs in A. (C) Gradient fractions from B were subjected to IP with αGFP, and the number of unspliced viral RNA copies per 1000 cells in IP eluates from each fraction was determined and normalized to inputs in A. Error bars show SEM from duplicate samples. Data in each column are from a single experiment that is representative of three independent replicate experiments.(TIF)Click here for additional data file.S3 Fig(A) COS-1 cells were transfected to express WT Gag GFP or Gag W184A/M185A GFP, and the V1B genome (Set II constructs in (B) Cells were harvested following PuroHS treatment, and equivalent aliquots of cell lysates were analyzed by WB for Gag, as were VLPs harvested from the corresponding cell supernatants. Graph shows the copy number of unspliced viral RNA in cell lysate or VLP aliquots corresponding to the equivalent of 1000 cells, as determined by RT-qPCR. (C) Lysates of cells transfected as in A were subjected to IP with αGFP or non-immune (N) antibody followed by Gag WB (left). IP eluates were also analyzed by RT-qPCR for copies of unspliced HIV-1 RNA, with NI values subtracted (graph). Error bars show SEM from duplicate samples. Data are representative of two independent replicate experiments. (D) Previously we have shown that Gag W184A/M185A is arrested at a membrane-targeted ~80S assembly intermediate when expressed from a proviral construct [in trans, cells expressing these constructs were analyzed by velocity sedimentation followed by WB for Gag. WB of equivalent aliquots of cell lysates (upper right) shows that Gag W184A/M185A GFP was expressed at higher levels than WT Gag.onstruct . To conf(TIF)Click here for additional data file.S4 Fig(A) Human H9 T cells that are chronically infected with HIV-1 were harvested without PuroHS treatment and analyzed by velocity sedimentation to separate complexes in the ~10S to ~750S range. Cell lysates and pooled fractions containing complexes of specific sizes  were analyzed by IP with antibody to Gag  or with a nonimmune control antibody, with unspliced HIV-1 RNA in IP eluates quantified by RT-qPCR. The ~10S pool contains fractions 1–4; the ~80S pool contains fractions 5–11; and the ~500S pool contains fractions 12–18 from a gradient similar to that shown in 3 copies per 1000 cells) corresponds to the limit of detection of unspliced HIV-1 RNA in standard curves. (B) In the same experiment, unfractionated cell lysate was also analyzed by IP with beads but no antibody (No Ab Control). Values for unspliced HIV-1 RNA from the input and IP from the ~10S fraction were below the limit of detection.(TIF)Click here for additional data file.S5 Fig(A) COS-1 cells were co-transfected to express WT Gag GFP with either the WT or ΔΨ V1B genomic construct (Set II constructs in (B) Cell lysates and VLPs were harvested from COS-1 cells transfected with the indicated Gag and GagZip constructs along with the WT V1B genomic construct (Set II constructs in (C) COS-1 cells were transfected to express WT Gag GFP or Gag Zip GFP and the V1B genome (Set II constructs in (D) Lysate from 293T cells (top) or H9 cells (bottom) was analyzed by velocity sedimentation followed by WB of gradient fractions to define the migration of endogenous ABCE1 and DDX6 in human cells.(TIF)Click here for additional data file.S6 Fig(A) Experimental schematic showing the positive and negative control conditions for cells expressing WT provirus. A schematic for the mock-transfected control is not shown. (B) The average number of PLA spots per cell was determined for all Gag-positive cells in five randomly chosen fields and normalized to Gag levels. Quantification was only performed for the Gag-transfected positive and negative controls, since there were no comparable Gag transfected cells to analyze in the mock-transfected control, but representative images show almost no signal in mock-transfected controls, as shown in C. (C) Shown are representative images for the Gag-transfected positive and negative controls (top three rows), and for the mock-transfected control (bottom row). From left to right for each construct: Gag IF (green) with DAPI-stained nuclei (blue), Gag-DDX6 PLA signal (red) with DAPI-stained nuclei (blue), and a merge of all three. Merge demonstrates that PLA spots are mainly in Gag-expressing cells for the positive control, and PLA spots are largely absent in NI controls. Inset in PLA panel shows a high magnification view of a cell to the left of the inset. Scale bars, 5 μm for main panels, 2.5 μm for inset. Data are representative of three independent replicate experiments. Error bars show SEM (n = 5 cells).To quantify background signal that is generated by nonspecific binding of either the Gag or DDX6 antibodies used for Gag-DDX6 PLA, positive and negative controls were generated in 293T cells transfected with the HIV-1 provirus expressing WT Gag (Set I construct in (TIF)Click here for additional data file.S7 Fig(A) Experimental schematic showing the DDX6 IF and secondary only conditions, both with Gag IF, for cells expressing WT provirus. (B) The green signal intensity in red Gag-positive cells was quantified for secondary-only (“Background”); this signal was also quantified in cells that were labeled with primary and secondary as total DDX6 IF signal intensity  or total DDX6 IF with high intensity P body signal subtracted . These signal intensities are shown as percent of mean total DDX6 IF intensity in Gag-positive cells from fields labeled with primary and secondary antibody. (C) Shown are representative images for the Gag-transfected cells with DDX6 IF (top three rows), and secondary-only (bottom row). From left to right for each construct: Gag IF (red) with DAPI-stained nuclei (blue), DDX6 IF signal (green) with DAPI-stained nuclei (blue), and a merge of all three. Inset in DDX6 IF central panel shows a high magnification view of a cell above the upper left corner of the inset. (D) Histogram shows the distribution of DDX6 signal intensity by pixel number, from the fields used to obtain values shown in B . Low intensity signal is seen on the left and high intensity signal is seen on the right. The threshold for high intensity P body signal is shown, as is the cut-off for background signal. Scale bars, 5 μm for main panel and inset. Data are representative of two independent replicate experiments. Error bars show SEM (n = 5 fields). ++ indicates a significant difference between WT Gag and Gag Zip (p<0.005).To quantify background signal that is generated by nonspecific binding of the fluorescent-linked secondary antibodies used for DDX6 IF, positive and negative controls were generated in 293T cells transfected with the HIV-1 provirus expressing WT Gag (Set I construct in (TIF)Click here for additional data file.S8 Fig(A) HeLa cells expressing MCP-GFP were transfected with V1B genomes that contain MS2 binding sites and express WT Gag, G2A, Gag Zip, or MACA (Set IV constructs in (B) Equivalent aliquots of cell lysates were analyzed by WB for Gag, as were VLPs harvested from the corresponding cell supernatants. Graph shows the number of unspliced viral RNA copies in cell lysate or VLP aliquots corresponding to the equivalent of 1000 cells, as determined by RT-qPCR. Error bars show SEM from duplicate samples. Data are representative of two independent replicate experiments.(TIF)Click here for additional data file.S1 TableND indicates % was not calculated because the number of assembly sites was < 1.0 per 25 μm PM. SEM was used to calculate significances for labeling as % of total using a two-tailed Student’s t-test. Shaded columns are shown in the graph in Early PM Assembly Sites are defined as PM assembly sites displaying less than half a bud, while Late PM Assembly Sites are defined as PM assembly sites displaying half a bud or greater. Shown as number of sites per 25 μm PM per cell (n = 10 cells) are the following: Early + Late PM Sites, Early PM Assembly Sites alone, or Late PM Assembly Sites alone. Labeling sites  are shown as number of assembly sites per 25 μm PM per cell (top number), and as % of total PM assembly sites for that group . (TIF)Click here for additional data file.


Apple stem grooving virus (ASGV)–Pyrus pyrifolia pathosystem to explore the possible roles of vsiRNA in thermotherapy.Heat treatment (known as thermotherapy) together with in vitro culture of shoot meristem tips is a commonly used technology to obtain virus-free germplasm for the effective control of virus diseases in fruit trees. RNA silencing as an antiviral defense mechanism has been implicated in this process. To understand if high temperature-mediated acceleration of the host antiviral gene silencing system in the meristem tip facilitates virus-derived small interfering RNAs (vsiRNA) accumulation to reduce the viral RNA titer in the fruit tree meristem tip cells, we used the PpDCL2,4, PpAGO1,2,4 and PpRDR1 genes, and found their expression levels were up-regulated in the ASGV-infected pear shoots at 37 °C.At first we determined the full-length genome sequence of the ASGV-Js2 isolate and then profiled vsiRNAs in the meristem tip of in vitro-grown pear (cv. ‘Jinshui no. 2’) shoots infected by ASGV-Js2 and cultured at 24 and 37 °C. A total of 7,495 and 7,949 small RNA reads were obtained from the tips of pear shoots cultured at 24 and 37 °C, respectively. Mapping of the vsiRNAs to the ASGV-Js2 genome revealed that they were unevenly distributed along the ASGV-Js2 genome, and that 21- and 22-nt vsiRNAs preferentially accumulated at both temperatures. The 5′-terminal nucleotides of ASGV-specific siRNAs in the tips cultured under different temperatures had a similar distribution pattern, and the nucleotide U was the most frequent. RT-qPCR analyses suggested that viral genome accumulation was drastically compromised at 37 °C compared to 24 °C, which was accompanied with the elevated levels of vsiRNAs at 37 °C. As plant Dicer-like proteins (DCLs), Argonaute proteins (AGOs), and RNA-dependent RNA polymerases (RDRs) are implicated in vsiRNA biogenesis, we also cloned the partial sequences of Collectively, these results showed that upon high temperature treatment, the ASGV-infected meristem shoot tips up-regulated the expression of key genes in the RNA silencing pathway, induced the biogenesis of vsiRNAs and inhibited viral RNA accumulation. This study represents the first report on the characterization of the vsiRNA population in pear plants infected by ASGV-Js2, in response to high temperature treatment.The online version of this article (doi:10.1186/s12985-016-0625-0) contains supplementary material, which is available to authorized users. Virus-induced gene silencing is an antiviral defense mechanism in plants, in which the vital elements involved are virus-derived small interfering RNAs (vsiRNAs) which are mainly 20–24 nucleotides (nt) in length , 2. The Arabidopsis, among the four relatively well-studied DCLs, DCL4 and DCL2 function in RNA silencing against RNA viruses by producing 21- and 22-nt vsiRNAs, respectively. It has been demonstrated that the activity of DCL2 in producing specific 22-nt vsiRNAs derived from Turnip crinkle virus (TCV) in Arabidopsis is enhanced by higher temperatures [Potato virus X (PVX)-infected Arabidopsis plants [N. benthamiana, silencing of RDR6 increases viral RNA accumulation and facilitates viruses to invade the meristem tissue [Chinese wheat mosaic virus (CWMV) accumulation and vsiRNA biogenesis at higher temperatures [It is well known that DCLs, AGOs, and the RNA-dependent RNA polymerases (RDRs) participate in the antiviral silencing pathways as key silencing factors, and their RNA silencing activities are varied under different temperatures , 12–15. eratures , 17. DCLeratures . Recent s plants , 20. In s plants . In N. beratures , 25.Apple stem grooving virus (ASGV), a member of the genus Capillovirus in the family Betaflexiviridae [iviridae . The ASGiviridae . ORF2 isiviridae . MP and iviridae , 29. Phyiviridae , 31. ASGiviridae –35. In aiviridae . In peariviridae . In the iviridae , 38. Higiviridae , 40. Theiviridae , 41–43. iviridae , 44. In iviridae . These dPpDCL2,4, PpAGOs, and PpRDR1 mRNA sequences and assessed their relative accumulation levels in the ASGV-infected pear shoot tips at 24 and 37 °C. Collectively, these results assist in a better understanding of the roles of vsiRNAs in ASGV infection in vitro-grown pear meristem tips in response to high temperature.To explore the possible roles of vsiRNA in the interaction of ASGV and pear plants at higher temperatures, we determined the complete genomic sequence of the ASGV-Js2 isolate and profiled the vsiRNAs in the meristem tip of in vitro-cultured pear shoots at 24 and 37 °C. We analyzed the distribution of vsiRNAs along the viral genome. The corresponding accumulation levels in relation to viral titer and the expression profiles of vsiRNA were also characterized in the shoot tip tissues by RT-qPCR. Furthermore, we determined the P. pyrifolia cv. ‘Jinshui no. 2’ shoots infected with ASGV or ASGV-free, were confirmed by RT-PCR  genome database [PpDCL2,4, PpAGO1,2,4, and PpRDR1 sequences were amplified by RT-PCR using specific primers from total RNA as template. The PCR products were purified and cloned. The PpDCL2,4, PpAGO1,2,4, and PpRDR1 DNA sequences were deposited into GenBank and their accession numbers are given in Additional file Primers used for amplification were designed based on the pear  method incorporated in the programs Clustal X 1.83 and MEGA6 software , 51.mp, PpDCL2,4, PpAGO1,2,4, and PpRDR1 were analyzed by real-time PCR. Two or three sets of primers were designed and estimated for each gene. The best primer set for each gene is provided in Additional file Actin gene was used as an internal reference gene for normalization of ASGV mp, PpDCL2,4, PpAGOs, and PpRDR1 gene expression levels. The RT-qPCR for each gene was performed for three replicates. The experimental data were analyzed essentially as described previously [To detect the vsiRNA expression levels obtained from the high-throughput sRNA sequencing, real-time quantitative PCR (RT-qPCR) was performed by One Step microRNA Prime-Script sRNA cDNA Synthesis Kit with Poly (A) tails and a special oligo-dT adaptor according to supplier’s instructions (Takara) as described by Liu et al. . Total Reviously , 53.To profile vsiRNAs derived from the ASGV-Js2 isolate in pear, we needed to determine the full-length genome sequence of the ASGV-Js2 isolate. The full-length ASGV-Js2 genome was cloned and sequenced as described in Methods , vsiRNA2931(+), and vsiRNA4379(+), respectively. Similarly, some reads were mapped to the negative strand at positions 5839–5859 and 4625–4645, designated vsiRNA5839(−) and vsiRNA4625(−), respectively. RT-qPCR results discovered that the expression levels of vsiRNA2931(+) and vsiRNA4625(−) in the shoot meristem tip tissue at 37 °C were nearly equal to those at 24 °C . The relative abundance of CLCV-derived siRNA is apparently positively correlated with viral titers in pumpkin [Arabidopsis, tobacco, and rice [PpDCL2, PpDCL4, PpAGO1, PpAGO2, PpAGO4, and PpRDR1 and determined their relative expression levels (Figs. Real-time PCR analysis using primers specific for the MP coding region Fig.  or RdRp  pumpkin . A possi pumpkin , 57, 64.and rice , 65–67. ls Figs.  and 9. Omp gene and vsiRNAs were characterized by RT-qPCR. We also cloned PpDCL2,4, PpAGO1,2,4 and PpRDR1 partial sequences and examined their expression patterns, and found their expression levels were up-regulated in the ASGV-infected pear shoots at 37 °C. This up-regulation was accompanied with the reduced level of viral RNA and the elevated levels of vsiRNAs. Taken together these data suggest that high temperature may induce and enhance the RNA silencing capacity in the pear meristem tissue.This study represents the first report on the characterization of vsiRNA in the in vitro-grown ASGV-infected pear shoots in response to high temperature treatment. The profiles of vsiRNAs showed an uneven distribution along the ASGV-Js2 genome, and that 21- and 22-nt vsiRNAs preferentially accumulated when cultured at higher temperature. ASGV-specific siRNAs from all libraries had a similar distribution of 5'-terminal nucleotides. U was the most frequent among the 5’ terminal nucleotides, and its frequency was slightly higher at 37 °C. The expression levels of the viral


However, the degree to which choice is exercised when entering an informal caring role is open to debate.In this study, we examined the degree of choice and constraint in entering a caring role, and the relationship between choice and carers’ well‐being.Data were derived from 1100 responses to a postal survey conducted in a British city. Statistical tests of association and multivariable regression modelling were applied to study the factors associated with choice in entering a caring role and the association that choice in entering a caring role had with carers’ well‐being.We found that informal care was generally perceived to be a free choice, albeit in most cases, a choice was also constrained by duty, financial or social resources. Having a sense of free choice in entering care was strongly and positively associated with the carer's well‐being.The study findings are consistent with a view that enabling individuals to have more choice in their caring roles may be beneficial. Rising healthcare costs means that governments increasingly look to the family for care for individuals unable to look after themselves The focus in this study is on choice in taking on the caring role. The issue of degree of choice within the caring role, for example in relation to combining work and care, is discussed elsewhere obligation alimentaire system, would oblige people to provide elder care, although this could be shared amongst siblings or one or more siblings could take overall responsibility. The responsibility view suggests that demand will create its own supply and this perspective is supported to some extent by evidence that the proportion of women providing intensive (>15 hours per week) out‐of‐home care in the USA is identical across prebaby boomer and baby boomer cohorts The reasons why so many individuals decide to engage in informal care, particularly when it is very time intensive, are not well understood. At one end of the spectrum, there is the view that people who become carers do so because they feel obliged to act as a carer when a family member becomes ill Carers may be constrained by factors other than duty. Carers are often in poor financial circumstances At the other end of this spectrum, the view is that a decision to undertake care is essentially a rational choice. This decision reflects individual cost benefit calculations linked to factors such as loss of income, ill health or increased stress due to caring responsibilities and any avoidance of guilt, satisfaction or ‘process utility’ derived from the provision of informal care A pragmatic approach would suggest that the degree of choice available to potential carers will differ depending on their individual circumstances and those of the people needing care; for some, the degree of choice may be more constrained than for others. The nature of these circumstances might also change over time and as people age. The critical feminist economics discourse on altruism, reciprocity and norms of responsibility provides some insights into these potential influences. Folbre , p.75 deTo date, there has been relatively limited empirical study of the degree to which family members feel they are exercising choice in taking on a caring role The high proportion of carers who feel constrained in their caring role is underscored by related research on the reasons for caring. A study of Dutch carers found that the most common reason for caring was ‘duty’ and that other constraints such as being ‘the only one…available’ were also mentioned as important factors The evidence on caring and well‐being suggests that, in general, more intensive caring roles, and specifically transitions into caring roles, are associated with lower levels of well‐being Our study addresses a gap by examining both choice and constraints in relation to entering caring. This is important because choice and constraints are likely to act simultaneously and may play a large role in the carer's subjective experience of caring. Our objectives were to (i) establish the degree to which individuals perceive caring to be choice or a constraint; (ii) identify whether the degree of choice varies according to carers’ characteristics and caring role; and (iii) estimate the association between choice in caring and the carer's well‐being. We investigated these issues using data from a survey of individuals living in a large city in the UK.This study is a secondary analysis of cross‐sectional data collected through a local government survey of residents’ quality of life. The survey covered a range of aspects of individuals’ lives, including a module of questions about the provision of informal care. The informal care questions covered the individual's decision to provide care; these survey data therefore offered an opportunity to study the decision to care amongst a heterogeneous group of carers in a community setting. Alongside data on informal care, data were collected on individuals’ socio‐demographic circumstances and well‐being, and these data were used to examine the factors associated with choice in caring and the relationship between well‐being and perceived choice in caring. Details of the survey, the construction of the choice in caring variable and the subsequent analysis are reported below.The data used in the study came from the 2009 Bristol City Council Quality of Life Survey How many hours of care are provided per week? (< 20 hours/20‐49 hours/50 hours +)How old is the care recipient? (0‐17 years/18‐64 years/65 years and over)What is the health of the care recipient like? (good/fair/bad)Are you the main carer for this person? (yes/no)Do you help with personal care? (yes/no)Do the following features of care provision apply? ○ I provide care because it is my duty? (yes/no)○ I had a free choice to provide care? (yes/no)○ There was no one else to provide care? (yes/no)○ There was no money for paid care? (yes/no)Respondents also completed the Carer Experience Scale  ‐ a six‐item scale measuring carer quality of life. No questions were included on other aspects of informal care provision, for example, on the duration of caring, the carer–recipient relationship, the carer's marital status, whether they co‐resided with the care recipient or whether they had multiple caring roles.5771 individuals responded to the survey, of whom 1 100 (19%) indicated that they provided some informal care in a typical week. This survey met the Local Authority standards of conduct, was compliant with UK law on data protection, and participation was both anonymous and voluntary.We combined the responses to the four questions concerning individuals’ decision to care to simultaneously examine carers’ perceptions of choice and constraints in caring. This allowed for a more nuanced analysis than simply studying the responses to the four questions in isolation. To investigate the degree to which caring was perceived to be a free choice, carers were categorised into three groups. In the first group were carers who reported caring was a free choice, and not constrained by duty, finances or social support (‘free‐choice’ carers). A second group comprised those carers who reported caring was a free choice, but also reported that at least one of the constraints was also present (‘constrained choice’ carers). The third group comprised carers who indicated that caring was not a free choice (‘unfree’ carers).The analysis involved two stages. The first investigation focused on identifying whether perceived choice in caring (as measured by the new three category variable) was affected by socio‐demographic factors and the nature of the caring role entered into. To examine the effect of socio‐demographic factors, we examined variables related to demography , culture  and empowerment . For caring role, we examined variables related to the nature of caring role (primary or secondary), the provision of personal care, hours of caring, the health status of the care recipient and the age of the care recipient. We used cross‐tabulations to explore the magnitude of associations between perceived choice in caring and these variables and, given the ordinal nature of the perceived choice variable, used Kruskal–Wallis tests to identify statistical significance of any associations.The second stage of the investigation focused on the relationship between perceived choice in caring and carers’ subsequent well‐being. We examined individuals’ hedonic well‐being To analyse the relationship between the measures of well‐being and perceived choice in caring and well‐being, we used Kruskal–Wallis (nonparametric) tests. We then used multivariable regression modelling, to allow for the fact that the relationship between well‐being and perceived choice in caring may be confounded by other factors. This involved modelling well‐being responses as a function of the degree of choice in caring  controlling for socio‐demographic and care‐related factors. We estimated the regression model where the dependent (well‐being) variable was (i) life satisfaction; (ii) happiness; (iii) capability (ICECAP‐O score); and (iv) caring experience (CES) score.Of the 1100 carers who responded to the quality‐of‐life survey, 798 (73%) answered the question about whether their decision to care was a free choice. The analyses that follow focus on these individuals. To set the sample of carers in context, Table Table Following the creation of the new variable see Fig. , around Table Table noncarers in terms of life satisfaction (p = 0.06), happiness (p = 0.09) and capability (p = 0.02). Conversely, carers who report a lack of free choice in caring report levels of life satisfaction (p < 0.01), happiness (p < 0.01) and capability (p < 0.01) below the level reported by noncarers.Table The regression models express well‐being in terms of carers’ life satisfaction, capability and caring experience Table  and careControlling for other factors, having a constrained choice in caring (relative to no free choice) was associated, on average, with the following:Higher life satisfaction ;Higher capability ;Better caring experience ;Lower odds of reporting being unhappy (odds ratio 0.68).Controlling for other factors, having a free and unconstrained choice in caring (relative to no free choice) was associated with even higher life satisfaction, capability, caring experience and happiness. The magnitude of these associations with well‐being can be put into context by comparing them with other factors associated with well‐being. For example, in terms of life satisfaction, the positive impact of free (unconstrained) choice is greater than the positive impact of having educational qualifications or home ownership. In terms of capability, the positive impact of free choice is comparable to the positive association with home ownership.both a choice and a constraint. In this survey, around half the sample of UK carers described their decision to provide informal care as a free choice but constrained by a sense of duty, financial resource or lack of social support. Most socio‐demographic factors were not related to the perception of choice in caring. Conversely, aspects of the caring role linked to caring intensity  were strongly associated with a perception of less choice. The perception of choice was strongly related to carers’ subsequent well‐being, controlling for the fact that those carers who perceived free choice tended to have less intensive caring roles.This study suggests that people often see participation in informal care as The finding that many people perceived some form of constraint in caring is not surprising, given previous research perceiving it as being chosen from free will. Psychological theory and research suggests and demonstrates that people are meaning‐making beings and that subjective meanings often mediate between a situation and a person's reaction to it It is also possible that while the decision to take on caring is constrained by economic, social and normative pressures and might not appear by others to be chosen, nonetheless, the person taking on this role benefits from The lack of association between perceived choice and socio‐demographic characteristics was unexpected. We did not find that perceived choice was any lower, for example, for women or those with less financial capability. This may be because the perception of choice to provide care is highly subjective. It might also be because choice in caring is more strongly influenced by other factor. For example, studying the kin relationship may reveal less perceived freedom in the decision to undertake spousal or parental care than care for an elderly relative or friend. Likewise studying the degree of prior attachment may help explain why a daughter who feels close to her elderly mother may choose to take on the role of caring, over and above other siblings who do not feel as emotionally close.We also found that choice in caring was strongly linked with the entry into secondary caring roles, especially where the recipient was not in ‘bad’ health and when no personal care was provided. One interpretation is that the perception of choice about the decision to provide care is evaluated in the context of the intensity of ongoing care provision, as much as in relation to the initial decision to provide care. In situations where a care role is less intensive, the carer takes on the role knowing he or she retains some freedom to live life other than through caring. In circumstances where care needs are more intensive, becoming the primary carer will constrain or force other roles to be abandoned or neglected. Furthermore, the carer may not be in a position to withdraw if the one cared for is in poor health. In these circumstances, it becomes harder to generate a narrative of choice, hence the association of lower choice with being a primary carer, providing personal care and caring for someone in poor health.While a positive relationship between choice in caring and well‐being was expected, the magnitude of the effect found in this study is worth emphasising. Choice in caring seems as important in terms of the carer's well‐being, if not more so, than more tangible factors, for example whether the carer provides personal care or whether the care recipient is in good health. Choice in caring may therefore be a valuable target for policymakers concerned about improving carer's well‐being. Future research might focus on developing an understanding of the degree to which social policy can expand perceived choice in caring and which aspects of choice can and ought to be targeted. It is important to highlight that choice is important not only in entry to the caring role, but also within the caring role. As a result, there may be gains to carers from expanding choice in the amount and type of social care support It is important, however, not to draw definitive conclusions about a causal link between choice in caring and well‐being. This is a cross‐sectional study, and it could be the case that carers with better well‐being were more able to exercise choice about whether to take on a caring role, or their higher well‐being made them more likely to feel as if they had choice to take on the role. Furthermore, the lack of some key variables means that we are unable to describe the sample as comprehensively as we would have liked, and examine other potentially relevant factors in relation to carers’ choices and constraints. A range of factors, in addition to duty, family support and financial resources, are likely to motivate and constrain decisions in relation to providing informal care. For example, we did not explore some of the positive motivations for care, such as emotional bonds, that have been highlighted in other studies This study suggests that, for many, entry into caring is perceived as both a choice and a constraint. Perception of choice in entering caring is positively associated with well‐being. Further research is needed to specify aspects of caring where free choice generates higher well‐being and whether these can be enhanced by social policy. Nevertheless, this study suggests, in general terms, that there may be significant benefits to carers from enhancing their choice on entry to a caring role.HA conducted the data analysis and wrote the first draft of the manuscript. All authors contributed to the study design, study conduct and later drafts of the manuscript.The study is based on data collected as part of a local government survey of quality of life of local residents. The local government organisation has an internal ethical review process for their survey work.Hareth Al‐Janabi was supported through this work by an MRC early career fellowship in economics of health (G1002334).Appendix S1. Key questions from the Quality of Life surveyClick here for additional data file.