Coat Protein Binding Site Research Articles

Characterization of sulfur compounds and antiviral activity against Tomato brown rugose fruit virus (ToBRFV) of Italian “Vessalico” garlic compared to other cultivars and landrace

In this study the Italian garlic “Aglio di Vessalico” was compared with the French cultivars Messidrôme and Messidor, whose cloves are used every year by the growers of Vessalico (Imperia, Italy) for sowing, and with the geographically adjacent “Aglio di Caraglio”. UHPLC-Q-trap analyses of the extracts of these commercial garlic accessions showed similar profiles for sulfur compounds, highlighting the presence of typical molecules of stored garlic. HC, PCA and SOM, applied to the LC/MS data, allowed to separate the garlic accessions, confirming similarities between Vessalico and French accessions, while Caraglio formed a more compact group. All garlic extracts showed ability to deactivate Tomato brown rugose fruit virus (ToBRFV) infectivity, as a possible consequence of disassembly of the virus coat protein (CP). Molecular docking showed a strong interaction of the sulfur compounds characteristic of aged extracts with a high number of residues into ToBRFV CP binding site, interfering with virulence progress.

Formation of synthetic RNA protein granules using engineered phage-coat-protein -RNA complexes

Liquid-solid transition, also known as gelation, is a specific form of phase separation in which molecules cross-link to form a highly interconnected compartment with solid – like dynamical properties. Here, we utilize RNA hairpin coat-protein binding sites to form synthetic RNA based gel-like granules via liquid-solid phase transition. We show both in-vitro and in-vivo that hairpin containing synthetic long non-coding RNA (slncRNA) molecules granulate into bright localized puncta. We further demonstrate that upon introduction of the coat-proteins, less-condensed gel-like granules form with the RNA creating an outer shell with the proteins mostly present inside the granule. Moreover, by tracking puncta fluorescence signals over time, we detected addition or shedding events of slncRNA-CP nucleoprotein complexes. Consequently, our granules constitute a genetically encoded storage compartment for protein and RNA with a programmable controlled release profile that is determined by the number of hairpins encoded into the RNA. Our findings have important implications for the potential regulatory role of naturally occurring granules and for the broader biotechnology field.

Impact of the Potential m6A Modification Sites at the 3'UTR of Alfalfa Mosaic Virus RNA3 in the Viral Infection.

We have previously reported the presence of m6A in the AMV (Alfamovirus, Bromoviridae) genome. Interestingly, two of these putative m6A-sites are in hairpin (hp) structures in the 3’UTR of the viral RNA3. One site (2012AAACU2016) is in the loop of hpB, within the coat protein binding site 1 (CPB1), while the other (1900UGACC1904) is in the lower stem of hpE, a loop previously associated with AMV negative-strand RNA synthesis. In this work, we have performed in vivo experiments to assess the role of these two regions, containing the putative m6A-sites in the AMV cycle, by introducing compensatory point mutations to interfere with or abolish the m6A-tag of these sites. Our results suggest that the loop of hpB could be involved in viral replication/accumulation. Meanwhile, in the 1900UGACC1904 motif of the hpE, the maintenance of the adenosine residue and the lower stem hpE structure are necessary for in vivo plus-strand accumulation. These results extend our understanding of the requirements for hpE in the AMV infection cycle, indicating that both the residue identity and the base-pairing capacity in this structure are essential for viral accumulation.

Structure and Dynamics of the Tetra-A Loop and (A-A)-U Sequence Motif within the Coliphage GA Replicase RNA Operator.

The three-dimensional structure of a RNA hairpin containing the RNA operator binding site for bacteriophage GA coat protein is presented. The phage GA operator contains the asymmetric (A-A)-U sequence motif and is capped by a four-adenine (tetra-A) loop. The uridine of the (A-A)-U motif preferentially pairs with the 5'-proximal cross-strand adenine, and the 3'-proximal adenine stacks into the helix. The tetra-A loop is well-ordered with adenine residues 2-4 forming a 3' stack. This loop conformation stands in contrast to the structure of the 5'-AUUA loop of the related phage MS2 operator in which residues 1 and 2 form a 5' stack. The context dependence of the (A-A)-U sequence motif conformation was examined using structures of 76 unique occurrences from the Protein Data Bank. The motif almost always has one adenine bulged and the other adenine adopting an A-U base pair. In the case in which the (A-A)-U motif is flanked by only one Watson-Crick base pair, the adenine adjacent to the flanking base pair tends to bulge; 80% of motifs with a 3' flanking pair have a 3' bulged adenine, and 84% of motifs with a 5' flanking pair have a 5' bulged adenine. The frequencies of 3'- and 5'-proximal adenines bulging are 33 and 67%, respectively, when the (A-A)-U motif is flanked by base pairs on both sides. Although a 3' flanking cytidine correlates (88%) with bulging of the 5'-proximal adenine, no strict dependence on flanking nucleotide identity was identified for the 5' side.

Generation and characterization of novel DNA aptamers against coat protein of grouper nervous necrosis virus (GNNV) with antiviral activities and delivery potential in grouper cells

Nervous necrosis virus (NNV) infected larvae and juveniles of more than 50 fish species, resulting in mortality rates of greater than 95%. However, there is no efficient method to control NNV infections. Aptamers generated by selective evolution of ligands by exponential enrichment (SELEX) are short, single-stranded nucleic acid oligomers. They display a high degree of affinity and specificity for many targets, such as viruses and viral proteins. In this study, three novel DNA aptamers (A5, A10, and B11) that specifically target the coat protein (CP) of grouper nervous necrosis virus (GNNV) were selected using SELEX. Secondary structures and minimum free energy (ΔG) predictions indicated that these aptamers could form stable, secondary stem-loop structures. Electrophoretic mobility shift assays, enzyme-linked immunosorbent assays, Kd measurements, the co-localization of tetramethylrhodamine (TAMRA) labeled-aptamers with the CP and flow cytometry analysis revealed that these aptamers could specifically bind the CP with high (nanomolar) affinities. In addition, competition analysis suggested the aptamers shared some common CP binding sites with the anti-CP antibody. Moreover, all three aptamers did not show any cytotoxic effects in vitro or in vivo, and anti-viral analysis indicated the selected aptamers could inhibit NNV infection in vitro and in vivo. Compared with controls, mortality of GNNV-infected fish decreased by 40% and 80% after 10 days infection, when the GNNV was pre-incubated with the 1000 nM A10 and B11, respectively. TAMRA-labeled aptamers could bind to NNV virions and directly enter NNV-infected cells, suggesting they could be used as tracers to study the mechanism of viral infection, as well as for targeted therapy. This is the first time that aptamers targeting a viral protein of marine fish have been generated and characterized. These aptamers hold promise as diagnostic, therapeutic, and targeted drug delivery agents for controlling NNV infections.

RNA imaging: seeing is believing.

RNA imaging: seeing is believing.

Affinity purification of long noncoding RNA-protein complexes from formaldehyde cross-linked mammalian cells.

Long noncoding RNAs (lncRNAs) are a class of recently identified untranslated RNA molecules that have been shown to function in diverse cellular processes. The purification and analysis of lncRNA-protein (lncRNP) complexes is critical toward understanding the normal physiological function of these molecules. Here, we describe the purification of lncRNP complexes from human cells using a FLAG-tagged MS2-phage coat protein (MS2 CP) that binds in sequence-specific fashion to MS2-phage coat protein-binding sites (MS2bs) with high affinity. In these experiments, a FLAG-tagged MS2 CP is transiently co-expressed with a version of the lncRNA into which 12 copies of the MS2bs have been inserted near its 3'-end. The lncRNA-FLAG-tagged MS2 CP complex is then isolated using an anti-FLAG antibody, allowing for characterization of associated cellular proteins and RNAs.

Sequence analysis of RNA 1 of lilac leaf chlorosis virus supports a close relationship to subgroup 3 ilarviruses

Analysis of the nucleotide sequence of the RNA 1 of lilac leaf chlorosis virus (LLCV) supports a close relationship with subgroup 3 ilarviruses. LLCV RNA 1 consists of 3404 nucleotides (nt) and encodes a single open reading frame consisting of 3111 nt. The deduced protein (M(r) 117 kDa) contains the putative methyltransferase domain in the N-terminal region, and the NTPase/helicase domain in the C-terminal region. A conserved 41-nt region was identified at the distal end of the 3'UTR of all three genomic fragments of LLCV, with hairpin structures that may constitute putative coat protein binding sites.

M-TAG: a PCR-based genomic integration method to visualize the localization of specific endogenous mRNAs in vivo in yeast

This protocol describes m-TAG, a novel method for the visualization of endogenously expressed mRNAs in live yeast (Saccharomyces cerevisiae). First, a gene of interest is tagged with multiple binding sites for the RNA-binding MS2 coat protein (MS2-CP), using a PCR-based genomic-tagging strategy and homologous recombination. Next, MS2-CP fused to GFP(x3) is expressed in cells; binding of this fusion protein to the tagged mRNA yields an RNA granule that can be visualized by fluorescence microscopy. While existing methods necessitate cell fixation (for in situ hybridization) or the detection of exogenously expressed mRNAs (from plasmids), or give transient signals (i.e., with fluorescent hybridization probes), m-TAG allows for the robust and stable visualization of endogenously expressed mRNAs in vivo and facilitates the study of mRNA dynamics under different growth conditions. The m-TAG procedure is simple, easy to perform and takes <3 weeks to yield cultured yeast strains for mRNA visualization.

Combined Use of MS2 and PP7 Coat Fusions Shows that TIA‐1 Dominates hnRNP A1 for K‐SAM Exon Splicing Control

Splicing of the FGFR2 K-SAM exon is repressed by hnRNP A1 bound to the exon and activated by TIA-1 bound to the downstream intron. Both proteins are expressed similarly by cells whether they splice the exon or not, so it is important to know which one is dominant. To answer this question, we used bacteriophage PP7 and bacteriophage MS2 coat fusions to tether hnRNP A1 and TIA-1 to distinct sites on the same pre-mRNA molecule. hnRNP A1 fused to one coat protein was tethered to a K-SAM exon containing the corresponding coat protein's binding site. TIA-1 fused to the other coat protein was tethered to the downstream intron containing that coat protein's binding site. This led to efficient K-SAM exon splicing. Our results show that TIA-1 is dominant for K-SAM exon splicing control and validate the combined use of PP7 and MS2 coat proteins for studying posttranscriptional events.

The Three-dimensional Structure of Genomic RNA in Bacteriophage MS2: Implications for Assembly

Using cryo-electron microscopy, single particle image processing and three-dimensional reconstruction with icosahedral averaging, we have determined the three-dimensional solution structure of bacteriophage MS2 capsids reassembled from recombinant protein in the presence of short oligonucleotides. We have also significantly extended the resolution of the previously reported structure of the wild-type MS2 virion. The structures of recombinant MS2 capsids reveal clear density for bound RNA beneath the coat protein binding sites on the inner surface of the T = 3 MS2 capsid, and show that a short extension of the minimal assembly initiation sequence that promotes an increase in the efficiency of assembly, interacts with the protein capsid forming a network of bound RNA. The structure of the wild-type MS2 virion at ∼9 Å resolution reveals icosahedrally ordered density encompassing ∼90% of the single-stranded RNA genome. The genome in the wild-type virion is arranged as two concentric shells of density, connected along the 5-fold symmetry axes of the particle. This novel RNA fold provides new constraints for models of viral assembly.

A genomic integration method to visualize localization of endogenous mRNAs in living yeast

mRNA localization may be an important determinant for protein localization. We describe a simple PCR-based genomic-tagging strategy (m-TAG) that uses homologous recombination to insert binding sites for the RNA-binding MS2 coat protein (MS2-CP) between the coding region and 3' untranslated region (UTR) of any yeast gene. Upon coexpression of MS2-CP fused with GFP, we demonstrate the localization of endogenous mRNAs (ASH1, SRO7, PEX3 and OXA1) in living yeast (Saccharomyces cerevisiae).

Cryo Electron Microscopy Reconstructions of the Leviviridae Unveil the Densest Icosahedral RNA Packing Possible

We solved the structures of the single-stranded RNA bacteriophages Qβ, PP7 and AP205 by cryo-electron microscopy. On the outside, the symmetrized electron density maps resemble the previously described cryo-electron microscopy structure of MS2. RNA density is present inside the capsids, suggesting that the genomic RNA of Qβ, PP7 and AP205, analogous to MS2, contains many coat protein-binding sites in addition to the hairpin on which assembly and packaging are initiated. All four bacteriophages harbour the same overall arrangement of the RNA, which is a unique combination of both triangles and pentagons. This combination has not been found in other icosahedral viruses, in which the RNA structures are either triangular or pentagonal. Strikingly, the unique RNA packing of the Leviviridae appears to deploy the most efficient method of RNA storage by obeying icosahedral symmetry.

Replication of Alfamo- and Ilarviruses: Role of the Coat Protein

In the family Bromoviridae, a mixture of the three genomic RNAs of bromo-, cucumo-, and oleaviruses is infectious as such, whereas the RNAs of alfamo- and ilarviruses require binding of a few molecules of coat protein (CP) to the 3' end to initiate infection. Most studies on the early function of CP have been done on the alfamovirus Alfalfa mosaic virus (AMV). The 3' 112 nucleotides of AMV RNAs can adopt two different conformations. One conformer consists of a tRNA-like structure that, together with an upstream hairpin, is required for minus-strand promoter activity. The other conformer consists of four hairpins interspersed by AUGC-sequences and represents a strong binding site for CP. Binding of CP to this conformer enhances the translational efficiency of viral RNAs in vivo 40-fold and blocks viral minus-strand RNA synthesis in vitro. AMV CP is proposed to initiate infection by mimicking the function of the poly(A)-binding protein.

Molecular Genetics of Bacteriophage P22 Scaffolding Protein's Functional Domains

The assembly intermediates of the Salmonella bacteriophage P22 are well defined but the molecular interactions between the subunits that participate in its assembly are not. The first stable intermediate in the assembly of the P22 virion is the procapsid, a preformed protein shell into which the viral genome is packaged. The procapsid consists of an icosahedrally symmetric shell of 415 molecules of coat protein, a dodecameric ring of portal protein at one of the icosahedral vertices through which the DNA enters, and approximately 250 molecules of scaffolding protein in the interior. Scaffolding protein is required for assembly of the procapsid but is not present in the mature virion. In order to define regions of scaffolding protein that contribute to the different aspects of its function, truncation mutants of the scaffolding protein were expressed during infection with scaffolding deficient phage P22, and the products of assembly were analyzed. Scaffolding protein amino acids 1-20 are not essential, since a mutant missing them is able to fully complement scaffolding deficient phage. Mutants lacking 57 N-terminal amino acids support the assembly of DNA containing virion-like particles; however, these particles have at least three differences from wild-type virions: (i) a less than normal complement of the gene 16 protein, which is required for DNA injection from the virion, (ii) a fraction of the truncated scaffolding protein was retained within the virions, and (iii) the encapsidated DNA molecule is shorter than the wild-type genome. Procapsids assembled in the presence of a scaffolding protein mutant consisting of only the C-terminal 75 amino acids contained the portal protein, but procapsids assembled with the C-terminal 66 did not, suggesting portal recruitment function for the region about 75 amino acids from the C terminus. Finally, scaffolding protein amino acids 280 through 294 constitute its minimal coat protein binding site.

Efficient translation of alfamovirus RNAs requires the binding of coat protein dimers to the 3' termini of the viral RNAs.

The coat protein (CP) of Alfalfa mosaic virus (AMV) is required to initiate infection by the viral tripartite RNA genome whereas infection by the tripartite Brome mosaic virus (BMV) genome is independent of CP. AMV CP stimulates translation of AMV RNA in vivo 50- to 100-fold. The 3' untranslated region (UTR) of the AMV subgenomic CP messenger RNA 4 contains at least two CP binding sites. A CP binding site in the 3'-terminal 112 nucleotides of RNA 4 was found to be required for efficient translation of the RNA whereas an upstream binding site was not. Binding of CP to the AMV 3' UTR induces a conformational change of the RNA but this change alone was not sufficient to stimulate translation. CP mutant R17A is unable to bind to the 3' UTR and translation in vivo of RNA 4 encoding this mutant occurs at undetectable levels. Replacement of the 3' UTR of this mutant RNA 4 by the 3' UTR of BMV RNA 4 restored translation of R17A-CP to wild-type levels. Apparently, the BMV 3' UTR stimulates translation independently of CP. AMV CP mutant N199 is defective in the formation of CP dimers and did not stimulate translation of RNA 4 in vivo although the mutant CP did bind to the 3' UTR. The finding that N199-CP does not promote AMV infection corroborates the notion that the requirement of CP in the inoculum reflects its role in translation of the viral RNAs.

Natural minus-strand RNAs of alfalfa mosaic virus as in vitro templates for viral RNA polymerase. 3'-terminal non-coded guanosine and coat protein are insufficient factors for full-size plus-strand synthesis.

Replication complexes of alfalfa mosaic virus produce in vivo large quantities of plus-strand RNAs, but this production is fully dependent on the presence of coat protein. In order to study this process of RNA-dependent and coat protein-regulated RNA synthesis we have isolated the three natural minus-strand RNAs (containing any posttranscriptional modification that might have occurred) and have tested them for coat protein binding sites and template activity in an in vitro system with the viral RNA polymerase. The enzyme was prepared by an advanced isolation procedure. All three minus strands had a single non-coded G at their 3' terminus. They were not able to withdraw coat protein subunits from virions as free virion RNAs do. No sites protected by coat protein against ribonuclease T1 degradation were found. Two large T1 oligonucleotides from minus RNA 1 and one from minus RNA 3 were bound by coat protein to Millipore filters. Except for minus RNA 3 which caused a minute amount of full-size plus strand to be synthesized, the minus strands did not function as templates for full-size complementary strands. On the other hand, they gave rise to a number of well-defined shorter products, the synthesis of which was stimulated by the addition of coat protein. These products could not be elongated by a chase treatment and were probably the result of internal initiations. It is concluded that, although posttranscriptional modifications of the template and the presence of coat protein may be necessary factors for plus-strand RNA synthesis, they are certainly not sufficient. Our purified in vitro system needs further sophistication.

Localization and anchoring of mRNA in budding yeast

Background: Eukaryotic cells localize selected mRNAs to a region of the cell as a means to sequester proteins. Signals within the 3′ untranslated region (3′ UTR) facilitate mRNA localization by both actin and microtubule cytoskeletal systems. Recently, an mRNA in the yeast Saccharomyces cerevisiae, ASH1, was shown to coalesce into a discrete particle that is maintained at the bud tip. Mutations in five genes, SHE1–SHE5, cause defects in particle formation and/or localization of the ASH1 transcript. Factors at the destination of the mRNA transport remain to be identified.Results: We have developed a system to label mRNA in living yeast with green fluorescent protein (GFP) and follow the dynamics of mRNA movement and localization. Constitutively expressing an ASH1 mRNA containing the bacteriophage MS2 coat-protein binding site adjacent to the ASH1 3′ UTR allowed us to visualize ASH1 mRNA with an MS2-coat-protein–GFP fusion protein (together denoted ‘gRNAASH1’). The gRNAASH1 was restricted to the bud tip in small to large budded cells, migrated to the bud neck prior to cell separation and then rapidly relocalized to the incipient site of bud growth. It also localized to regions of polarized growth during mating. In cells lacking Bud6p/Aip3p or Bnilp/She5p, which are involved in polarity establishment and actin organization, gRNAASH1 migrated to the bud but failed to remain at the bud tip. These studies reveal discrete transport and anchoring steps in mRNA localization.Conclusions: The ASH1 mRNA was maintained at sites of polarized growth throughout the vegetative and mating cell cycles. Bud6p/Aip3p and Bni1p/She5p are required to maintain the transcript at the cortical bud cap.

NMR structure and dynamics of an RNA motif common to the spliceosome branch-point helix and the RNA-binding site for phage GA coat protein.

The RNA molecules that make up the spliceosome branch-point helix and the binding site for phage GA coat protein share a secondary structure motif in which two consecutive adenine residues occupy the strand opposite a single uridine, creating the potential to form one of two different A.U base pairs while leaving the other adenine unpaired or bulged. During the splicing of introns out of pre-mRNA, the 2'-OH of the bulged adenine participates in the transesterification reaction at the 5'-exon and forms the branch-point residue of the lariat intermediate. Either adenine may act as the branch-point residue in mammals, but the 3'-proximal adenine does so preferentially. When bound to phage GA coat protein, the bulged adenine loops out of the helix and occupies a binding pocket on the surface of the protein, forming a nucleation complex for phage assembly. The coat protein can bind helices with bulged adenines at either position, but the 3'-proximal site binds with greater affinity. We have studied this RNA motif in a 21 nucleotide hairpin containing a GA coat protein-binding site whose four nucleotide loop has been replaced by a more stable loop from the related phage Ms2. Using heteronuclear NMR spectroscopy, we have determined the structure of this hairpin to an overall precision of 2.0 A. Both adenine bases stack into the helix, and while all available NOE and coupling constant data are consistent with both possible A.U base pairs, the base pair involving the 5'-proximal adenine appears to be the major conformation. The 3'-proximal bulged adenine protonates at unusually high pH, and to account for this, we propose a model in which the protonated adenine is stabilized by a hydrogen bond to the uridine O2 of the A.U base pair. The 2'-OH of the bulged adenine adopts a regular A-form helical geometry, suggesting that in order to participate in the splicing reaction, the conformation of the branch-point helix in the active spliceosome may change from the conformation described here. Thus, while the adenine site preferences of the spliceosome and of phage GA may be due to protein factors, the preferred adenine is predisposed in the free RNA to conformational rearrangement involved in formation of the active complexes.

Auxiliary downstream elements are required for efficient polyadenylation of mammalian pre-mRNAs.

We have previously identified a G-rich sequence (GRS) as an auxiliary downstream element (AUX DSE) which influences the processing efficiency of the SV40 late polyadenylation signal. We have now determined that sequences downstream of the core U-rich element (URE) form a fundamental part of mammalian polyadenylation signals. These novel AUX DSEs all influenced the efficiency of 3'-end processing in vitro by stabilizing the assembly of CstF on the core downstream URE. Three possible mechanisms by which AUX DSEs mediate efficient in vitro 3'-end processing have been explored. First, AUX DSEs can promote processing efficiency by maintaining the core elements in an unstructured domain which allows the general polyadenylation factors to efficiently assemble on the RNA substrate. Second, AUX DSEs can enhance processing by forming a stable structure which helps focus binding of CstF to the core downstream URE. Finally, the GRS element, but not the binding site for the bacteriophage R17 coat protein, can substitute for the auxiliary downstream region of the adenovirus L3 polyadenylation signal. This suggests that AUX DSE binding proteins may play an active role in stimulating 3'-end processing by stabilizing the association of CstF with the RNA substrate. AUX DSEs, therefore, serve as a integral part of the polyadenylation signal and can affect signal strength and possibly regulation.