Role of the coat Protein-RNA interaction in the life cycle of bacteriophage MS2

The coat protein of the RNA bacteriophage MS2 interacts with viral RNA to translationally repress replicase synthesis. This protein-RNA interaction is also thought to play a role in genome encapsidation. In this study the strength of the interaction was perturbed by constructing a recombinant genome containing a super-repressing coat mutation. Because replicase synthesis is prematurely repressed, the mutant produces plaques about five orders of magnitude less efficiently than wild-type. The few plaques obtained are second-site revertants of the original coat mutation and fall into two categories. Those of the first type contain nucleotide substitutions within the translational operator that reduce or destroy its ability to bind coat protein, showing that this interaction is not necessary for genome encapsidation. Revertants of the second type are double mutants in which one substitution converts the coat initiator AUG to AUA and the other substitutes an A for the G normally present two nucleotides upstream of the coat start codon. The mutation of the coat protein gene AUG to AUA, by itself, reduces coat protein synthesis to a few percent of the wild-type level. The second substitution destabilizes the coat initiator stem-loop and restores coat protein synthesis to within a few fold of wild-type levels. Received: 17 June 1996 / Accepted: 5 December 1996     

The RNA binding site of bacteriophage MS2 coat protein.

The coat protein of the RNA bacteriophage MS2 binds a specific stem-loop structure in viral RNA to accomplish encapsidation of the genome and translational repression of replicase synthesis. In order to identify the structural components of coat protein required for its RNA binding function, a series of repressor-defective mutants has been isolated. To ensure that the repressor defects were due to substitution of binding site residues, the mutant coat proteins were screened for retention of the ability to form virus-like particles. Since virus assembly presumably requires native structure, this approach eliminated mutants whose repressor defects were secondary consequences of protein folding or stability defects. Each of the variant coat proteins was purified and its ability to bind operator RNA in vitro was measured. DNA sequence analysis identified the nucleotide and amino acid substitutions responsible for reduced RNA binding affinity. Localization of the substituted sites in the three-dimensional structure of coat protein reveals that amino acid residues on three adjacent strands of the coat protein beta-sheet are required for translational repression and RNA binding. The sidechains of the affected residues form a contiguous patch on the interior surface of the viral coat.     

Crystal Structure of the Bacteriophage Qβ Coat Protein in Complex with the RNA Operator of the Replicase Gene

The coat proteins of single-stranded RNA bacteriophages specifically recognize and bind to a hairpin structure in their genome at the beginning of the replicase gene. The interaction serves to repress the synthesis of the replicase enzyme late in infection and contributes to the specific encapsidation of phage RNA. While this mechanism is conserved throughout the Leviviridae family, the coat protein and operator sequences from different phages show remarkable variation, serving as prime examples for the co-evolution of protein and RNA structure. To better understand the protein–RNA interactions in this virus family, we have determined the three-dimensional structure of the coat protein from bacteriophage Qβ bound to its cognate translational operator. The RNA binding mode of Qβ coat protein shares several features with that of the widely studied phage MS2, but only one nucleotide base in the hairpin loop makes sequence-specific contacts with the protein. Unlike in other RNA phages, the Qβ coat protein does not utilize an adenine-recognition pocket for binding a bulged adenine base in the hairpin stem but instead uses a stacking interaction with a tyrosine side chain to accommodate the base. The extended loop between β strands E and F of Qβ coat protein makes contacts with the lower part of the RNA stem, explaining the greater length dependence of the RNA helix for optimal binding to the protein. Consequently, the complex structure allows the proposal of a mechanism by which the Qβ coat protein recognizes and discriminates in favor of its cognate RNA.     

Capsid protein synthesis from replicating RNA directs specific packaging of the genome of a multipartite, positive-strand RNA virus

Flock house virus (FHV) is a bipartite, positive-strand RNA insect virus that encapsidates its two genomic RNAs in a single virion. It provides a convenient model system for studying the principles underlying the copackaging of multipartite viral RNA genomes. In this study, we used a baculovirus expression system to determine if the uncoupling of viral protein synthesis from RNA replication affected the packaging of FHV RNAs. We found that neither RNA1 (which encodes the viral replicase) nor RNA2 (which encodes the capsid protein) were packaged efficiently when capsid protein was supplied in trans from nonreplicating RNA. However, capsid protein synthesized in cis from replicating RNA2 packaged RNA2 efficiently in the presence and absence of RNA1. These results demonstrated that capsid protein translation from replicating RNA2 is required for specific packaging of the FHV genome. This type of coupling between genome replication and translation and RNA packaging has not been observed previously. We hypothesize that RNA2 replication and translation must be spatially coordinated in FHV-infected cells to facilitate retrieval of the viral RNAs for encapsidation by newly synthesized capsid protein. Spatial coordination of RNA and capsid protein synthesis may be key to specific genome packaging and assembly in other RNA viruses.     

Control of Replication in RNA Bacteriophages

The rates of viral RNA and protein syntheses for wild-type RNA bacteriophages and their nonpolar, coat protein amber mutants were determined in amber suppressor (S26R1E, Su-1 and H12R8a, Su-3) and nonsuppressor (AB259, S26, and Q13) strains of Escherichia coli in the presence of rifamycin. It was demonstrated that the rates of synthesis of phage-specific replicase and RNA minus strands drop off concurrently in both wild-type and coat protein mutant-infected Su(-) and Su(+) cells after 10 and 15 min postinfection, respectively. The rate of synthesis of RNA plus strands started to decline 5 to 10 min later in both cases. Excessive synthesis of replicase in the coat protein mutant-infected cells was accompanied by a similar overproduction of RNA minus strands, but not of plus strands. Partial suppression of protein synthesis in wild-type phage-infected cells abolishing coat protein control over replicase accumulation led to prolongation of replicase synthesis. Such an effect was observed also in coat protein mutant-infected cells, indicating that the excess of replicase itself may be capable of suppression of replicase synthesis in the absence of coat protein. The prolongation of replicase synthesis was followed by the prolonged synthesis of RNA minus strands in both cases. Moreover, replicase and minus strands were formed in nearly equal amounts when protein synthesis was partially inhibited. Assuming functional instability of phage RNAs, the observed coupling of replicase and minus-strand RNA synthesis offers a possibility for control of viral RNA replication by means of control of replicase synthesis on the translational level. A hypothesis is put forward to explain the molecular mechanism of such coupling between the syntheses of replicase and RNA minus strands.     

An examination of the electrostatic interactions between the N-terminal tail of the Brome Mosaic Virus coat protein and encapsidated RNAs

The coat protein of positive-stranded RNA viruses often contains a positively charged tail that extends toward the center of the capsid and interacts with the viral genome. Electrostatic interaction between the tail and the RNA has been postulated as a major force in virus assembly and stabilization. The goal of this work is to examine the correlation between electrostatic interaction and amount of RNA packaged in the tripartite Brome Mosaic Virus (BMV). Nanoindentation experiment using atomic force microscopy showed that the stiffness of BMV virions with different RNAs varied by a range that is 10-fold higher than that would be predicted by electrostatics. BMV mutants with decreased positive charges encapsidated lower amounts of RNA while mutants with increased positive charges packaged additional RNAs up to ～900 nt. However, the extra RNAs included truncated BMV RNAs, an additional copy of RNA4, potential cellular RNAs, or a combination of the three, indicating that change in the charge of the capsid could result in several different outcomes in RNA encapsidation. In addition, mutant with specific arginines changed to lysines in the capsid also exhibited defects in the specific encapsidation of BMV RNA4. The experimental results indicate that electrostatics is a major component in RNA encapsidation but was unable to account for all of the observed effects on RNA encapsidation. Thermodynamic modeling incorporating the electrostatics was able to predict the approximate length of the RNA to be encapsidated for the majority of mutant virions, but not for a mutant with extreme clustered positive charges. Cryo-electron microscopy of virions that encapsidated an additional copy of RNA4 revealed that, despite the increase in RNA encapsidated, the capsid structure was minimally changed. These results experimentally demonstrated the impact of electrostatics and additional restraints in the encapsidation of BMV RNAs, which could be applicable to other viruses.     

Coordinate replication of alfalfa mosaic virus RNAs 1 and 2 involves cis- and trans-acting functions of the encoded helicase-like and polymerase-like domains

RNAs 1 and 2 of the tripartite genome of alfalfa mosaic virus encode the replicase proteins P1 and P2, respectively, whereas RNA 3 encodes the movement protein and coat protein. Transient expression of wild-type (wt) and mutant viral RNAs and proteins by agroinfiltration of plant leaves was used to study cis- and trans-acting functions of the helicase-like domain in P1 and the polymerase-like domain in P2. Three mutations in conserved motifs of the helicase-like domain of P1 affected one or more steps leading to synthesis of minus-strand RNAs 1, 2, and 3. In leaves containing transiently expressed P1 and P2, replication of wt but not mutant RNA 1 was observed. Apparently, the transiently expressed P1 could not complement the defect in replication of the RNA 1 mutant. Moreover, the transiently expressed wt replicase supported replication of RNA 2, but this replication was blocked in trans by coexpression of mutant RNA 1. However, expression of mutant RNA 1 did not interfere with the replication of RNA 3 by the wt replicase. Similarly, a mutation in the GDD motif encoded by RNA 2 could not be complemented in trans and affected the replication of RNA 1 by a wt replicase, while replication of RNA 3 remained unaffected. In competition assays, the transient wt replicase preferentially replicated RNA 3 over RNAs 1 and 2. The results indicate that one or more functions of P1 and P2 act in cis and point to the existence of a mechanism that coordinates the replication of RNAs 1 and 2.     

A physical interaction between viral replicase and capsid protein is required for genome-packaging specificity in an RNA virus

Genome packaging is functionally coupled to replication in RNA viruses pathogenic to humans (Poliovirus), insects (Flock house virus [FHV]), and plants (Brome mosaic virus [BMV]). However, the underlying mechanism is not fully understood. We have observed previously that in FHV and BMV, unlike ectopically expressed capsid protein (CP), packaging specificity results from RNA encapsidation by CP that has been translated from mRNA produced from replicating genomic RNA. Consequently, we hypothesize that a physical interaction with replicase increases the CP specificity for packaging viral RNAs. We tested this hypothesis by evaluating the molecular interaction between replicase protein and CP using a FHV-Nicotiana benthamiana system. Bimolecular fluorescence complementation in conjunction with fluorescent cellular protein markers and coimmunoprecipitation assays demonstrated that FHV replicase (protein A) and CP physically interact at the mitochondrial site of replication and that this interaction requires the N-proximal region from either amino acids 1 to 31 or amino acids 32 to 50 of the CP. In contrast to the mitochondrial localization of CP derived from FHV replication, ectopic expression displayed a characteristic punctate pattern on the endoplasmic reticulum (ER). This pattern was altered to relocalize the CP throughout the cytoplasm when the C-proximal hydrophobic domain was deleted. Analysis of the packaging phenotypes of the CP mutants defective either in protein A-CP interactions or ER localization suggested that synchronization between protein A-CP interaction and its subcellular localization is imperative to confer packaging specificity.     

Translational repression by bacteriophage MS2 coat protein expressed from a plasmid. A system for genetic analysis of a protein-RNA interaction

The coat protein of bacteriophage MS2 is a translational repressor. It inhibits the synthesis of the viral replicase by binding a specific RNA structure that contains the replicase translation initiation region. In order to begin a genetic dissection of the repressor activity of coat protein, a two-plasmid system has been constructed that expresses coat protein and a replicase-beta-galactosidase fusion protein from different, compatible plasmids containing different antibiotic-resistant determinants. The coat protein expressed from the first plasmid (pCT1) represses synthesis of a replicase-beta-galactosidase fusion protein encoded on the other plasmid (pRZ5). Mutations in the translational operator or in coat protein result in constitutive synthesis of the enzyme. This permits the straightforward isolation of mutations in the coat sequence that affect repressor function. Because of the potential importance of cysteine residues for RNA binding, mutations were constructed that substitute serines for the cysteine residues normally present at positions 46 and 101. Both of these mutations result in translational repressor defects. Chromatographic and electron microscopic analyses indicate that the plasmid-encoded wild-type coat protein forms capsids in vivo. The ability of the mutants to adopt and/or maintain the appropriate conformation was assayed by comparing them to the wild-type protein for their ability to form capsids. Both mutants exhibited evidence of improper folding and/or instability as indicated by their aberrant elution behavior on a column of Sepharose CL-4B. Methods were developed for the rapid purification of plasmid-encoded coat protein, facilitating future biochemical analyses of mutant coat proteins.     

The regulatory region of MS2 phage RNA replicase cistron. Functional activity of individual MS2 RNA fragments

The present work deals with the structural-functional organization of regulatory regions of messenger RNAs. Some principles of the action of a translational repressor (coat protein) and the formation of the ribosomal initiation complex at the replicase cistron have been studied with MS2 phage RNA. When the complex of MS2 RNA with the coat protein is treated with T₁ ribonuclease, the coat protein selectively protects mainly two fragments (59 and 103 nucleotides in length) from digestion; these fragments contain the intercistronic regulatory region and the beginning of the MS2 replicase cistron. These polynucleotides have been isolated in a pure state and their primary structure has been established.It has been established that both MS2 RNA fragments contain all the necessary information for specific interaction with MS2 coat protein and form a complex with it with an efficiency close to that observed in the case of native MS2 RNA. They also provide the normal polypeptide chain initiation at the replicase cistron. Enzymatic binding of the second aminoacyl-tRNA and electrophoretic analysis of N-terminal dipeptides prove that only the true initiator codon of the replicase cistron is recognized by a ribosome despite the presence of a few additional AUG triplets within the polynucleotides. Under conditions of limited hydrolysis by T₁ ribonuclease, the beginning of the replicase cistron has been removed from the shortest polynucleotide leading to a complete loss of its ability to bind both the coat protein and a ribosome.Some principles of the functioning of the regulatory region in MS2 RNA as well as the nature of the initiator signal of protein biosynthesis are discussed.     

RNA Recognition by the MS2 Phage Coat Protein

The interaction of MS2 coat protein and its translational operator hairpin is a very well-characterized RNA–protein complex. The recent high-resolution cocrystal structure successfully explains many biochemical experiments measuring the affinity of the protein–RNA interaction for mutant proteins and chemically modified RNAs. However, an analysis of a tight binding variant of the RNA suggests that the conformation of the free RNA is also an important determinant of the affinity.     

Sequence and organization of barley yellow dwarf virus genomic RNA.

The nucleotide sequence of the genomic RNA of barley yellow dwarf virus, PAV serotype was determined, except for the 5'-terminal base, and its genome organization deduced. The 5,677 nucleotide genome contains five large open reading frames (ORFs). The genes for the coat protein (1) and the putative viral RNA-dependent RNA polymerase were identified. The latter shows a striking degree of similarity to that of carnation mottle virus (CarMV). By comparison with corona- and retrovirus RNAs, it is proposed that a translational frameshift is involved in expression of the polymerase. An ORF encoding an Mr 49,797 protein (50K ORF) may be translated by in-frame readthrough of the coat protein stop codon. The coat protein, an overlapping 17K ORF, and a 3'6.7K ORF are likely to be expressed via subgenomic mRNAs.     

Encapsidation of turnip crinkle virus is defined by a specific packaging signal and RNA size.

A protoplast infection assay has been used to reliably examine the viral RNA encapsidation of turnip crinkle virus (TCV). Analysis of the encapsidation of various mutant viral RNAs revealed that a 186-nucleotide (nt) region at the 3' end of the coat protein (CP) gene, with a bulged hairpin loop of 28 nt as its most essential element, was indispensable for TCV RNA encapsidation. When RNA fragments containing the 186-nt region were used to replace the CP gene of a different virus, tomato bushy stunt virus, the resulting chimeric viral RNAs were encapsidated into TCV virions. Furthermore, analysis of the encapsidated chimeric RNA species established that the RNA size was an important determinant of the TCV assembly process.     

Coat protein binds to the 3'-terminal part of RNA 4 of alfalfa mosaic virus.

All four RNAs of alfalfa mosaic virus contain a limited number of sites with a high affinity for coat protein [Van Boxsel, J. A. M. (1976), Ph.D. Thesis, University of Leiden]. In order to localize these sites in the viral RNAs, RNA 4 Tthe subgenomic messenger for coat protein) was subjected to a very mild digestion with ribonucleast T1. The ten major fragments, apparently resulting from five preferential hits, were separated and tested for messenger activity in a wheat germ cell-free system, as well as for the capacity to withdraw coat protein from intact particles. Fragments which stimulated amino acid incorporation were assumed to contain the 5 terminus. Strong evidence was obtained for the location of sites with a high affinity for coat protein near the 3' terminus. The smallest fragment which has the 3'-terminal cytosine comprises only 10% of the length of intact RNA 4 but still possesses these sites. Evidence is presented that the complete coat protein cistron is in the complementing 90% fragment. Possibly, the high-affinity sites are entirely located in the 3'-terminal extracistronic part of RNA 4. They will have the same position in RNA 3 and, possibly, also in the other parts of the genome of alfalfa mosaic virus. The need of this genome for coat protein in order to become infectious may therefore find its explanation in the fact that a conformational change at the 3' ends of the genome parts brought about by the coat protein is required for recognition by the viral replicase.     

The interaction between bamboo mosaic virus replication protein and coat protein is critical for virus movement in plant hosts

Bamboo mosaic virus (BaMV) is a positive-sense RNA virus belonging to the genus Potexvirus. Open reading frame 1 (ORF1) encodes the viral replication protein that consists of a capping enzyme domain, a helicase-like domain (HLD), and an RNA-dependent RNA polymerase domain from the N to C terminus. ORF5 encodes the viral coat protein (CP) required for genome encapsidation and the virus movement in plants. In this study, application of a yeast-two hybrid assay detected an interaction between the viral HLD and CP. However, the interaction did not affect the NTPase activity of the HLD. To identify the critical amino acids of CP interacting with the HLD, a random mutational library of CP was created using error-prone PCR, and the mutations adversely affecting the interaction were screened by a bacterial two-hybrid system. As a result, the mutations A209G and N210S in CP were found to weaken the interaction. To determine the significance of the interaction, the mutations were introduced into a BaMV infectious clone, and the mutational effects on viral replication, movement, and genome encapsidation were investigated. There was no effect on accumulations of BaMV CP and genomic RNAs within protoplasts; however, the virus cell-to-cell movement in plants was restricted. Sequence alignment revealed that A209 of BaMV CP is conserved in many potexviruses. Mutation of the corresponding residue in Foxtail mosaic virus CP also reduced the viral HLD-CP interaction and restricted the virus movement, suggesting that interaction between CP and a widely conserved HLD in the potexviral replication protein is crucial for viral trafficking through plasmodesmata.     

Dissecting the key recognition features of the MS2 bacteriophage translational repression complex.

The MS2 RNA operator capsid offers an unparalleled opportunity to study sequence-specific protein-protein and RNA-protein interactions in molecular detail. RNA molecules encompassing the minimal translational operator recognition elements can be soaked into crystals of RNA-free coat protein shells, allowing the RNA to access the interior of the capsids and make contact with the operator binding sites. Correct interpretation of these structural studies depends critically on functional analysis in solution to confirm that the interactions seen in the crystal are not an artefact of the unusual approach used to generate the RNA-protein complexes. Here we present a series of in vivo and in vitro functional assays, using coat proteins carrying single amino acid substitutions at residues which either interact with the operator RNA or are involved in stabilizing the conformation of the FG loop, the site of the major quasi-equivalent conformational change. Variant operator RNAs have been assayed for coat protein affinity in vitro. The results reveal the robustness of the operator-coat protein interaction and the requirement for both halves of a protein dimer to contact RNA in order to achieve tight binding. They also suggest that there may be a direct link between the conformation of the FG loop and RNA binding.     

An RNA mutation that increases the affinity of an RNA-protein interaction.

The introduction of a cytidine in place of one of the two single stranded uridines in the R17 replicase translational operator results in a much tighter binding to R17 coat protein. The complex containing the variant RNA is stable to gel electrophoresis and has a binding constant about 50 times greater than the one with wild type RNA. The nearly thirty percent increase in the free energy of binding for the variant RNA is primarily due to a more favorable enthalpy of interaction. A possible explanation for this surprising result is that the U to C change leads to a greater extent of formation of a transient covalent complex between the protein and the RNA.     

Mutations that increase the affinity of a translational repressor for RNA.

The coat protein of the RNA bacteriophage MS2 is a specific RNA binding protein that represses translation of the viral replicase gene during the infection cycle. As an approach to characterizing the RNA-binding site of coat protein we have isolated a series of coat mutants that suppress the effects of a mutation in the translational operator. Each of the mutants exhibits a super-repressor phenotype, more tightly repressing both the mutant and wild-type operators than does the wild-type protein. The variant coat proteins were purified and subjected to filter binding assays to determine their affinities for the mutant and wild-type operators. Each protein binds the operators from 3 to 7.5-fold more tightly than normal coat protein. The amino acid substitutions seem to extend the normal binding site by introducing new interactions with RNA.     

Translational control by a long range RNA-RNA interaction; a basepair substitution analysis.

One of the two mechanisms that regulate expression of the replicase cistron in the single stranded RNA coliphages is translational coupling. This mechanism prevents ribosomes from binding at the start of the replicase cistron unless the upstream coat cistron is being translated. Genetic analysis had identified a maximal region of 132 nucleotides in the coat gene over which ribosomes should pass to activate the replicase start. Subsequent deletion studies in our laboratory had further narrowed down the regulatory region to 12 nucleotides. Here, we identify a long-distance RNA-RNA interaction of 6 base pairs as the basis of the translational polarity. The 3' side of the complementarity region is located in the coat-replicase intercistronic region, some 20 nucleotides before the start codon of the replicase. The 5' side encodes amino acids 31 and 32 of the coat protein. Mutations that disrupt the long-range interaction abolish the translational coupling. Repair of basepairing by second site base substitutions restores translational coupling.     

Specific RNA binding by amino-terminal peptides of alfalfa mosaic virus coat protein.

Specific RNA-protein interactions and ribonucleoprotein complexes are essential for many biological processes, but our understanding of how ribonucleoprotein particles form and accomplish their biological functions is rudimentary. This paper describes the interaction of alfalfa mosaic virus (A1MV) coat protein or peptides with viral RNA. A1MV coat protein is necessary both for virus particle formation and for the initiation of replication of the three genomic RNAs. We have examined protein determinants required for specific RNA binding and analyzed potential structural changes elicited by complex formation. The results indicate that the amino-terminus of the viral coat protein, which lacks primary sequence homology with recognized RNA binding motifs, is both necessary and sufficient for binding to RNA. Circular dichroism spectra and electrophoretic mobility shift experiments suggest that the RNA conformation is altered when amino-terminal coat protein peptides bind to the viral RNA. The peptide--RNA interaction is functionally significant because the peptides will substitute for A1MV coat protein in initiating RNA replication. The apparent conformational change that accompanies RNA--peptide complex formation may generate a structure which, unlike the viral RNA alone, can be recognized by the viral replicase.