Influence of adaptive mutations, from thermal adaptation experiments, on the infection cycle of RNA bacteriophage Qß.

A population's growth rate is determined by multiple 'life history traits'. To quantitatively determine which life history traits should be improved to allow a living organism to adapt to an inhibitory environment is an important issue. Previously, we conducted thermal adaptation experiments on the RNA bacteriophage Qß using three independent replicates and reported that all three end-point populations could grow at a temperature (43.6°C) that inhibited the growth of the ancestral strain. Even though the fitness values of the endpoint populations were almost the same, their genome sequence was not, indicating that the three thermally adapted populations may have different life history traits. In this study, we introduced each mutation observed in these three end-point populations into the cDNA of the Qß genome and prepared three different mutants. Quantitative analysis showed that they tended to increase their fitness by increasing the adsorption rate to their host, shortening their latent period (i.e., the duration between phage infection and progeny release), and increasing the burst size (i.e., the number of progeny phages per infected cell), but all three mutants decreased their thermal stability. However, the degree to which these traits changed differed. The mutant with the least mutations showed a smaller decrease in thermal stability, the largest adsorption rate to the host, and the shortest latent period. These results indicated that several different adaptive routes exist by which Qß can adapt to higher temperatures, even though Qß is a simple RNA bacteriophage with a small genome size, encoding only four genes.

Protein-RNA Interactions in the Single-Stranded RNA Bacteriophages.

Bacteriophages of the Leviviridae family are small viruses with short single-stranded RNA (ssRNA) genomes. Protein-RNA interactions play a key role throughout the phage life cycle, and all of the conserved phage proteins - the maturation protein, the coat protein and the replicase - are able to recognize specific structures in the RNA genome. The phage-coded replicase subunit associates with several host proteins to form a catalytically active complex. Recognition of the genomic RNA by the replicase complex is achieved in a remarkably complex manner that exploits the RNA-binding properties of host proteins and the particular three-dimensional structure of the phage genome. The coat protein recognizes a hairpin structure at the beginning of the replicase gene. The binding interaction serves to regulate the expression of the replicase gene and can be remarkably different in various ssRNA phages. The maturation protein is a minor structural component of the virion that binds to the genome, mediates attachment to the host and guides the genome into the cell. The maturation protein has two distinct RNA-binding surfaces that are in contact with different regions of the genome. The maturation and coat proteins also work together to ensure the encapsidation of the phage genome in new virus particles. In this chapter, the different ssRNA phage protein-RNA interactions, as well as some of their practical applications, are discussed in detail.

Crystal Structure of the Maturation Protein from Bacteriophage Qß.

Virions of the single-stranded RNA bacteriophages contain a single copy of the maturation protein, which is bound to the phage genome and is required for the infectivity of the particles. The maturation protein mediates the adsorption of the virion to bacterial pili and the subsequent release and penetration of the genome into the host cell. Here, we report a crystal structure of the maturation protein from bacteriophage Qß. The protein has a bent, highly asymmetric shape and spans 110Å in length. Apart from small local substructures, the overall fold of the maturation protein does not resemble that of other known proteins. The protein is organized in two distinct regions, an α-helical part with a four-helix core, and a ß stranded part that contains a seven-stranded sheet in the central part and a five-stranded sheet at the tip of the protein. The Qß maturation protein has two distinct, positively charged areas at opposite sides of the α-helical part, which are involved in genomic RNA binding. The maturation protein binds to each of the surrounding coat protein dimers in the capsid differently, and the interaction is considerably weaker compared to coat protein interdimer contacts. The coat protein- or RNA-binding residues are not preserved among different ssRNA phage maturation proteins; instead, the distal end of the α-helical part is the most evolutionarily conserved, suggesting the importance of this region for maintaining the functionality of the protein.

The capsid of the small RNA phage PRR1 is stabilized by metal ions.

Many nonenveloped virus particles are stabilized by calcium ions bound in the interfaces between the protein subunits. These ions may have a role in the disassembly process. The small RNA phages of the Leviviridae family have T=3 quasi-symmetry and are unique among simple viruses in that they have a coat protein with a translational repressor activity and a fold that has not been observed in other viruses. The crystal structure of phage PRR1 has been determined to 3.5 A resolution. The structure shows a tentative binding site for a calcium ion close to the quasi-3-fold axis. The RNA-binding surface used for repressor activity is mostly conserved. The structure does not show any significant differences between quasi-equivalent subunits, which suggests that the assembly is not controlled by conformational switches as in many other simple viruses.

Aggregation and surface properties of F-specific RNA phages: implication for membrane filtration processes.

We report an experimental investigation of the electrokinetic properties and size variations of four F-specific bacteriophages of the types MS2, GA, Qbeta and SP (21-30 nm in diameter) over a broad range of pH values (1.5-7.5) and NaNO3 electrolyte concentrations (1-100 mM). The results obtained by dynamic light scattering show that the aggregation of SP and GA particles takes place over the whole range of pH and ionic strength conditions examined. For MS2 phages, the aggregation of MS2 particles is not observed for pH higher than the isoelectric point (pI) and large ionic strengths for which interparticular repulsive electrostatic interactions are however expected to be sufficiently screened. Aggregation of the MS2 phages, dispersed in 1 and 100 mM electrolyte concentration, occurs at pH 4, which basically corresponds to the pI as determined by electrophoresis measurements. The Qbeta particles suspended in solutions of low electrolyte concentrations aggregate at low pH values (pI approximately 3) and, unlike MS2, at large ionic strengths over the whole range of pH conditions considered in this study. These elements allow the determination of the hydrophobic sequence for the four phages SP approximately GA>Qbeta>MS2. Close inspection of the electrokinetic results reveals small to significant variations of the pI values-depending on the phage considered-with respect to the concentration of indifferent NaNO3 electrolyte. This indicates that features other than chemical and electrostatic in nature play a key role in determining the pI and more generally the electrophoretic mobility mu of viral particles. A qualitative interpretation is given and is based on the consideration of inner electro-osmotic flow within the isolated or aggregated particles. The impact of the flow properties within the particles is further in agreement with recent theoretical formalism developed for the electrokinetics of soft multiplayer particles, the phages analyzed here being some illustrative examples. The determination and qualitative interpretation of the surface properties of the viral particles as reported in the current study are commented within the context of water treatment especially concerning viral removal by membrane filtration processes.

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 approximately 9 A resolution reveals icosahedrally ordered density encompassing approximately 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.

Membrane-containing viruses with icosahedrally symmetric capsids.

Viruses with an icosahedrally symmetric protein capsid and a membrane infect hosts from all three domains of life. Similar architectural principles are shared by different viral families, as exemplified by double-stranded DNA viruses such as PRD1 and STIV. During virus assembly, the membrane lipids are selectively acquired from the host cell. The X-ray structure of bacteriophage PRD1 revealed that the lipids are asymmetrically distributed between the two leaflets and facet length is controlled by a tape-measure protein. In most membrane-containing viruses, viral and host membranes fuse during viral entry. In the best-understood systems of the alphaviruses, flaviviruses and herpes viruses, fusion is mediated by viral glycoproteins. Recent structural advances reveal how very different protein architectures can be used to form trimeric extensions that extend into the target cell membrane and then fold back to mediate fusion of the target and viral membranes.

Structural constraints and mutational bias in the evolutionary restoration of a severe deletion in RNA phage MS2.

A 4-nucleotide (nt) deletion was made in the 36-nt-long intercistronic region separating the coat and replicase genes of the single-stranded RNA phage MS2. This region is the focus of several RNA structures conferring high fitness. One such element is the operator hairpin, which, in the course of infection, will bind a coat-protein dimer, thereby precluding further replicase synthesis and initiating encapsidation. Another structure is a long-distance base pairing (MJ) controlling replicase expression. The 4-nt deletion does not directly affect the operator hairpin but it disrupts the MJ pairing. Its main effect, however, is a frame shift in the overlapping lysis gene. This gene starts in the upstream coat gene, runs through the 36-nt-long intercistronic region, and ends in the downstream replicase cistron. Here we report and interpret the spectrum of solutions that emerges when the crippled phage is evolved. Four different solutions were obtained by sequencing 40 plaques. Three had cured the frame shift in the lysis gene by inserting one nt in the loop of the operator hairpin causing its inactivation. Yet these low-fitness revertants could further improve themselves when evolved. The inactivated operator was replaced by a substitute and thereafter these revertants found several ways to restore control over the replicase gene. To allow for the evolutionary enrichment of low-probability but high-fitness revertants, we passaged lysate samples before plating. Revertants obtained in this way also restored the frame shift, but not at the expense of the operator. By taking larger and larger lysates samples for such bulk evolution, ever higher-fitness and lower-frequency revertants surfaced. Only one made it back to wild type. As a rule, however, revertants moved further and further away from the wild-type sequence because restorative mutations are, in the majority of cases, selected for their capacity to improve the phenotype by optimizing one of several potential alternative RNA foldings that emerge as a result of the initial deletion. This illustrates the role of structural constraints which limit the path of subsequent restorative mutations.

Cooperative mechanism of RNA packaging motor.

P4 is a hexameric ATPase that serves as the RNA packaging motor in double-stranded RNA bacteriophages from the Cystoviridae family. P4 shares sequence and structural similarities with hexameric helicases. A structure-based mechanism for mechano-chemical coupling has recently been proposed for P4 from bacteriophage phi12. However, coordination of ATP hydrolysis among the subunits and coupling with RNA translocation remains elusive. Here we present detailed kinetic study of nucleotide binding, hydrolysis, and product release by phi12 P4 in the presence of different RNA and DNA substrates. Whereas binding affinities for ATP and ADP are not affected by RNA binding, the hydrolysis step is accelerated and the apparent cooperativity is increased. No nucleotide binding cooperativity is observed. We propose a stochastic-sequential cooperativity model to describe the coordination of ATP hydrolysis within the hexamer. In this model the apparent cooperativity is a result of hydrolysis stimulation by ATP and RNA binding to neighboring subunits rather than cooperative nucleotide binding. The translocation step appears coupled to hydrolysis, which is coordinated among three neighboring subunits. Simultaneous interaction of neighboring subunits with RNA makes the otherwise random hydrolysis sequential and processive.

Resolution of the 1H-1H NOE spectrum of RNA into three dimensions using 15N-1H two-bond couplings.

The feasibility of using two-bond 15N-1H couplings to resolve the 1H-1H nuclear Overhauser effect spectrum of RNA into a third dimension was investigated, using the 36-nucleotide gene 32 messenger RNA pseudoknot of bacteriophage T2 as an example. The two-bond 15N-1H couplings present in adenosine and guanosine were found to be suitable for generating a three-dimensional 1H-1H-15N NOESY-HSQC spectrum with reasonably good sensitivity, as well as favorable chemical shift dispersion in the nitrogen dimension. The described NMR experiment provides a tool that can be used to complement other heteronuclear methods in the analysis of RNA structure.

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.

The crystal structure of bacteriophage GA and a comparison of bacteriophages belonging to the major groups of Escherichia coli leviviruses.

The three-dimensional structure of the small T=3 RNA bacteriophage GA has been determined at 3.4 A resolution. The structure was solved by molecular replacement, using the phage MS2 as an initial model. A comparison of the protein shells of the four related phages GA, MS2, fr and Qbeta was carried out in order to define structural features of particular importance for their assembly and specific RNA interaction. A high degree of similarity was found in the RNA binding sites, whereas larger structural differences are located in the loop regions of the coat proteins, especially in the FG loops forming 5-fold and quasi-6-fold contacts. The overall arrangement of the protein subunits in the shells of these phages is very similar, although the details of the interactions differ. The few conserved interactions are suggested to govern the subunit packing during assembly.

[Thermal denaturation of molecular complexes formed by single stranded RNA with tilorone]. / Temperaturna denaturatsiia molekuliarnykh kompleksiv odnolantsiugovoï RNK tyloronom.

The study of thermal denaturation for molecular complexes formed during interaction between phage MS2 single-stranded RNA and tilorone has been carried out. Both certain increase of Tm (by on 1.1) and the narrowing of complexes melting interval, delta T, (by 5.6 degrees C), have been detected. The value of hyperchromic effect, H, increases twice. The conclusion is made concerning the formation of double-stranded regions in single-stranded RNA molecules in the process of RNA--tilorone interactions. These results are discussed as an additional proof of our previous presumption concerning stabilization of spontaneously forming double-stranded RNA regions by tilorone binding.

Proton NMR and structural features of a 24-nucleotide RNA hairpin.

The three-dimensional conformation of a 24-nucleotide variant of the RNA binding sequence for the coat protein of bacteriophage R17 has been analyzed using NMR, molecular dynamics, and energy minimization. The imino proton spectrum is consistent with base pairing requirements for coat protein binding known from biochemical studies. All 185 of the nonexchangeable protons were assigned using a variety of homonuclear 2D and 3D NMR methods. Measurements of nuclear Overhauser enhancements and two-quantum correlations were made at 500 MHz. New procedures were developed to characterize as many resonances as possible, including deconvolution and path analysis methods. An average of 21 distance constraints per residue were used in molecular dynamics calculations to obtain preliminary folded structures for residues 3-21. The unpaired A8 residue is stacked in the stem, and the entire region from G7 to C15 in the upper stem and loop appears to be flexible. Several of these residues have a large fraction of S-puckered ribose rings, rather than the N-forms characteristic of RNA duplexes. There is considerable variation in the low-energy loop conformations that satisfy the distance constraints at this preliminary level of refinement. The Shine-Dalgarno ribosome binding site is exposed, and only two apparently weak base pairs would have to break for the 16S ribosomal RNA to bind and the ribosome to initiate translation of the replicase gene. Although the loop form must be regarded as tentative, the known interaction sites with the coat protein are easily accessible from the major groove side of the loop.

Secondary structure model for the last two domains of single-stranded RNA phage Q beta.

We have determined the nucleotide sequence of three positive single-stranded RNA coliphages and have used this information, together with the known sequences of the related phages Q beta and SP, to construct a secondary structure model for the two distal domains of Q beta RNA. The 3' terminal domain, which is about 100 nucleotides long, contains most of the 3' untranslated region and folds into four short, regular hairpins. The adjacent 3' replicase domain contains about 1100 nucleotides. Hairpins in this protein-coding domain are much longer and more irregular than in the 3' untranslated region. Both domains are defined by long-distance interactions. The secondary structure is not a collection of hairpin structures connected by single-stranded regions. Rather, the RNA stretches between the stem-loop structures are all involved in an extensive array of long-distance interactions that contract the molecule to a rigid structure in which all hairpins are predicted to have a fixed position with respect to each other. A general feature of the model is that helices tend to be organized in four-way junctions with little or no unpaired nucleotides between them. As a result, there is a potential for coaxial stacking of adjacent stems. The essential features of the model are supported by the S1 nuclease cleavage pattern. Viral RNA sequences are strongly constrained by their coding function. As a result, structural evolution by simple base-pair substitution is not always possible, as this usually requires the juxtaposition of the codon wobble positions in stems. Rather, we often observe co-ordinate base substitutions that maintain the stem, but tend to change the position at which bulges or internal loops are found. Structures that differ in this way are apparently equally fit. Also, the relative position of hairpin loops can shift several nucleotides through an alignment based on maximal sequence identity i.e. amino acid homology. The fact that these structural irregularities do not occur at the 3' untranslated region suggests indeed that the coding function of the RNA constrains the secondary structure. Hairpins with the stable tetraloop motif GNRA and UNCG or their complement are over-represented. This suggests their involvement in segregation of plus and minus strand. The genome of the coliphages contains a well-defined high affinity binding site for the coat protein, which serves to suppress replicase translation and also acts as a nucleation point in capsid formation. Close to the 3' end we find an additional conserved helix that meets the described consensus criteria for coat-protein binding.

Crystal structure of the MS2 coat protein dimer: implications for RNA binding and virus assembly.

BACKGROUND: The coat protein in RNA bacteriophages binds and encapsidates viral RNA, and also acts as translational repressor of viral replicase by binding to an RNA hairpin in the RNA genome. Because of its dual function, the MS2 coat protein is an interesting candidate for structural studies of protein-RNA interactions and protein-protein interactions. In this study, unassembled MS2 coat protein dimers were selected to analyze repressor activity and virus assembly. RESULTS: The crystal structure of a mutant MS2 coat protein that is defective in viral assembly yet retains repressor activity has been determined at 2.0 A resolution. The unassembled dimer is stabilized by interdigitation of alpha-helices, and the formation of a 10-stranded antiparallel beta-sheet across the interface between monomers. The substitution of arginine for tryptophan at residue 82 results in the formation of two new inter-subunit hydrogen bonds that further stabilize the dimer. Residues that influence RNA recognition, identified by molecular genetics, were located across the beta-sheet. Two of these residues (Tyr85 and Asn87) are displaced in the unliganded dimer and are located in the same beta-strand as the Trp-->Arg mutation. CONCLUSIONS: When compared with the structure of the coat protein in the assembled virus, differences in orientation of residues 85 and 87 suggest conformational adjustment on binding RNA in the first step of viral assembly. The substitution at residue 82 may affect virus assembly by imposing conformational restriction on the loop that makes critical inter-subunit contacts in the capsid.

Crystal structure of bacteriophage fr capsids at 3.5 A resolution.

The structure of recombinant capsids of the bacterial virus fr has been determined by X-ray crystallography at 3.5 A resolution. The capsids were produced by expressing the fr coat protein in Escherichia coli, the natural host of the virus, and are probably essentially identical to the protein shell of the native virus. The structure was determined using molecular replacement with the protein shell of the related MS2 virus, and refined to a crystallographic R-factor of 0.228. A comparison of the protein shells of the viruses shows that they are very similar, and indicates that they may have a similar regulation of the assembly of the quasi-symmetrical protein shell.

NTP binding induces conformational changes in the double-stranded RNA bacteriophage ø6 subviral particles.

Bacteriophage ø6 is a double-stranded RNA virus consisting of a nucleocapsid (NC) surrounded by a membrane. Beneath the NC major coat protein, P8, resides the ø6 RNA polymerase complex which is composed of four early proteins P1, P2, P4, and P7. Protein P1 forms the dodecahedral framework with which the other three proteins are associated. We have developed a new method for the isolation of stable polymerase complex particles which retain their structural integrity and polymerase activity for several days. Purine nucleotides, especially GTP, dGTP, ddGTP, and GDP, stabilized the particle efficiently. Furthermore, binding of any NTP was shown to induce conformational changes in the NC structure, as detected by alterations in the binding properties of NC-specific monoclonal antibodies. In the presence of NTPs, most of the epitopes in protein P4 become more exposed than without NTPs, while the epitopes in protein P8 were either masked or unmasked due to NTP binding. Based on the accessibility of the epitopes of protein P1 on the NC, we postulate that at least part of this protein is also accessible on the NC surface.