Flaviviruses such as Yellow fever, Dengue, West Nile, and Zika generate disease-linked viral noncoding RNAs called subgenomic flavivirus RNAs. Subgenomic flavivirus RNAs result when the 5′–3′ progression of cellular exoribonuclease Xrn1 is blocked by RNA elements called Xrn1-resistant RNAs located within the viral genome’s 3′-untranslated region that operate without protein co-factors. Here, we show that Xrn1-resistant RNAs can halt diverse exoribonucleases, revealing a mechanism in which they act as general mechanical blocks that ‘brace’ against an enzyme’s surface, presenting an unfolding problem that confounds further enzyme progression. Further, we directly demonstrate that Xrn1-resistant RNAs exist in a diverse set of flaviviruses, including some specific to insects or with no known arthropod vector. These Xrn1-resistant RNAs comprise two secondary structural classes that mirror previously reported phylogenic analysis. Our discoveries have implications for the evolution of exoribonuclease resistance, the use of Xrn1-resistant RNAs in synthetic biology, and the development of new therapies. Subgenomic flavivirus RNAs are generated by a host exoribonuclease and play an important role in virus replication and pathogenesis. Here, the authors show the mechanism by which subgenomic flavivirus RNAs are generated and identify two structurally distinct sfRNA classes in flaviviruses. Infection by mosquito-borne flaviviruses (MBFVs) results in amplification of the genomic RNA (gRNA) and also production of noncoding subgenomic flaviviral RNAs (sfRNAs)8. sfRNAs accumulate to high levels, interacting with many cellular proteins to influence processes such as RNA interference, proper cellular RNA decay, the interferon response, and the process of transmission between mosquito vector and vertebrate host19. sfRNAs have been implicated in cytopathicity in cell culture and in pathogenicity in fetal mice20, thus they are directly related to disease symptoms and are potential therapeutic targets.Flaviviruses are single-stranded, (+)-sense RNA viruses with 10–11 kb-long genomes21. MBFV genomes contain discrete RNA structures in their 3′-untranslated region (UTR) that block the progression of Xrn1. These RNA elements are sufficient to block Xrn1 without the use of accessory proteins, thus they have been assigned the name ‘Xrn1-resistant RNAs’ (xrRNAs)27. xrRNAs halt the enzyme at a defined location such that the viral RNA located downstream of the xrRNAs is protected from degradation. These protected RNAs are sfRNAs, and in some but not all cases multiple xrRNA structures give rise to multiple sfRNA species  the conserved xrRNA structure and sequence could make specific contacts to Xrn1’s surface to prevent helicase activity or block enzyme conformational changes, ii) it could somehow alter the specific catalytic mechanism of Xrn1’s active site, iii) it could use nonspecific physical interactions with the enzyme, iv) it could present a general mechanical unfolding problem, or it could use some combination of these strategies.Xrn1 can unwind and degrade highly structured RNAs such as picornaviral IRES elementsure Fig. , and mod32, direct evidence is lacking for whether other non-arthropod-borne flaviviruses also form sfRNAs. These flaviviruses include members of the no known arthropod vector flaviviruses (NKVFVs) that infect small mammals and bats, and the insect-specific flaviviruses (ISFVs) that have no identified vertebrate host. While the sequences of MBFV xrRNAs contain a number of absolutely conserved nucleotides within a shared three-dimensional fold25, the non-MBFV putative xrRNAs do not have the same sequences as those found in MBFVs29. If these ISFVs and NKVFVs do form sfRNAs, this raises questions of whether they arise from folded RNA structures that function without protein co-factors, and how their structures compare with the MBFV xrRNAs.In addition, although the formation of sfRNA in the MBFVs and tick-borne flaviviruses (TBFVs) is well-establishedHere, we describe studies to address fundamental questions of the mechanism underlying Xrn1 resistance by MBFV xrRNAs and the existence and structure of xrRNAs from other flaviviruses. Using a biochemical approach, we discovered that xrRNAs can halt exoribonucleases unrelated to Xrn1 and that the nature of the interface between the RNA and the enzyme affects where the enzyme halts. This reveals that xrRNAs operate as general mechanical blocks to diverse molecular machines, with implications for the use of these RNAs in diverse contexts. Furthermore, we directly demonstrate that Xrn1 resistance can be achieved by sequences within the 3′-UTR of flavivirus RNAs outside of the MBFVs, but that in some groups the xrRNAs use a secondary structural strategy that differs from that of the MBFVs. By extension, structured exoribonuclease-resistant RNAs may be widespread, possibly conferring novel pathways for RNA maturation or regulation of mRNA decay.KUN) as a representative xrRNA and challenged it with pure bacterial RNase J1, a 5′–3′ exoribonuclease unrelated to Xrn1  of Xrn1’s halt site. Because these exoribonucleases have narrow entrance tunnels to their active sites that only allow single-stranded RNA to enter, the difference in halt sites likely correlates with the distance between the active site and the surface of each enzyme35. In contrast to RNase J1, the halt site of Dxo1 was somewhat poorly defined, with the enzyme appearing to ‘stutter’ as it interacts with the xrRNA , by infecting C6/36 cells. Northern blot analysis with a probe to the 3′-end of the viral RNA revealed a robust band consistent with the production of sfRNA, and reverse transcription showed the location in the 3′-UTR Fig. . To deteExamination of these secondary structures show that several tertiary structure interactions critical for maintaining the three-dimensional structure of the MBFV xrRNAs can also form in the CFAV xrRNAs Fig. . Specifi+•G-C base triple, which is isosteric with the U•A-U and thus can structurally replace it. To test this, we generated mutant versions of the xrRNA and tested them for Xrn1 resistance in vitro , resistance was recovered; this provides strong evidence for the existence of a base triple , reveals a sequence that matches the xrRNA patterns of the MBFVs25, but many other members do not have this sequence pattern. To examine sfRNA formation in these divergent NKVFVs, we infected BHK-21J cells with Modoc virus (MODV), Montana myotis leukoencephalitis virus (MMLV), Apoi virus (ApoiV), and Rio Bravo Virus (RBV); these represent various groups of NKVFVs. Northern blot analysis with probes to the 3′-ends of the viruses revealed the presence of sfRNAs in all  is the primary determinant of resistance; rather than thermodynamic stability, resistance is conferred by a specific RNA topology that the enzyme encounters as it approaches from the 5′ side, and this structure presents a specific barrier to progression.Combined with previous data, the mechanism that emerges is one in which xrRNAs resist progression of exoribonucleases using a specific three-dimensional RNA fold that braces against the surface of the enzyme and creates a general mechanical block to continued enzyme movement. Several existing pieces of evidence fit this mechanistic model. First, although the MBFV xrRNAs block Xrn1 approaching from the 5′ side, the viral RNA-dependent RNA polymerase can pass through from the 3′ side, as can the reverse transcriptase used in our experiments. Although the nature of these enzymes’ helicase activity may differ from Xrn1’s, this observation is consistent with resistance owing to an encounter with the 5′ side of the structure. Also, while the global structural stability of the xrRNA may play a role in resistance, Xrn1 passes through many very stable structured RNAs, suggesting thermodynamic stability is not the sole source of resistance18. Likewise, versions of Xrn1 capable of overcoming xrRNAs are unlikely to arise through a specific mutation within a species, eliminating one host-centered mechanism of evolving resistance to the virus. The ability to block diverse exoribonucleases also confers the potential to use processive 5′–3′ exoribonucleases other than Xrn1 to produce sfRNAs. Indeed, Xrn1 knockdown experiments in Zika virus suggested the presence of redundancy in the 5′–3′ decay machinery that could be exploited by xrRNAs that block diverse exoribonucleases27.The mechanism supported by the data has several implications for the function of these xrRNAs in flavivirus infection. The ability of the MBFV xrRNAs to resist diverse exoribonucleases may assist them in operating in a wide range of hosts and vectors. In other words, sequence differences in Xrn1 from different species are unlikely to result in an enzyme that is able to degrade through an xrRNA. Although viral tropism is dependent on many factors, at least the xrRNA is likely to be functional in a very wide range of species37. In addition, the distance (in terms of sequence length) between the sequences involved in a putative long-range base pairing interaction is longer than is typical for the MBFVs25. Likewise, the halt point for the exoribonuclease in the tested NKVFV and TBFV xrRNAs lies within a helical element and only 5–6 nucleotides upstream of the junction. This is potentially significant because as the enzyme approaches, it will unwind these helical elements, with the effect of further increasing the amount of single-stranded RNA between the structure and the long-range base pairing. Furthermore, at the moment the enzyme halts, the 5–6 nucleotides between the junction and the halt point will be single-stranded and inside the enzyme, suggesting a different RNA structure-based mechanism of halting Xrn1. Although it is possible that these divergent secondary structures and sequences may possess three-dimensional folds or topologies similar to those from the MBFV, it is premature to make this prediction; it is just as possible that they use a different tertiary structure and strategy to block Xrn1. These observations suggest there are at least two secondary structural classes of xrRNAs within the flavivirus genus: the well-characterized class typified by the MBFVs and ISFVs (Class 1) and the class found in the TBFV and some of the NKVFVs (Class 2)  Fig. .Flaviviridae family, the hepatitis C virus (a hepacivirus) and bovine viral diarrhea virus (a pestivirus) have been shown to block Xrn1 progression past a point proximal to the 5′-end of the viral genomic RNA, resulting in dysregulation of host RNA decay pathways40. However, Xrn1 resistance does not appear to be as robust as in the MBFVs and serves a somewhat different purpose in that it does not result in accumulation of a noncoding RNA that alters multiple host pathways during infection. Also, there is no evidence of a three-dimensional RNA structure that is similar to the xrRNAs found in the MBFV 3′-UTRs. Nonetheless, the presence of the ability to resist Xrn1 by these members of the Flaviviridae family outside the flavivirus genus show how useful this ability may be for viruses, inviting speculation that this function may exist throughout the viral RNA world. Indeed, exoribonuclease-resistant RNA sequences have been reported in some plant-infecting viruses that show no sequence similarity to the flavivirus xrRNAs41.It is also worth considering that outside the flavivirus genus but within the Flaviviridae and their use of different arthropod vectors  structural features. Consistent with this, MODV and MMLV are members of the NKVFVs that are overall more closely related to TBFVs, and their xrRNAs match that class. Hence, the point of divergence of Class 1 from Class 2 xrRNAs appears to be near the point where MBFVs and TBFVs phylogenetically diverge and where the diverse NKVFVs lay. We hesitate to make strong conclusions based on these observations; nonetheless it will be interesting to see if future identification of any arthropod vectors for these various NKVFVs shows that the class of xrRNAs correlates with the vector; that is, if Class 1 xrRNAs are exclusive to mosquitoes and Class 2 xrRNAs are exclusive to ticks.It is informative to examine the diverse RNA structure-based ways to halt Xrn1 in light of the phylogenetic relationship of the ors Fig. . In the Within the areas of RNA-based nanotechnology, synthetic biology, and RNA-based therapies, viral RNAs such as xrRNAs can provide elegant building blocks for diverse applications. One can imagine a wide range of applications where it would be advantageous to control the degradation of specific RNA species. Given that the flavivirus xrRNAs are active against a range of exoribonucleases from different species, they might also be utilized as tools within a range of organisms to protect specific RNAs from 5′–3′ decay. The widespread presence of xrRNAs in the flaviviruses, the diversity of sequences and structures capable of mechanically blocking different exoribonucleases, and the apparent usefulness of this strategy for viral infection suggests that RNA structures capable of interfering with exoribonuclease activity may be more widespread than is currently appreciated.22. The RNase J1 expression vector was a kind gift of Ciaran Condon, and the Dxo1 expression vector was a kind gift of Liang Tong. RNase J1 was expressed in BL21 (DE3) cells in LB containing kanamycin and chloramphenicol at 30 °C. Expression was induced with 0.1 mM IPTG at OD600 = 0.6 and protein was expressed at 30 °C for 3 h. The cells were harvested by centrifugation at 5488×g for 10 minutes and the cell pellets were stored at −80 °C until further use. Dxo1 was expressed in Rosetta (DE3) cells in LB with kanamycin. The cells were grown at 37 °C to OD600 = 0.6–0.9. Expression was induced with 0.25 mM IPTG, the temperature was lowered to 16 °C and culture continued overnight before harvesting and storing similar to RNase J1 (above). Cell pellets were lysed by sonication for 2 minutes processing time and centrifuged at 31,000×g for 30 minutes to clear the lysate. The protein was purified using Ni-NTA resin (Thermo) in a gravity flow column followed by size exclusion chromatography with either a Superdex 75 or Superdex 200 column. The final product was stored in buffer containing 20 mM Tris pH 7.3, 300 mM NaCl, 1 mM DTT or 2 mM BME, and 10% glycerol (1 mM EDTA was added to the RNase J1 sample) at −80 °C. The purity was assessed by SDS-PAGE and Coomassie staining.The RppH and Xrn1 enzymes were expressed and purified as describedEscherichia coli (DH5α) and their sequences verified. To generate DNA template for transcription reactions, the desired region of the plasmid was amplified by PCR, then used in 5 mL in vitro transcription reactions and purified as previously described22.Plasmids encoding the RNA sequences of interest under control of a T7 promoter were generated using standard molecular biology techniques. In brief, gene fragments (gBlocks) synthesized by Integrated DNA Technologies (IDT) were ligated into pUC19 vector (NEB) between the BamHI and EcoRI sites. Resultant plasmids were amplified in 2, 1 mM DTT. This solution was incubated at 85 °C for 3 min, followed by 20 °C for 5 min, then held at 4 °C. Then, 0.8 µg of His-Xrn1 and 0.08 µg of His-BdRppH were added and the mixture was incubated at 37 °C for 2 h. The resulting RNA products were analyzed by denaturing PAGE22.The exoribonuclease resistance assays were carried out by first folding 1–2 µg of 3′-UTR RNA and 1 µg of the 24-mer control RNA in a buffer composed of 50 mM Tris, pH 7.9, 100 mM NaCl, 10 mM MgCl22. The RT products were resolved on a 12% denaturing polyacrylamide sequencing gel. The gels were visualized by phosphorimaging using a Typhoon 9400 Imager (Molecular Dynamics) and visualized with ImageQuant software.Exoribonuclease halt sites were mapped by gel purifying His-Xrn1 digested 3′-UTR flavivirus RNA using the same protocol as mentioned above but at a larger scale . Then RT-PCR was performed using unique 5′-end-labeled primers for each flavivirus and GoScript Reverse Transcriptase (Promega)42. In brief, RNAs were transcribed as described above, and purified using Agencourt AMPure XP magnetic beads. To anneal the RNA, it was heated to 90 °C for 2 minutes, transferred to ice for 2 minutes, and then placed at room temperature. RNA was modified via exposure to 24 mg/mL NMIA for 30 minutes before halting the reaction with an acid quench solution 42. Modified RNAs were then reverse transcribed at 42 °C for 45 minutes with a universal fluorescently labeled primer (IDT) (/5–6FAM/ AAAAAAAAAAAAAAAAAAAAGTTGTTGTTGTTGTTTCTTT). Labeled cDNA products were eluted in HiDi formamide and Gene Scan ROX 350 (Thermo) for capillary electrophoresis analysis on an Applied Biosystems 3500 XL Genetic Analyzer42. All reactions were performed in triplicate, as were “no modification” control reactions and ddNTP ladders. Analysis of capillary electrophoresis data was performed using the HiTRACE MATLAB toolkit (MathWorks) and 1D analysis pipeline developed by the Das and Yoon labs46. For further information, the website and tutorial for these programs and protocols can be found at: https://hitrace.github.io/HiTRACE/.We utilized the one-dimensional (1D) chemical probing method described by Cordero et al. to conduct our SHAPE experiments and analysis49. Infections were performed as previously described48. Total RNA was isolated with Trizol (Invitrogen) at 30 h.p.i. from BHK-21J cells infected with MODV, APOIV, MMLV, or RBV , or at 36 h.p.i. from CFAV-infected C6/36 cells .The origin and culture conditions of the BHK-21J  and C6/36 (ATCC CRL-1660) cells have been described2O and 5 μg was used for RT-PCR to amplify the Xrn1 stalling site-containing region of either MODV (nts. 10247–10343) or MMLV (nts. 10265–10367). Primers for these reactions can be found in Supplementary Table 50  was dissolved in 30 ml H50. Approximately 1.5 × 106 transfected BHK-21J cells were seeded in a 35 mm plate. Total RNA was isolated from the transfected cells at 8 h post-electroporation using Trizol (Invitrogen). For Northern blotting, 7.5–10 μg of total RNA isolated from either infected or electroporated cells was denatured using formaldehyde and separated on a formaldehyde-containing 1.5% agarose gel and blotted to Hybond-N+ 51. The blots were hybridized using 32P-labeled oligonucleotides as probes (Supplementary Table BHK-21J cells were transfected with 5 μg of in vitro transcribed Sinrep5 recombinant RNAs as describedSequences were aligned using CLUSTALW though the online server. Accession numbers of sequences are contained in the appropriate figure legends.All data are available from the authors upon request.Supplementary InformationPeer Review File


The Rac1 GTPase plays key roles in cytoskeletal organization, cell motility and a variety of physiological and disease-linked responses. Wild type Rac1 signaling entails dissociation of the GTPase from cytosolic Rac1-Rho GDP dissociation inhibitor (GDI) complexes, translocation to membranes, activation by exchange factors, effector binding, and activation of downstream signaling cascades. Out of those steps, membrane translocation is the less understood. Using transfections of a expression cDNA library in cells expressing a Rac1 bioreporter, we previously identified a cytoskeletal feedback loop nucleated by the F-actin binding protein coronin 1A (Coro1A) that promotes Rac1 translocation to the plasma membrane by facilitating the Pak-dependent dissociation of Rac1-Rho GDI complexes. This screening identified other potential regulators of this process, including WDR26, basigin, and TMEM8A. Here, we show that WDR26 promotes Rac1 translocation following a Coro1A-like and Coro1A-dependent mechanism. By contrast, basigin and TMEM8A stabilize Rac1 at the plasma membrane by inhibiting the internalization of caveolin-rich membrane subdomains. This latter pathway is F-actin-dependent but Coro1A-, Pak- and Rho GDI-independent. Lysates were then kept on ice for 10 min, centrifuged at 14,000 rpm for 10 min at 4°C to remove unbroken cells and nuclei. The supernatants obtained in that step were transferred into polycarbonate tubes (Beckman) and subjected to centrifugation at 100,000 xg for 1 hour in a refrigerated ultracentrifuge (Beckman). After the centrifugation, the cytosol-containing supernatant was removed and the crude membrane pellet gently washed with hypotonic lysis buffer and recollected by ultracentrifugation. Membrane fractions were then assayed for total protein content using the Bradford method (Bio-Rad) and subjected to immunoblotting. As control, we analyzed by aliquots of cell lysates taken before the ultracentrifugation step as above.Exponentially growing COS1 cells cultured in 100 mm dishes were transfected using Lipofectamine 2000 according to the manufacturer’s recommendations (Thermo Fisher Scientific). To this end, 2 μg of the appropriate plasmid DNA and 6 μl of Lipofectamine 2000 were diluted separately into 250 μl serum-free DMEM, the two solutions were then mixed, incubated for 30 min at room temperature, and added onto cells. After culturing for 24–36 hours, cells were washed with chilled phosphate buffered saline solution, re-suspended in 1 ml of ice-cold hypotonic solution buffer . Membranes were blocked in either 5% non-fat dry milk (when immunoblotted with standard antibodies) or 5% bovine serum albumin  in 25 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.1% Tween-20 (TBS-T) for 1 hour and then incubated overnight at 4°C with the appropriate primary antibody diluted in blocking buffer. After three washes with TBS-T, membranes were incubated with horseradish peroxidase-conjugated to either protein A or the appropriate secondary antibody  for 1 hour at room temperature. Immunoreacting bands were developed using a standard chemoluminescent method .WDR26   of Rac1) of Pak1 eliminates the WDR26-mediated translocation of Rac1 in COS1 cells . As expected [Fig 5A and 5B). These effects are specific, because the expression of the two Pak1 mutant proteins has no effect on the translocation of Rac1 mediated by both Bsg and TMEM8A . Despite their effect on Rac1 translocation, the Pak1 mutant proteins do not affect the subcellular localization of Coro1A and WDR26 . Further underscoring the concurrent action of WDR26 and Coro1A in the same pathway, we found that Myc-Pak1  and endogenous Rho GDI  can co-immunoprecipitate with EGFP-WDR26 in COS1 cells. Conversely, we could find EGFP-WDR26  and Myc-Pak  in the immunoprecipitates obtained with antibodies to endogenous Rho GDI. Confirming the specificity of these associations, we could not detect them when EGFP-WDR26 was replaced by the non-chimeric EGFP in these co-immunoprecipitation experiments . They are also Coro1A-dependent, because we could not observe them when performing co-immunoprecipitation experiments in Coro1A-depleted (ACC2-2) COS1 cells . Further confirming the Coro1A-WDR26 interconnection, we found that these two proteins can co-immunoprecipitate when co-expressed in COS1 cells . As control, such interaction does not occur when performing co-immunoprecipitations with a control protein . This association is independent on downstream elements of the Coro1A translocation pathway [Fig 5D, compare lanes 3 and 4). All together, these results indicate that: (i) WDR26 and Coro1A work in the same translocation pathway in cells. (ii) They can form heteromolecular complexes prior to the subsequent binding to the Pak-ArhGEF7 complexes that are involved in the Coro1A-mediated translocation of Rac1 [The foregoing results suggest that WDR26 mediates Rac1 translocation in a Coro1A- , we next analyzed the potential implication of Rho GDI in this pathway. Since Pak1 mediates Rac1-Rho GDI disassembly through Rho GDI phosphorylation on two serine residues (Ser101 and Ser174) present in the vicinity of the hydrophobic pocket where the GTPase prenyl group binds to [Fig 6A and 6B). This is due to Rac1 sequestration, because such an inhibition does not occur when expressing an inactive Rho GDI protein (Δ1–60 mutant)  [S101A+S174A cannot inhibit the WDR26- and Coro1A-mediated translocation of a Rac1 mutant version (R66E) that cannot form complexes with Rho GDIs [Fig 6C and 6D). The overexpression of Rho GDIs only induces a marginal effect on the Bsg-EGFP-elicited translocation of Rac1  . LikewisRho GDIs   but not in Coro1A-depleted  cells. The ectopic expression of EGFP-WDR26 does not rescue normal dissociation rates in the latter cells . However, as expected [Fig 6C, top panel, compare lanes 7 and 8). We confirmed the similar immunoprecipitation of endogenous Rho GDI in these experiments by incubating the same blots with antibodies to this protein . The appropriate expression of the proteins used in these experiments was confirmed by Western blot analysis using aliquots of the total cellular lysates utilized in the immunoprecipitation step .Given the implication of WDR26 and Coro1A in the same regulatory pathway, we next investigated whether the overexpression of WDR26 could rescue the defective release of Rac1 from Rac1-Rho GDI complexes previously described in a stable clone of Coro1A-deficient COS1 cells . To thisexpected , this reFig 6D) leads to lower rates of translocation of EGFP-Rac1 to the plasma membrane upon the EGF stimulation of serum-starved COS1 cells . These data indicate that WDR26 and Coro1A play coordinated rather than redundant functions in this regulatory step, a result that is in agreement with the lack of WDR26-mediated Rac1 translocation previously observed in Coro1A-deficient cells . These observations also indicate that this pathway entails the Pak-mediated disassembly of Rac1-Rho GDI complexes in cells.Since the depletion of Coro1A abrogates the translocation of Rac1 induced upon the EGF stimulation of COS1 cells, we finally investigated whether endogenous WDR26 was also involved in this response. Consistent with this idea, we found that the effective siRNA-mediated depletion of this protein  and the level of dependency on both Rac1  and Pak  signaling was a reflection of two non-overlapping mechanisms of translocation of Rho GTPases. Given the implication of Bsg [Fig 3A) and F-actin  in COS1 cells . In agreement with this, we observed that the expression of a HA-tagged version of Bsg can promote the localization at the plasma membrane of an EGFP version containing at this C-terminus the polybasic region of Rac1 (referred to hereafter as EGFP-PBRΔCAAX) . Since this sequence lacks the CAAX box, the translocation of this fusion protein has to be directly mediated by the Rac1 polybasic tail using a prenylation- and Rho GDI-independent mechanism. The same effect is observed when EGFP-PBRΔCAAX is coexpressed with TMEM8A-FLAG . These effects are specific, because the ectopic expression of either Bsg or TMEM8A cannot change the subcellular localization typically displayed by the non-chimeric EGFP  and EGFP-RhoA . Based on the foregoing observations, we decided to investigate whether Bsg and TMEM8A could play an integrin-like role in the regulation of the dynamics of cholesterol-enriched plasma membrane subdomains. To this end, we evaluated the effect of the ectopic expression of these two proteins on the internalization of these structures in cells that had been maintained in suspension. It is known that these experimental conditions promote the internalization of these membrane subdomains in a Cav1- and dynamin 2 (Dnm2)-dependent mechanism due to loss of integrin signaling [K44A-HA-expressing cells . By contrast, Coro1A-mRFP does not have any influence in the internalization of those membrane subdomains under the same experimental conditions . Collectively, these results indicate that β-propeller domain proteins promote increased amounts of Rac1 at the plasma membrane by regulating in a Pak-dependent manner the release of Rac1 from Rac1-Rho GDI complexes. By contrast, Bsg and TMEM8A do so by maintaining the steady state amount of Cav1-enriched microdomains at the plasma membrane . These two pathways share similarities  but differ in other key biological properties .We surmised that the different behavior of the β-propeller and transmembrane proteins in terms of spectrum of GTPases they could translocate . Consistent with this, these two proteins promote the translocation of a similar spectrum of GTPases (Rac1 and the highly related RhoG) in a cholesterol-enriched membrane subdomain-, F-actin-, Pak-, and Rho GDI-dependent manner . Our data also indicate that these two proteins work in a concerted rather than redundant manner in this process. We have postulated in a previous study that the Coro1A pathway is involved in the generation of secondary waves of Rac1 translocation and activation during cell signaling that are triggered upon the reorganization of the F-actin cytoskeleton by an initial pool of stimulated Rac1 proteins  [Fig 7E). Interestingly, we have recently described that Coro1A is involved in a myosin II-dependent step downstream of Rac1. This role is signaling branch-specific, since it only affects the responses associated with the engagement of the Rac1-Pak-ArhGEF7 pathway [We have described here the characterization of three proteins  that, together with Coro1A , were is . This is . It is l pathway . Due to  pathway . In this pathway –69. It wFig 7E). A direct translocation effect of these proteins cannot be formally ruled out, as inferred by the observation that Bsg and TMEM8A can “attract” a prenylation-defective Rac1 mutant to the plasma membrane. It is likely that this effect is mediated by a clustering effect of these proteins on Rac1-docking-competent subdomains at the plasma membrane, as evidenced by the ability shown by these transmembrane proteins to translocate an EGFP fused to the C-terminal polybasic region of Rac1.By contrast, we have observed that the role of Bsg and TMEM8A in this GTPase translocation step is probably an indirect effect derived from their implication in the stabilization of Cav1-enriched plasma membrane subdomains. Consistent with this mode of action, we have seen that their effect on Rac1 localization is highly dependent on the presence of cholesterol in the plasma membrane and the F-actin cytoskeleton, two elements that contribute to the formation and stability of Cav1-enriched subdomains at the plasma membrane, respectively. Moreover, unlike the case of Coro1A and WDR26, their activities are independent of both Pak1 signaling and the dissociation of the Rac1-Rho GDI complexes. These results indicate that these two transmembrane proteins contribute to the stabilization of the steady state pool of Rac1 molecules already present at the membrane rather than being involved in the Coro1A-dependent amplification phase of the Rac1 stimulation cycle (Further work will be required to fully assess the biological relevance and cell context in which these regulatory mechanisms operate. We have seen that the depletion of the endogenous Coro1A  and WDR2


Here, we examine the potential of using mass spectrometry-based proteomics data to further annotate the function of SNVs in cancer samples. RNA-seq and whole genome sequencing (WGS) data from Jurkat cells were used to construct a custom database of single amino acid variant (SAAV) containing peptides and identified over 1,000 such peptides in two Jurkat proteomics datasets. The analysis enabled the detection of a truncated form of splicing regulator YTHDC1 at the protein level. To extend the functional annotation further, a Jurkat phosphoproteomics dataset was analysed, identifying 463 SAAV containing phosphopeptides. Of these phosphopeptides, 24 SAAVs were found to directly impact the phosphorylation event through the creation of either a phosphorylation site or a kinase recognition motif. We identified a novel phosphorylation site created by a SAAV in splicing factor SF3B1, a protein that is frequently mutated in leukaemia. To our knowledge, this is the first study to use phosphoproteomics data to directly identify novel phosphorylation events arising from the creation of phosphorylation sites by SAAVs. Our study reveals multiple functional mutations impacting the splicing pathway in Jurkat cells and demonstrates potential benefits of an integrative proteogenomics analysis for high-throughput functional annotation of SNVs in cancer.Massively parallel DNA sequencing enables the detection of thousands of germline and somatic single nucleotide variants (SNVs) in cancer samples. The functional analysis of these mutations is often carried out through  This serves to reduce the total numbers of SNVs that are considered in downstream data analysis. However, experimental validation of the role that these SNVs have on protein structure and function remains a major challenge and therefore is not typically performed in routine analyses.DNA sequencing technologies have enabled the rapid identification of single nucleotide variants (SNVs) within cancer genomes. Hundreds to thousands of non-synonymous SNVs can now be routinely identified within gene coding regions of individual cancers through whole genome or exome sequencing (WGS/WXS) , and expThe detection of a SNV translated at the protein level is often the first step taken to infer whether the SNV is functional. Mass spectrometry-based proteomics experiments are now capable of detecting thousands of proteins in a single experiment. Recent studies have shown that by integrating SNV calls from WXS or RNA-seq data, it is possible to generate a sample-specific reference protein database that can be used to query a sample-matched proteomics dataset. For example, RNA-seq data from two colon cancer cell lines have been used to generate customised databases containing single amino acid variants (SAAVs) which can be applied to search the respective proteomics data . This stThese studies have clearly demonstrated the ability to detect and quantify SAAV containing peptides. Nevertheless, how the detection of SAAVs at the protein level can benefit downstream analyses of protein function remains to be fully demonstrated. In this study, we first assess the potential of using an ‘ultra-deep’ Jurkat proteomics dataset to increase the number of SAAV containing peptides identified. We use several strategies to evaluate the accuracy of these SAAV containing peptides. Finally, to further demonstrate the potential of proteogenomics, we use a Jurkat phosphoproteomics dataset to show that this data can be used to directly identify SNVs that create novel protein phosphorylation events. Through this analysis, we highlight and discuss candidate SNVs that may play a role in the leukaemogensis of Jurkat cells.A flowchart of the data analysis pipeline used is shown in Figure As the aim of this study was to identify functional SNVs, we examined how the use of higher depth proteomics data can increase the number of candidates. To achieve this, the Mertins dataset, which has over 2 million (i.e. 500%) more tandem mass spectra than the Sheynkman dataset, was analysed similarly. In total, 1,048 SAAV containing peptides, consisting of 929 unique SAAVs, were identified . The majVariant calling using RNA-seq data can result in not detecting all variants in exonic regions since it is only able to detect SNVs in expressed genomic regions. Furthermore, studies have shown that RNA-seq data can also have higher false positive detection rates when compared with DNA sequencing due to mismatches arising from RNA to cDNA conversion during RNA sequencing library preparation as well as the complexities associated with precise alignment of reads spanning spliced junctions. To determine whether RNA-seq SNV detection is adequate, with regard to the goal of identifying SAAV containing peptides, we sought to compare the use of SNVs from RNA-seq and WGS.The genome of the Jurkat dataset was recently whole genome-sequenced at 60X coverage [To determine whether the higher number of variants from the WGS data would result in a greater number of SAAV peptides being discovered, we searched the Mertins proteomics dataset using a custom database generated using the WGS SNVs. Despite the larger search database, only 686 SAAVs could be identified using the WGS custom SAAV peptide database compared with 930 identified from the corresponding RNA-seq custom SAAV peptide database Figure . The oveTo demonstrate that the SAAV containing peptides that we identified are true positives, we further performed a series of control analyses. Firstly, the SAAV containing peptides common to both RNA-seq and WGS SNV databases were classified as “heterozygous” or “homozygous” based on the variant allele frequency of the corresponding SNV derived from the WGS data. In total, we detected 274 “homozygous”  = 1.0) and 516 “heterozygous” SAAV containing peptides. Of the 274 “homozygous” SAAV containing peptides, we found 7 peptides with a corresponding reference peptide detected. This is unexpected as only a single sequence should be present at homozygous alleles. Further examination of the WGS sequence coverage showed that these SNVs had low coverage  had the corresponding reference sequence detectable . The disThe detection of the 355 reference peptides with a corresponding SAAV containing peptide allows the abundance of the corresponding peptides to be directly compared. Given that the corresponding SNVs are heterozygous, it would be expected that the abundance of the two alleles would be similar. Indeed, a clear correlation between the abundance of the reference and the corresponding SAAV containing peptide can be seen  of all non-synonymous and nonsense SNVs detected by RNA-seq, we compared the read depth of SNVs corresponding to SAAV containing peptides with all remaining SNVs. The read depth in RNA-seq of identified SNVs was significantly higher than unidentified SNVs  Figure . This suAccurate protein quantification relies on the assumption that all proteolytic peptides are being detected and quantified. Standard database searches do not detect and include SAAV containing peptides in protein quantification. Thus, we examined whether the inclusion of SAAV containing peptides in the calculation of protein abundance may impact upon protein quantification accuracy. We used the Sheynkman dataset for this analysis, as the RNA-seq and proteomics data were generated from the same cells . Using tt-test) increase in correlation coefficient  increase in correlation coefficient, from R = 0.6978 to R = 0.7154  across all proteins  with the corresponding mRNA expression value showed a modest but significant  4 Figure . Althoug59 4 FiguIn order to determine whether the use of proteomics data can help the identification of candidate cancer driver mutations, we sought to prioritise SNVs that are most likely to alter protein function. Examination of the list of SAAVs  reveals YTHDC1 from the Catalog of Somatic Mutations in Cancer (COSMIC) database shows a relatively large number of stop gain mutations over the protein  of mutations are stop-gain mutations which are significantly more than in all genes in general , 29.3%  and 20.3% , respectively. While, to our knowledge, YTHDC1 mutations have not previously been implicated in leukaemia, mutations affecting splice factors are common in leukaemia [* stop gain mutation in YTHDC1 may contribute to leukaemogenesis in Jurkat cells by impacting the RNA splicing pathway.Based on the RNA-seq data, the variant is heterozygous  but is c protein . Of all n Figure . Tumour eukaemia , 17 and eukaemia . Thus, tHaving established that potential cancer-associated SNVs can be inferred from proteomics data, we further investigated the use of phosphoproteomics data in more directly identifying SNVs that can alter the phosphorylation state of a protein. Using the SAAV peptide database, we applied MaxQuant  to analyThe inclusion of SAAVs within the search space can influence the detection of different phosphorylation events. For example, (1) the SAAV may create an amino acid that allows a novel phosphorylation site to form, (2) the SAAV may create a motif that allows a novel phosphorylation event to take place, and (3) the SAAV may not impact the formation of the phosphorylation event, but instead may simply add the SAAV into the protein database, thus allowing the SAAV-containing phosphopeptide to be detected. To this end, the phosphopeptides were examined further to determine to which of the three categories described above, each one belonged. In this case, to determine motif creation, we only considered proline-directed kinases such as mitogen-activated protein kinases (MAPK) and cyclin-dependent protein kinases (CDK)  as theseBased on the 3 categories outlined above, each of the 462 SAAV phosphopeptides was categorised. We found that only a minority  of SAAVs created a phosphorylation event, whereas the majority simply enabled the detection of an existing phosphorylation event Figure . A summaYTHDC1, analysis of the allele frequency of this SF3B1 SNV in the ExAc database [SF3B1 is involved in the regulation of splicing as part of the splice factor complex. SF3B1 is a known phosphoprotein  and the database  shows thin silico methods have been applied to predict the gain of phosphorylation from somatic mutations [SNVs that lead to the creation of novel phosphorylation sites have previously been described in several studies. For example, the germline R405S variant in androgen receptor (AR) has been shown to result in the phosphorylation of S405 and this post-translational modification results in the disruption of protein-protein interactions between AR and EP300 . In the utations . HoweverIn summary, by integrating RNA-seq and proteomics data, we have shown that the translation of some SNVs can be detected at the protein level, and this can allow more accurate inference to the functional impact of mutations. Although our analysis does not enable direct inference of whether particular variants play a direct role in causing cancer, we have demonstrated that it can be used to further prioritise candidate SNVs. In this case, we found two SNVs which both affect the RNA splicing pathway in Jurkat cells. As splicing factor mutations are common in blood disorders –18, thesThe Jurkat (T-cell acute lymphocytic leukaemia) cell line RNA-seq data  were obtThe Jurkat RNA-seq data in SRA format were converted to fastq, trimmed using sickle (version 1.33) and aligned using tophat (version 1.4.0) . Samtoolhttps://github.com/gpertea/gffread) was used to translate the transcripts defined in the GTF file into protein sequences.To facilitate simple and accurate mapping of SNPs to SAAVs in proteins, the human Refseq protein annotations were used, as both the human proteome database, and for variant annotation. Annotation of SNPs against Refseq was performed using ANNOVAR . To creaIn order to generate the custom SAAV database, a Python script was written to directly map SAAVs from the ANNOVAR annotated SNPs and to output theoretical tryptic peptides up to 1 missed cleavage either side of the SAAV.LC-MS/MS data in RAW format were analysed using MaxQuant (version 1.3.5.8) . The datFor protein quantitative analysis, the peptide ion intensity and the iBAQ quantitative value were obtained from MaxQuant. To calculate new updated protein iBAQ values incorporating SAAV containing peptides, the ion intensity of SAAV containing peptides was added to the respective protein's total ion intensity. The updated iBAQ value was calculated by dividing the updated total intensity value by the number of theoretically detectable peptides.t-test, calculated with the R package cocor [For the comparison of RNA expression to protein abundance, RNA expression was measured as Reads Per Kilobase of transcript per Million mapped reads (RPKM). To compare the significance of the change in the correlation of RNA and protein abundance before and after inclusion of SAAV containing peptides, significance was determined using the Williams’ (1959) ge cocor .


Gag oligomerization and genomic RNA packaging are essential elements that facilitate HIV assembly. However, mechanistic details of these interactions are not clearly defined. Here, we overcome previous limitations in producing large quantities of full-length recombinant Pr55Gag that is required for isothermal titration calorimetry (ITC) studies, and we have revealed the thermodynamic properties of HIV assembly for the first time. Thermodynamic analysis showed that the binding between RNA and HIV Pr55Gag is an energetically favourable reaction (ΔG<0) that is further enhanced by the oligomerization of Pr55Gag. The change in enthalpy (ΔH) widens sequentially from: (1) Pr55Gag-Psi RNA binding during HIV genome selection; to (2) Pr55Gag-Guanosine Uridine (GU)-containing RNA binding in cytoplasm/plasma membrane; and then to (3) Pr55Gag-Adenosine(A)-containing RNA binding in immature HIV. These data imply the stepwise increments of heat being released during HIV biogenesis may help to facilitate the process of viral assembly. By mimicking the interactions between A-containing RNA and oligomeric Pr55Gag in immature HIV, it was noted that a p6 domain truncated Pr50Gag Δp6 is less efficient than full-length Pr55Gag in this thermodynamic process. These data suggest a potential unknown role of p6 in Pr55Gag-Pr55Gag oligomerization and/or Pr55Gag-RNA interaction during HIV assembly. Our data provide direct evidence on how nucleic acid sequences and the oligomeric state of Pr55Gag regulate HIV assembly.The interactions that occur during HIV Pr55 Gag proteins have not been available in the past 30 years due to technical limitation, a comprehensive thermodynamic analysis of full-length HIV Pr55Gag has not been possible. Here, we have generated sufficient amount of full-length recombinant HIV Pr55Gag protein for isothermal titration calorimetry analysis. Our analyses have shown that the major interactions amongst HIV proteins and RNA sequences during viral assembly are energetically favourable reactions. In other words, HIV Pr55Gag proteins and viral RNA have evolved to overcome the energy barrier for virus formation by utilising energy obtained from protein-RNA interactions in order to facilitate the viral assembly process. Furthermore, HIV also use the oligomeric states of HIV Pr55Gag proteins and the RNA sequences as means to regulate the viral assembly process.Formation of any virus particle will require energy, yet the precise biophysical properties that drive the formation of HIV particles remain undefined. Isothermal titration calorimetry (ITC) is a biophysical technique that is the gold standard to reveal parameters governing biochemical and biophysical reaction. However, ITC requires large amount of proteins for analysis. As large quantities of full-length recombinant HIV Pr55 Gag). As the major structural protein forming the virus, it is essential that Pr55Gag packs tightly with other Pr55Gag molecules to shape the virus shell while also encapsulating the genomic RNA required for its replication. Fittingly, Pr55Gag is comprised of multi-functional domains that facilitate key interactions involved in the early stages of the assembly process. The nucleocapsid (NC) domain is responsible for genomic RNA packaging and the capsid (CA) domain mediates the key interactions to promote Pr55Gag-Pr55Gag oligomerization (for review see  as measured by the maximum heat energy release during ITC titrations of Psi SL3 RNA into the Pr55Gag protein . The reaction is also thermodynamically favourable with ΔG<0. The detected energy release in these reactions is likely to be the sum of both Pr55Gag-RNA interaction and Pr55Gag-Pr55Gag oligomerization. To estimate the fraction of energy released that is contributed by Pr55Gag-RNA vs Pr55Gag-Pr55Gag oligomerization, we have used a mutant, Pr55Gag WM316-7AA (or Pr55Gag WM)  , but it was only 60% of the energy release of the wild type Pr55Gag-RNA interaction . These data implied that the oligomerization of Pr55Gag during Pr55Gag-RNA interaction contributed to at least 40% of energy detected in the ITC experiment, and the Pr55Gag- Pr55Gag interactions increased its favourable binding enthalpy with Psi SL3 RNA. In support of this, interaction of Psi SL3 RNA with both processed NC proteins (p15NC-SP2-p6 and p7NC that lack the CA-CA interaction domains for oligomerization) also displayed less favourable binding enthalpies [ΔH ~ -5-6 kcal mol-1] compared to the full-length Gag proteins Pr55Gag.Using ITC, we first characterized the binding between Pr55affinity , 27. Rep5Gag WM)  that is hexamers . As measGag and Pr50Δp6Gag with Psi SL3 RNA however had a more unfavourable entropic contribution compared to that of Pr55Gag WM . As entropy is a measure of the disorder of the reaction , Pr50Δp6Gag [Kd = 0.098 ± 0.003 μM], Pr55Gag WM316-7AA [Kd = 0.088 ± 0.035 μM], p15NC-SP2-p6 [Kd = 0.057 ± 0.011 μM] and p7NC [Kd = 0.048 ± 0.023μM] did not differ significantly . These data imply that the binding between Pr55Gag and SL3 RNA for genomic RNA selection during viral assembly is independent of the oligomerization state of Pr55Gag. Our ITC results demonstrate that the binding of Psi SL3 RNA to Pr55Gag is energetically favourable (ΔG<0). Bindings of Psi SL3 to p15NC-SP2—p6 and p7NC are results of both favourable entropic and enthalpic contributions, suggesting the involvement of both polar and hydrophobic interactions  , which is comparable to that released in the binding to processed NC proteins (p15NC-SP2-p6 and p7NC) . In contrast, higher enthalpy was released in the binding of the same 20mer GU-containing RNA motif 4x 5’-GAUGG-3’ RNA with the oligomeric forms of Gag (Pr55Gag and Pr50GagΔp6) with means of ΔH ~-45 kcal mol-1 and -35 kcal mol-1, respectively  , suggesting that oligomerization contributes at least in part to the favourable enthalpy release of Pr55Gag binding with GU-containing RNA. These data are consistent with those observed with Psi SL3 RNA based Pr55Gag-RNA ITC studies . In support of this, more enthlapy release was also observed in the interaction between the 20 mer of A-containing RNA motif 4x 5’-GAGAA-3’ RNA and the oligomeric forms of Gag (Pr55Gag and Pr50GagΔp6), with means of ΔH ~-80 kcal mol-1  , over the interaction between the 20 mer of A-containing RNA motif 4x 5’-GAGAA-3’ RNA and the oligomeric-deficient forms of Gag (Pr55Gag WM)[ΔH ~ -60 kcal mol-1]  . Agreeably, ITC analyses repeated with the second set of GU-containing and A-containing RNA sequence motifs  produced a similar trend in binding enthalpies measured across the Gag constructs . Our results showed that the favourable enthalpy and Gibbs Free energy release from oligomerization can potentially be a general feature of RNA-Pr55Gag protein interaction during viral assembly.To investigate whether the thermodynamic relationship with Pr55AAGGA3’) . BindingFig 3A and 3C). This result suggests that the type of RNA sequence interacting with the Gag protein also contributes to the favourable release of enthalpy. Additionally, the type of RNA sequence may also be a determinant of how tightly packed Gag can bind. An analysis of the stoichiometry of binding showed a trend that 1 A-containing RNA molecule would bind to 5 Gag molecules (RNA:Gag N~0.2) in comparison to 1 GU-containing RNA molecule would bind to 3 Gag molecules (RNA:Gag N~0.33) . However, it is important to acknowledge that the binding stoichiometry data are related to a single type of GU-containing RNA motif and a single type of A-containing RNA motif, and these motifs are artificially presented in 4 consecutive repeats within a synthetic RNA. The potential importance of different RNA-Gag binding stoichiometry must be validated using multiple different natural A-containing RNA motifs and natural GU-containing RNA motifs within the context of HIV RNA genome.Conversely, across the Gag constructs tested independent of their size and oligomeric capacity, thermodynamic analysis also indicated that the interaction of the A-containing RNA motifs with Gag were characterized by about 2-times more favourable enthalpy compared to that of the GU-containing RNA motifs , suggesting that these designated A-containing RNA motifs might in part better support tight packing of Pr55Gag-RNA and/or Pr55Gag-Pr55Gag interaction during the biological process, leading to greater loss of conformational freedom. Nevertheless, the larger favourable binding enthalpy associated with binding of A-containing RNA motifs to Pr55Gag overcomes the unfavourable entropy to drive the reaction forward. These differential thermodynamic relationships between Pr55Gag and A-containing RNA motifs in immature virus vs Pr55Gag and GU-containing RNA motifs in cytoplasm have provided insight on how viral particle formations are regulated. The Gibb’s free energy (ΔG) consistently remained at ~-10 kcal mol-1 , indicating the process is an energetically favourable spontaneous event. We have also performed additional control experiments showing that less than 5% of materials can be pelleted from solution after our ITC experiments using Gag and RNA . Furthermore, similar amount of pelletable materials (<5%) were collected in parallel ITC experiments when nucleic acid free buffer was injected into the ITC chamber containing recombinant Gag . These data implied that no virus-like-particles were generated in these ITC reactions under our experimental conditions when ‘low’ concentrations of Pr55Gag and RNA were used .Furthermore, a larger unfavourable entropy was detected when A-containing RNA motifs were used in ITC experiments compared to GU-containing RNA motifs (kd) from ITC A-containing and GU-containing RNA binding curves revealed that oligomeric capable Pr55Gag displayed ~3-times stronger binding affinity for the immature virus A-containing RNA motif over the cytosolic GU-containing RNA motif . This occurrence was not observed when oligomerization-impaired forms of Gag (Pr55Gag WM) were used in parallel experiments . Unexpectedly, Pr50Δp6Gag, which is capable of high-order Gag oligomerization, also did not display binding preference toward A-containing RNA motif . It is possible that the p6 domain in Pr55Gag plays a part in facilitating the binding of A-containing RNA to oligomeric forms of Pr55Gag, implying a previously unknown role of p6 late domain in the packing of Pr55Gag-Pr55Gag and/or Pr55Gag-RNA interaction at the late stage of HIV assembly.In addition to the more favourable binding enthalpy of A-containing RNA to various Gag constructs, calculated binding affinities , Pr55Gag [CA Helix 6] , Pr55Gag [CA Helix 10] (Gag [CA All 4]) (Gag (CA dimerization interface WM 316–7 AA), Pr55Gag (CA helix 6 TTSTLQ 239–44 AASALA), Pr55Gag (CA helix 10 D329A), and Pr55Gag CA major homology region (MHR K290A)  . These data are consistent with that of the previously described oligomerization impaired mutant, Pr55Gag WM , showing that oligomerization of Pr55Gag can help to increase the level of energy being released during Pr55Gag-RNA interaction in HIV assembly.To independently assess whether cytosolic low-order Gag oligomer binding with A-containing RNA is a less energetically favourable process than the interaction between high-order Gag oligomer and A-containing RNA, three additional Gag oligomerization defective mutants were engineered for analysis. These Gag oligomierization-impaired mutants are: (1) Pr55R K290A) . These rR K290A) , 31, likGag, Pr50GagΔp6, and Pr55Gag WM. The His-Tag was removed via TEV protease digestion, and the His-Tag free recombinant Gag proteins  were subsequently column purified for analysis. ITC analyses of Pr55Gag-TEV [Fig 6B left panel], Pr50GagΔp6-TEV [Fig 6B middle panel], and Pr55Gag WM-TEV [Fig 6B right panel] with either GU-containing RNA (4x 5’-GAUGG-3’) or A-containing RNA (4x 5’-GAGAA-3’) resulted in energy release ranging from ΔH ~ -8 kcal mol-1 to ~ -50kcal mol-1  ~-10 kcal mol-1 for Pr50Gag Δp6-TEV-GU-containing RNA binding ; (3) ~-10 kcal mol-1 for Pr55Gag WM-TEV-GU-containing RNA binding ; (4) ~-45 kcal mol-1 for Pr55Gag-TEV-A-containing RNA binding ; (5) ~-25 kcal mol-1 for Pr50Gag Δp6-TEV-A-containing RNA binding ; (3) ~-25 kcal mol-1 for Pr55Gag WM-TEV-A-containing RNA binding . Analogous to the ITC results using the His-Tag containing Gag  , Gag binding with A-containing RNA (4x 5’-GAGAA-3’) were consistently shown to be a more energetically favourable reaction than parallel analyses that used GU-containing RNA (4x 5’-GAUGG-3’) as substrates . Furthermore, the binding between Pr55Gag-TEV and A-containing RNA was a more thermodynamically favourable reaction than that between Pr50GagΔp6-TEV and A-containing RNA, highlighting the potential role of p6 in this process. Although the C-terminus His-Tag has no selective bias on the overall data interpretation, it is important to acknowledge that reduced levels of enthalpy (ΔH) were also detected with the Gag-RNA binding when the C-terminus His-Tag was removed from the recombinant Gag , implying that the C-terminus His-Tag has consistently increased the amounts of enthalpy release during Gag-RNA interaction.Given that the C-terminus His-Tag is present in all of the recombinant Gag used in this study thus far, it is unlikely that the His-Tag would selectively interfere with the binding for one of these three groups of RNA  with Gag. However, to directly rule out any potential selective bias that might be introduced by the C-terminus His-Tag, a Tobacco Etch Virus (TEV) protease cleavage site was engineered in between the C-terminus end of Gag sequences and the His Tag for Pr55Gag protein during viral assembly, a 37mer RNA fragment representing the coding region of HIV reverse transcriptase (RNA positions 2671–2707) was used for ITC analyses. Using CLIP-sequencing, this RNA sequence (HIVNL4.3RNA2671-2707), consisting 3 independent A-containing RNA motifs, has been previously identified to be important for Pr55Gag binding in immature HIV  in comparison to the wild type Pr55Gag and A-containing RNA ITC analyses. The nucleic acid sequence dependence of the binding enthalpy is likely related to the process of HIV assembly and virion genesis. Early work by Campbell and Rein  when it binds to A-containing RNA motifs over GU-containing RNA motifs, and it is remains to be determined why HIV Pr55Gag has a binding preference with GU-containing RNA motifs in the cytoplasm and the plasma membrane , Pr55Gag [CA Helix 10 D329A], Pr55Gag [CA All 4]) and processed NC proteins  were expressed with C-term His-tag and purified as previously described /[Gag] molar ratio. The corrected data were analyzed to determine number of binding sites (Ka):The quantity r is defined as the moles of nucleic acid [D] bound per mole of protein [P] with an association constant  gives:Solving the quadratic for the fractional occupancy  at fractional saturation r is given by The total heat content (Q) of the solution in the volume of the sample cell . In order to correct for the displaced volume (Vi), the change in heat content Q(i) at the end of the ith injection is defined by n, Ka, and ΔH by standard Marquardt methods until no further significant improvement in fit occurs with continued iteration.The total heat content, Q can be calculated as function of 0) was calculated from the fundamental equation of thermodynamics The Gibbs free energy .100,000 g for 1 hr . The supernatant was removed and the pellet was resuspended in 50μl of TBS. Protein estimation was done using UV-Vis (A280) on both the supernatant and the pelletable materials  via Bradford Protein Assay  and (B) approximate sequence positions A-containing motifs [4x 5’-GAGAA-3’(blue) and 4x 5’-AAGGA-3’(dark blue)] within the complete HIV genome (NCBI Reference Sequence: NC_001802.1).Schematic of HIV-1 genome with (TIF)Click here for additional data file.S4 Fig(A) A-containing (4x 5’-GAGAA-3’) RNA showing that >90% of the initial input protein still remained soluble. Control ITC experiment where RNA free buffer was injected into ITC chambers was also carried out simultaneously (B) as a parallel comparison. n = 3Using spectroscopic analytical procedure as well as UV-Vis Spectrophotometry, the pelletable materials is expressed as percentage of the total initial input protein were estimated post-ITC reactions. Following ITC analyses using (TIF)Click here for additional data file.


Mussismilia hispida and the surrounding water. This survey was performed on five reefs along a transect from the coast (Reef 1) to offshore (Reef 5), representing a gradient of influence of the river mouth, for almost 12 months (4 sampling times), in the dry and rainy seasons. A clear increasing gradient of organic-pollution proxies  was observed from Reef 1 to Reef 5, during both seasons, and was highest at the Buranhém River mouth (Reef 1). Conversely, a clear inverse gradient of the network analysis of the whole bacterial communities also revealed more-complex network relationships at Reef 5. Our data also indicated a higher relative abundance of members of the bacterial core, dominated by Acinetobacter sp., at Reef 5, and higher diversity of site-stable bacterial populations, likely related to the higher abundance of total coliforms and N content (proxies of sewage or organic pollution) at Reef 1, during the rainy season. Thus, the less “polluted” areas may show a more-complex network and a high relative abundance of members of the bacterial core , resulting in a more-homogeneous and well-established bacteriome among sites/samples, when the influence of the river is stronger (rainy seasons).Acclimatization via changes in the stable (core) or the variable microbial diversity and/or abundance is an important element in the adaptation of coral species to environmental changes. Here, we explored the spatial-temporal dynamics, diversity and interactions of variable and core bacterial populations associated with the coral  Host-bacteriome relationships and the effects of these interactions on the metaorganism development, ecology and evolution in coral reefs are currently under active study . HoweverThe coral microbiome can be modulated by the host , and is Considering the complex nature of the coral and its associated bacterial-microbiome relationships, the core bacteriome, i.e., the set of bacterial species shared among multiple microbial assemblages , as wellMussismilia hispida  to explore possible local adaptations of the bacterial core.Our group has previously described the bacterial core associated with mucus of the coral  hispida , showing hispida . Here, wPermission for sampling was obtained from the Brazilian Institute of Environment and Renewable Natural Resources (IBAMA)/Chico Mendes Institute for Biodiversity Conservation (ICMBio), permanent license number 16942-1, in accordance with Normative Instruction No. 03/2014 of the System of Authorization and Information on Biodiversity (SISBIO), and from local authorities of the Municipality Environmental Agency (SMMA), Porto Seguro, Bahia, Brazil. Permission for the microbial survey was obtained from CNPq .Supplementary Figure S1), fragments of visually healthy M. hispida colonies  and 1 L of the surrounding seawater (∼40 cm distant from the colonies) were sampled using sterilized bottles at five reefs located close to a protected marine area  of Porto Seguro, Bahia, Brazil. Four of these reefs are located approximately 2 km from each other , starting 2 km off the mouth of the Buranhém River and ending at a patch of reef near the southeastern part of the Recife de Fora. Reef 5 is located in the northeastern area of the “Parque Natural Municipal do Recife de Fora,” theoretically away from the direct influence of the river mouth.Every 2–3 months between August 2013 and May 2014  . All samples  were immediately frozen in liquid nitrogen and stored at –80°C in the laboratory.® DNA Isolation Kit  following a modification of the method described by Coral samples  and seawater material scraped off the filters were homogenized, and the DNA was extracted using the PowerSoil1 was used to process the raw sequence data .The remaining high-quality sequences were binned into operational taxonomic units (OTUs) at 99% sequence identity using vSEARCH . A repreHere, we considered the “core” as a subset of bacterial taxa universally present in all samples . ConsideM. hispida. Significant correlations between OTUs with a minimum occurrence of 8 were determined using Pearson’s correlation and Spearman’s correlation  using the CoNet app  was obtained using the web-based tool InteractiVenn .n = 4, 15 ml each) was filtered (0.45 μm), acidified (1% HNO3), protected from light with aluminum foil, and kept at -20°C until analysis. The dissolved organic carbon (DOC) concentration in seawater was determined in a Total Organic Carbon (TOC) analyzer . Ion concentrations  were determined in seawater samples via flame photometry.Sediment samples (∼0.3 mg) were dried (60°C) and ground, and the percentages of N, C, and carbonate were determined via Costech EA-Delta XP IRMS. Seawater from each collection site  according to the manufacturer’s instructions. The matrices were ordered by non-metric dimensional scaling (NMDS)  using a Estimates of α-diversity and β-diversity were based on an evenly rarified OTU abundance matrix. For β-diversity metrics, we analyzed the differences among the profiles and composition of bacterial communities, using a Principal coordinates analysis (PCoA), with a Bray-Curtis distance matrix with PRIMER6 . To asseThe OTUs associated with the bacterial family Enterobacteriaceae, i.e., potentially including coliforms, were used as a proxy for anthropogenic pollution.Statistical differences of α-diversity matrices , nitrogen content, Enterobacteriaceae, core and variable OTUs, and site-stable bacterial populations (persistent OTUs found at each reef/sampling point) among the samples were determined using a Mann–Whitney test in Statistica software (v. 7.1). Pearson’s correlation was used to test for a relationship between nitrogen content and abundance of Enterobacteriaceae OTUs.M. hispida colonies and the surrounding seawater showed that those communities were dynamic, changing in time and space . For all sampling times (rainy and dry seasons), the bacterial communities from the surrounding seawater differed among the sampling sites, according to the PERMANOVA  . The same pattern was observed for the bacterial communities of M. hispida according to the PERMANOVA  . However, the coral bacterial community from Reef 3 seemed to show an intermediate profile. Depending on the season, this community seemed to be more similar to the nearshore reefs, Reefs 1 and 2 (p > 0.05) , or to the offshore reefs, Reefs 4 and 5 (p > 0.05) . In contrast, the microbial communities from Reefs 4 and 5 differed significantly from Reef 1 at all sampling times (p < 0.05) .Non-metric dimensional scaling of the 16S rRNA gene-based PCR-DGGE profile of bacterial communities from For the subsequent analysis of the bacterial diversity (Illumina Sequencing), Reef 1 (closest to the coast and to the Buranhém River mouth), Reef 3 (midway between Reefs 1 and 5) and Reef 5 (farthest from the Buranhém River mouth) were chosen, based on the DGGE profiles observed at the four sampling times.M. hispida coral and the surrounding seawater were significantly different from each other  . In addition to differences in composition, the seawater bacterial communities were three times richer than those found in association with the coral  . According to sequencing data, M. hispida bacterial communities were dynamic , varying mainly according to the sampling location . Additionally, only samples from Reefs 1 and 5 showed distinct profiles . The coral bacterial communities sampled at Reef 3 could not be discriminated from samples from Reef 1  or Reef 5 . The bacterial communities associated with M. hispida also varied significantly over the four sampling times  . No significant differences were observed between the location and sampling period for any of the diversity indexes tested (Table 1) . In addition, the rarefaction curves from all samples reached a plateau .Principal coordinates analysis based on sequencing data revealed two main distinct groups, showing that the bacterial communities associated with p < 0.05) .The OTUs associated with the family Enterobacteriaceae showed a significant gradient in abundance, with the highest prevalence at Reef 1, closest to the Buranhém River mouth , showing a significant gradient of the N content, where Reef 1 was again the site of the highest concentration. No significant differences in salinity, C, carbonate, and DOC were detected among the different sampling sites (Supplementary Table S1).The functional correlation between the abundances of Enterobacteriaceae OTUs from the coral microbiome and the seawater N content was also positive (Pearson′s r = 0.165) , the site-stable bacterial populations (persistent OTUs found at each reef/sampling point) and the variable bacterial populations  were evaluated . During rainy seasons, at Reef 5, the coral bacteriome was composed of a higher proportion (relative abundance) of the bacterial core (74–96%) . Significant differences among the site-stable bacterial populations (persistent OTUs found at each reef/sampling point) of the reefs were observed only during rainy season 2. Therefore, there was a proportionately higher predominance of the site-stable bacterial populations at R3 (15%), followed by R1 (5%) and R5 (<1%), respectively . On the other hand, Reef 3 showed different proportions of the variable and core bacteriomes  depending on the sampling period.In order to explore the dynamics of variable and permanent bacterial populations associated with p < 0.05) .During dry seasons, there were no significant differences in the proportions of the variable or core bacterial microbiomes among the reefs. However, in dry season 1, Reef 1 showed a higher proportion of site-stable bacterial populations compared to Reefs 3 and 5 . The following bacterial populations were identified as core members: Acinetobacter sp., Comamonadaceae II, Neisseriaceae, Staphylococcus sp., Enhydrobacter sp., and Peptostreptococcaceae. In general, the bacteriome core was dominated by Acinetobacter sp. and Comamonadaceae II. Neisseriaceae was the third most prevalent OTU at Reef 1 in rainy-season samples. Although differences were detected, the distribution of the core bacterial members was homogeneous during the dry season.The abundance of each core member varied among the different reefs at different sampling times . The site-stable bacterial population from Reef 1 was composed of 9 different OTUs . The site-stable bacterial populations associated with Reef 3 were represented by five OTUs . Finally, the site-stable bacterial populations from Reef 5 were composed of four OTUs  .Overall, the site-stable bacterial populations from dry-season samples were more similar to each other .Based on a Venn diagram, most of the site-stable bacteria were exclusive to each reef. However, Comamonadaceae I occurred at Reefs 1 and 5, and also Figure 5). The overall topological properties of these networks were also calculated in order to distinguish the OTU correlations at the different reefs (Table 2). For the number of nodes, edges and correlations (positive and negative), Reef 5 showed the highest values and Reef 1 the lowest , indicating that a more-complex network topology existed at Reef 5 than at Reefs 3 and 1 (Table 2).Co-occurrence networks were generated for each reef individually, based on their 150 most abundant OTUs . Some site-stable bacterial populations (persistent OTUs found at each reef/sampling point) were also observed, such as Mycobacterium sp., Streptococcus sp., Achromobacter sp., Comamonadaceae I, Corynebacterium spp. I and II, and Sphingopyxis alaskensis. Close relationships among Acinetobacter sp., Comamonadaceae II, and Staphylococcus sp. were observed in all network analyses .Bacterial core members were easily found in network interactions from Reefs 1, 3, and 5, including M. hispida bacteriome  showed significantly different profiles, as previously reported for different coral species  and alsocteriome , 2018.M. hispida, sampled on different reefs for nearly 1 year, were extremely dynamic. In particular, six members composed the core bacteriome of M. hispida, considering all sampled reefs and times , and comprised ∼20–96% of the total bacterial community members. In some cases, Acinetobacter sp. appeared as the predominant member, such as during the rainy season at Reef 5. This genus has been described as being vertically transmitted from parent to offspring in M. hispida . In addition, the site-stable bacterial diversity was proportionally higher at the nearshore Reef 1 (closest to the Buranhém River mouth) during the rainy seasons, suggesting a local bacterial adaptation due to environmental changes and impacts. Our data also indicated a higher diversity of OTUs representing the site-stable bacterial populations, which could be related to the higher abundance of total coliforms and higher N content (proxies for sewage or organic pollution) found at Reef 1.Several studies on coral bacteriomes have revealed that these organisms can quickly respond when they are faced with environmental impacts, such as pollution and climate change . SimilarFigure 5), considering all sampling times, the same gradient as observed for the proxies of organic pollution. This lack of complexity of the network relationships on the reefs that were more exposed to organic pollution, leading to a more susceptible bacteriome  among the microbial players . In genecteriome , could ecteriome .Pseudomonas sp. This genus was previously described as a member of the M. hispida bacterial core .The raw data from each sample are available in the NCBI Sequence Read Archive (SRA) under the Accession Number , JM, and AB : acquisition of data . RP, DL, JE, and JS: analyses and interpretation of data. RP and DL: drafting of the manuscript. RP, JS, and CC: financial support. All authors: critical revision of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer DTS and handling Editor declared their shared affiliation.


Reef restoration activities have proliferated in response to the need to mitigate coral declines and recover lost reef structure, function, and ecosystem services. Here, we describe the recent shift from costly and complex engineering solutions to recover degraded reef structure to more economical and efficient ecological approaches that focus on recovering the living components of reef communities. We review the adoption and expansion of the coral gardening framework in the Caribbean and Western Atlantic where practitioners now grow and outplant 10,000’s of corals onto degraded reefs each year. We detail the steps for establishing a gardening program as well as long-term goals and direct and indirect benefits of this approach in our region. With a strong scientific basis, coral gardening activities now contribute significantly to reef and species recovery, provide important scientific, education, and outreach opportunities, and offer alternate livelihoods to local stakeholders. While challenges still remain, the transition from engineering to ecological solutions for reef degradation has opened the field of coral reef restoration to a wider audience poised to contribute to reef conservation and recovery in regions where coral losses and recruitment bottlenecks hinder natural recovery. Acropora cervicornis, A. palmata, and Orbicella spp.  and staghorn (A. cervicornis) corals  the  Species ; and 3) ) corals .Acropora and Orbicella are especially problematic to restore as recruitment failure of these reef-building species prevents natural recovery , ProQuest ASFA (Aquatic Sciences and Fisheries Abstracts), and Google Scholar databases since 1980. A total of 268 papers were identified, with a steady increase in the number of publications over time. The period prior to 2000 had 29 publications, 2001–2005 had 37, 2006–2010 had 99, 2011–2015 had 103. The most publications in a single year (36) were recorded in 2015. In addition to an increasing trend in the number of publications, the proportion of publications reporting on engineering compared to ecological reef restoration solutions has changed over time, showing a clear shift of emphasis in the field. Prior to 2000, 51% of the publications were on engineering reef restoration, compared to 35% in 2001–2005, 18% in 2006–2010, and only 4% in 2011–2015. The increase in the number of citations is also reflected in an increase in the number of projects and programs. Engineering solutions may still be needed in cases where the substrate remains unstable and thus inadequate for successful transplantation or natural or assisted coral recruitment. Even in these instances, the recent development of ecological methods to stabilize loose rubble by deploying reef sponges may replace the use of cement as a binding agent . The prohttp://www.secore.org), following earlier successful outcomes in the Pacific . By supplementing gardening activities with restoration using coral larvae, a greater impact on genetic diversity can be achieved. Moreover, the gametes and larvae reared from nursery stocks can provide key resources to support novel research activities such as coral hardening and assisted evolution http://wwOrbicella  , both taxa recently added to the US ESA, as well as Pseudodiploria (Florida)   and DendFlorida)  and otheAcropora thickets that would otherwise take decades to form .In addition to supplying corals for restoration, coral gardening programs provide a range of secondary benefits to ecosystems and local economies . Coral g to form ; 2) crea to form ; 3) impr to form ; 5) conthttp://www.puntacana.org/) that was initiated by A. Bowden-Kerby and expanded by V. Galvan, J. Kheel, and D. Lirman that has been in place for > 10 years. The program has outplanted > 15,000 staghorn corals onto reefs where this species had been eradicated due to algal overgrowth, disease, pollution, and coastal development. This program has also enhanced the local economy by: 1) restoring reefs that have become preferred dive sites used by local operators and hotels; 2) developing a “Coral First Aid” PADI dive specialty course taught by local dive shops to tourists; and 3) training local fishermen to become “coral gardeners” by providing them SCUBA certifications and employment opportunities to guide ecotourism excursions to nurseries and restoration sites. Transitioning fishermen from harvesting to gardening has the added benefit of reducing the impacts of unsustainable fishing practices on the reefs being restored. It is estimated that each fishermen hired as a coral gardener keeps an estimated 12.5 lbs of parrotfish per day on the local reefs, further improving reef conditions . The loss of resources or interest after the initial stages of a new program has resulted in “orphan” nurseries where corals continue to grow but are not maintained or, worse, never outplanted. In these cases, the nursery platforms collapse resulting in mortality of threatened/endangered corals . UntendeAn important aspect of responsible coral gardening is to ensure that the scale of the nursery is commensurate with the resources available. The relative ease and low cost of nursery construction, coupled with the high survivorship and fast growth of corals within nurseries  can creaEven the best-planned restoration programs can lose funding, momentum, or interest. Some of the factors that have contributed to this scenario include: 1) lack of sustained funding beyond the nursery stage; 2) turnover in dive shop and hotel ownership and personnel; 3) vandalism and physical damage to nursery resources; and 4) loss of local support. In these cases, a clear exit strategy is needed to prevent the proliferation of orphan nurseries. An exit strategy should be an explicit part of the planning process and should clearly identify the scenarios that would trigger the interruption of a project and the steps needed to terminate the restoration project responsibly. At a minimum, an adequate exit strategy would require the outplanting of all nursery corals onto suitable reef habitat and the removal of all nursery materials from the site to prevent these materials from damaging nearby reef resources. This common-sense approach would mitigate negative perceptions of coral gardening in the local community and allow for the re-initiation of future projects in the same area if resources become available or conditions improve.The first advances in coral propagation within nurseries and the outplanting of nursery-grown corals were achieved by trial-and-error. With the maturation and expansion of this field, a number of programs have developed strong, science-based methods now published in the peer-reviewed literature and as manuals available online  that canHermodice concentrate on newly deployed staghorn outplants . Outplanted corals also face potentially detrimental water chemistry conditions where ocean acidification has created reef environments with low aragonite saturation states , and an increasing number of species now routinely propagated. The next step, outplanting nursery-grown corals onto wild reefs, is still experiencing mixed results, with variable performance of outplants . These cn states .An example of the challenges faced by outplanted corals was provided by the unprecedented, back-to-back bleaching events recorded in the Florida Keys in 2014 and 2015. During these events, both outplanted and wild colonies showed similar patterns of bleaching and mortality that were highly influenced by coral genotype and location  . Such diConsidering the relatively young age of the gardening activities in the Caribbean and Western Atlantic, data are still lacking on the long-term survivorship of outplants. However, in Culebra, Puerto Rico, the site of the oldest gardening program in the Caribbean, staghorn outplants deployed in 2003 are still alive today . Staghorn outplants have survived > 7 years in the Dominican Republic and outplants of both staghorn and elkhorn corals have survived > 6 years in Belize . In Mexico, thriving first-generation elkhorn outplants are > 5 years old . In Florida, staghorn outplants have been shown to survive > 5 years, during which colonies have grown considerably, fragmented, and created new colonies. In addition, one extremely positive outcome of the gardening activities has been the observation of successful spawning of nursery and outplanted staghorn corals in Florida and the Caribbean  in Florida and elsewhere (The use of coral gardening methods for species and reef recovery are not without potential negative impacts that need to be considered. The two main ecological concerns, in our opinion, are disease impacts within nurseries and outplanted populations and genetic impacts on the extant populations. Diseases have been a major source of mortality to corals (particularly lsewhere . By proplsewhere  suggestilsewhere . Neverthlsewhere  also raiAnother concern for gardening programs is the role that coral outplants can play on the genetic and genotypic diversity of wild populations, especially considering that the coral species being restored have experienced recent drastic bottlenecks in coral abundance. These concerns include the introduction of genotypes into novel environments where the fitness of a restored population may decline due to founder effects, genetic swamping, and inbreeding/outbreeding depression . In the As reef restoration activities and programs in the Caribbean and Western Atlantic have transitioned from costly engineering projects into efficient ecological approaches, the coral gardening framework has “come of age” in the past decade and is now at the forefront of this important and emerging field. While challenges and data gaps remain, coral propagation and outplanting within a gardening framework conducted at meaningful scales and supported by strong science can play a significant role in the restoration of coral reef communities, the restitution of ecologic and economic services, and the recovery of threatened coral taxa. As the support for coral gardening grows, the next major step will be the documentation of benchmarks that can be used by practitioners to determine the efficacy of their efforts and impacts at the species and community levels.While clearly beneficial for all regions of the world, coral gardening is especially important in the Caribbean and Western Atlantic where reef-building taxa are experiencing reproductive bottlenecks . Coral gFinally, it is important to temper expectations and note that no amount of coral gardening can fully recover a depleted species or ecosystem, especially when environmental and climate challenges remain. The goal of these activities should instead be to foster the natural recovery by re-establishing spatially connected populations with high genotypic diversity that can promote the successful sexual reproduction and natural recovery of the targeted species. Similarly, coral gardening and reef restoration cannot be the only tools employed. Reef restoration practitioners have recognized that local management tools such as watershed management, sustainable fishing practices, and the establishment of MPAs, among others, should be concurrently implemented to foster reef resilience.