Impact of increased mutagenesis on adaptation to high temperature in bacteriophage Qß.

RNA viruses replicate with very high error rates, which makes them more sensitive to additional increases in this parameter. This fact has inspired an antiviral strategy named lethal mutagenesis, which is based on the artificial increase of the error rate above a threshold incompatible with virus infectivity. A relevant issue concerning lethal mutagenesis is whether incomplete treatments might enhance the adaptive possibilities of viruses. We have addressed this question by subjecting an RNA virus, the bacteriophage Qß, to different transmission regimes in the presence or the absence of sublethal concentrations of the mutagenic nucleoside analogue 5-azacytidine (AZC). Populations obtained were subsequently exposed to a non-optimal temperature and analyzed to determine their consensus sequences. Our results show that previously mutagenized populations rapidly fixed a specific set of mutations upon propagation at the new temperature, suggesting that the expansion of the mutant spectrum caused by AZC has an influence on later evolutionary behavior.

Structural basis for RNA-genome recognition during bacteriophage Qß replication.

Upon infection of Escherichia coli by bacteriophage Qß, the virus-encoded ß-subunit recruits host translation elongation factors EF-Tu and EF-Ts and ribosomal protein S1 to form the Qß replicase holoenzyme complex, which is responsible for amplifying the Qß (+)-RNA genome. Here, we use X-ray crystallography, NMR spectroscopy, as well as sequence conservation, surface electrostatic potential and mutational analyses to decipher the roles of the ß-subunit and the first two oligonucleotide-oligosaccharide-binding domains of S1 (OB1-2) in the recognition of Qß (+)-RNA by the Qß replicase complex. We show how three basic residues of the ß subunit form a patch located adjacent to the OB2 domain, and use NMR spectroscopy to demonstrate for the first time that OB2 is able to interact with RNA. Neutralization of the basic residues by mutagenesis results in a loss of both the phage infectivity in vivo and the ability of Qß replicase to amplify the genomic RNA in vitro. In contrast, replication of smaller replicable RNAs is not affected. Taken together, our data suggest that the ß-subunit and protein S1 cooperatively bind the (+)-stranded Qß genome during replication initiation and provide a foundation for understanding template discrimination during replication initiation.

Changes in protein domains outside the catalytic site of the bacteriophage Qß replicase reduce the mutagenic effect of 5-azacytidine.

UNLABELLED: The high genetic heterogeneity and great adaptability of RNA viruses are ultimately caused by the low replication fidelity of their polymerases. However, single amino acid substitutions that modify replication fidelity can evolve in response to mutagenic treatments with nucleoside analogues. Here, we investigated how two independent mutants of the bacteriophage Qß replicase (Thr210Ala and Tyr410His) reduce sensitivity to the nucleoside analogue 5-azacytidine (AZC). Despite being located outside the catalytic site, both mutants reduced the mutation frequency in the presence of the drug. However, they did not modify the type of AZC-induced substitutions, which was mediated mainly by ambiguous base pairing of the analogue with purines. Furthermore, the Thr210Ala and Tyr410His substitutions had little or no effect on replication fidelity in untreated viruses. Also, both substitutions were costly in the absence of AZC or when the action of the drug was suppressed by adding an excess of natural pyrimidines (uridine or cytosine). Overall, the phenotypic properties of these two mutants were highly convergent, despite the mutations being located in different domains of the Qß replicase. This suggests that treatment with a given nucleoside analogue tends to select for a unique functional response in the viral replicase. IMPORTANCE: In the last years, artificial increase of the replication error rate has been proposed as an antiviral therapy. In this study, we investigated the mechanisms by which two substitutions in the Qß replicase confer partial resistance to the mutagenic nucleoside analogue AZC. As opposed to previous work with animal viruses, where different mutations selected sequentially conferred nucleoside analogue resistance through different mechanisms, our results suggest that there are few or no alternative AZC resistance phenotypes in Qß. Also, despite resistance mutations being highly costly in the absence of the drug, there was no sequential fixation of secondary mutations. Bacteriophage Qß is the virus with the highest reported mutation rate, which should make it particularly sensitive to nucleoside analogue treatments, probably favoring resistance mutations even if they incur high costs. The results are also relevant for understanding the possible pathways by which fidelity of the replication machinery can be modified.

Experimental selection reveals a trade-off between fecundity and lifespan in the coliphage Qß.

Understanding virus evolution is key for improving ways to counteract virus-borne diseases. Results from comparative analyses have previously suggested a trade-off between fecundity and lifespan for viruses that infect the bacterium Escherichia coli (i.e. for coliphages), which, if confirmed, would define a particular constraint on the evolution of virus fecundity. Here, the occurrence of such a trade-off is investigated through a selection experiment using the coliphage Qß. Selection was applied for increased fecundity in three independent wild-type Qß populations, and the ability of the virions to remain viable outside the host was determined. The Qß life-history traits involved in the evolution of fecundity and the genetic changes associated with this evolution were also investigated. The results reveal that short-term evolution of increased fecundity in Qß was associated with decreased viability of phage virions. This trade-off apparently arose because fecundity increased at the expense of reducing the amount of resources (mainly time) invested per produced virion. Thus, the results also indicate that Qß fecundity may be enhanced through increases in the rates of adsorption to the host and progeny production. Finally, genomic sequencing of the evolved populations pinpointed sequences likely to be involved in the evolution of Qß fecundity.

Involvement of type I and type II mechanisms on the photoinactivation of non-enveloped DNA and RNA bacteriophages.

Microbial photodynamic inactivation (PDI), involving the use of a photosensitizer (PS), light and molecular oxygen, with the subsequent production of reactive oxygen species (ROS), has been considered a promising and effective technology for viral inactivation. Although singlet oxygen is generally accepted as the main damaging species in PDI, ROS like free radicals may also be involved in the process, inducing damages to proteins, lipids, nucleic acids and other molecular structures. In this study, the relative importance of each mechanism (type I and type II) on the photoinactivation of non-enveloped DNA (T4-like phage) and RNA (Qß phage) viruses was evaluated. For this purpose, two cationic porphyrins (Tri-Py(+)-Me-PF and Tetra-Py(+)-Me) and four different ROS scavengers were used. The scavenging effect of sodium azide and L-histidine (singlet oxygen quenchers) and of D-mannitol and L-cysteine (free radical scavengers) was assessed by exposure of both phages (T4-like and Qß) to each cationic porphyrin (5.0µM for T4-like phage and 0.5µM for Qß phage) and white light (40Wm(-2)) in the presence of different concentrations of the scavengers (5, 10, 50 and 100mM). Sodium azide and L-histidine gave the best protection, reducing the phototoxic effect of Tri-Py(+)-Me-PF on T4-like phage respectively by 80% and 72% and in the presence of Tetra-Py(+)-Me by 90% and 78%. Free radical scavengers D-mannitol and L-cysteine did not significantly reduce the rate of T4-like phage photoinactivation (around 20% protection, for both PS). The sodium azide protection on Qß phage photoinactivation, in the presence of Tri-Py(+)-Me-PF, was lower (39%) when compared with T4-like phage. D-mannitol did not exert on Qß phage any protective effect after 90min of irradiation. The effect of the simultaneous presence of singlet oxygen and free radicals scavengers at 100mM confirmed that singlet oxygen (type II mechanism) is clearly the main ROS involved in T4-like and Qß phages photoinactivation by these two cationic PS. As RNA-type phages are more easily photoinactivated when compared with DNA-type ones, the protection conferred by the scavengers during the PDI process is lower and this should be taken into account when the main mechanism involved in PDI of different viruses is to be studied.

A(2) expression and assembly regulates lysis in Qß infections.

The capsids of ssRNA phages comprise a single copy of an ~45 kDa maturation protein that serves to recognize the conjugative pilus as receptor, to protect the ends of the viral RNA and also to escort the genomic RNA into the host cytoplasm. In the Alloleviviridae, represented by the canonical phage Qß, the maturation protein A(2) also causes lysis. This is achieved by inhibiting the activity of MurA, which catalyses the first committed step of murein biosynthesis. Previously, it was shown that Qß virions, with a single copy of A(2), inhibit MurA activity. This led to a model for lysis timing in which, during phage infection, A(2) is not active as a MurA inhibitor until assembled into virion particles, thus preventing premature lysis before a sufficient yield of viable progeny has accumulated. Here we report that MurA inactivates purified Qß particles, casting doubt on the notion that A(2) must assemble into particles prior to MurA inhibition. Furthermore, quantification of A(2) protein induced from a plasmid indicated that lysis is entrained when the amount of the lysis protein is approximately equimolar to that of cellular MurA. Qß por mutants, isolated as suppressors that overcome a murA(rat) mutation that reduces the affinity of MurA for A(2), were shown to be missense mutations in A(2) that increase the translation of the maturation protein. Because of the increased production of A(2), the por mutants have an attenuated infection cycle and reduced burst size, indicating that a delicate balance between assembled and unassembled A(2) levels regulates lysis timing.

Viral genomic single-stranded RNA directs the pathway toward a T=3 capsid.

The molecular mechanisms controlling genome packaging by single-stranded RNA viruses are still largely unknown. It is necessary in most cases for the protein to adopt different conformations at different positions on the capsid lattice in order to form a viral capsid from multiple copies of a single protein. We showed previously that such quasi-equivalent conformers of RNA bacteriophage MS2 coat protein dimers (CP(2)) can be switched by sequence-specific interaction with a short RNA stem-loop (TR) that occurs only once in the wild-type phage genome. In principle, multiple switching events are required to generate the phage T=3 capsid. We have therefore investigated the sequence dependency of this event using two RNA aptamer sequences selected to bind the phage coat protein and an analogous packaging signal from phage Qbeta known to be discriminated against by MS2 coat protein both in vivo and in vitro. All three non-cognate stem-loops support T=3 shell formation, but none shows the kinetic-trapping effect seen when TR is mixed with equimolar CP(2). We show that this reflects the fact that they are poor ligands compared with TR, failing to saturate the coat protein under the assay conditions, ensuring that sufficient amounts of both types of dimer required for efficient assembly are present in these reactions. Increasing the non-cognate RNA concentration restores the kinetic trap, confirming this interpretation. We have also assessed the effects of extending the TR stem-loop at the 5' or 3' end with short genomic sequences. These longer RNAs all show evidence of the kinetic trap, reflecting the fact that they all contain the TR sequence and are more efficient at promoting capsid formation than TR. Mass spectrometry has shown that at least two pathways toward the T=3 shell occur in TR-induced assembly reactions: one via formation of a 3-fold axis and another that creates an extended 5-fold complex. The longer genomic RNAs suppress the 5-fold pathway, presumably as a consequence of steric clashes between multiply bound RNAs. Reversing the orientation of the extension sequences with respect to the TR stem-loop produces RNAs that are poor assembly initiators. The data support the idea that RNA-induced protein conformer switching occurs throughout assembly of the T=3 shell and show that both positional and sequence-specific effects outside the TR stem-loop can have significant impacts on the precise assembly pathway followed.

Quantitative analysis of the bacteriophage Qbeta infection cycle.

In this study, the infection cycle of bacteriophage Qbeta was investigated. Adsorption of bacteriophage Qbeta to Escherichia coli is explained in terms of a collision reaction, the rate constant of which was estimated to be 4x10(-10) ml/cells/min. In infected cells, approximately 130 molecules of beta-subunit and 2x10(5) molecules of coat protein were translated in 15 min. Replication of Qbeta RNA proceeded in 2 steps-an exponential phase until 20 min and a non-exponential phase after 30 min. Prior to the burst of infected cells, phage RNAs and coat proteins accumulated in the cells at an average of up to 2300 molecules and 5x10(5) molecules, respectively. An average of 90 infectious phage particles per infected cell was released during a single infection cycle up to 105 min.

A cryptic lysis gene near the start of the Qbeta replicase gene in the +1 frame.

The maturation/lysis (A2) protein encoded by the group B single-stranded RNA bacteriophage Qbeta mediates lysis of host Escherichia coli cells. We found a frameshift mutation in the replicase (beta-subunit) gene of Qbeta cDNA causes cell lysis. The mutant has a single base deletion 73 nucleotides (nt) 3' from the start of the replicase gene with consequent translation termination at a stop codon 129-131 nt further 3'. The 43-amino acid C-terminal part of the 67-amino acid product encoded by what in WT (wild-type) is the +1 frame, is rich in basic amino acids This 67-aa protein can mediate cell lysis whose characteristics indicate that the protein may cause lysis by a different mechanism and via a different target, than that caused by the A2 maturation/lysis protein. Synthesis of a counterpart of the newly discovered lysis product in wild-type phage infection would require a hypothetical ribosomal frameshifting event. The lysis gene of group A RNA phages is also short, 75 codons in MS2, and partially overlaps the first part of their equivalently located replicase gene, raising significant evolutionary implications for the present finding.

Evolution of bacteriophage in continuous culture: a model system to test antiviral gene therapies for the emergence of phage escape mutants.

The emergence of viral escape mutants is usually a highly undesirable phenomenon. This phenomenon is frequently observed in antiviral drug applications for the treatment of viral infections and can undermine long-term therapeutic success. Here, we propose a strategy for evaluating a given antiviral approach in terms of its potential to provoke the appearance of resistant virus mutants. By use of Q beta RNA phage as a model system, the effect of an antiviral gene therapy, i.e., a virus-specific repressor protein expressed by a recombinant Escherichia coli host, was studied over the course of more than 100 generations. In 13 experiments carried out in parallel, 12 phage populations became resistant and 1 became extinct. Sequence analysis revealed that only two distinct phage mutants emerged in the 12 surviving phage populations. For both escape mutants, sequence variations located in the repressor binding site of the viral genomic RNA, which decrease affinity for the repressor protein, conferred resistance to translational repression. The results clearly suggest the feasibility of the proposed strategy for the evaluation of antiviral approaches in terms of their potential to allow resistant mutants to appear. In addition, the strategy proved to be a valuable tool for observing virus-specific molecular targets under the impact of antiviral drugs.

Functional replacement of the Escherichia coli hfq gene by the homologue of Pseudomonas aeruginosa.

The 102 aa Hfq protein of Escherichia coli (Hfq(Ec)) was first described as a host factor required for phage Qbeta replication. More recently, Hfq was shown to affect the stability of several E. coli mRNAs, including ompA mRNA, where it interferes with ribosome binding, which in turn results in rapid degradation of the transcript. In contrast, Hfq is also required for efficient translation of the E. coli and Salmonella typhimurium rpoS gene, encoding the stationary sigma factor. In this study, the authors have isolated and characterized the Hfq homologue of Pseudomonas aeruginosa (Hfq(Pa)), which consists of only 82 aa. The 68 N-terminal amino acids of Hfq(Pa) show 92% identity with Hfq(Ec). Hfq(Pa) was shown to functionally replace Hfq(Ec) in terms of its requirement for phage Qbeta replication and for rpoS expression. In addition, Hfq(Pa) exerted the same negative effect on E. coli ompA mRNA expression. As judged by proteome analysis, the expression of either the plasmid-borne hfq(Pa) or the hfq(Ec) gene in an E. coli Hfq(-) RpoS(-) strain revealed no gross difference in the protein profile. Both Hfq(Ec) and Hfq(Pa) affected the synthesis of approximately 26 RpoS-independent E. coli gene products. These studies showed that the functional domain of Hfq resides within its N-terminal domain. The observation that a C-terminally truncated Hfq(Ec) lacking the last 27 aa [Hfq(Ec(75))] can also functionally replace the full-length E. coli protein lends further support to this notion.

Phylogeny, genome evolution, and host specificity of single-stranded RNA bacteriophage (family Leviviridae).

Bacteriophage of the family Leviviridae have played an important role in molecular biology where representative species, such as Q beta and MS2, have been studied as model systems for replication, translation, and the role of secondary structure in gene regulation. Using nucleotide sequences from the coat and replicase genes we present the first statistical estimate of phylogeny for the family Leviviridae using maximum-likelihood and Bayesian estimation. Our analyses reveal that the coliphage species are a monophyletic group consisting of two clades representing the genera Levivirus and Allolevivirus. The Pseudomonas species PP7 diverged from its common ancestor with the coliphage prior to the ancient split between these genera and their subsequent diversification. Differences in genome size, gene composition, and gene expression are shown with a high probability to have changed along the lineage leading to the Allolevivirus through gene expansion. The change in genome size of the Allolevivirus ancestor may have catalyzed subsequent changes that led to their current genome organization and gene expression.

Replication of coliphage Q beta as affected by host cell number, nutrition, competition from insusceptible cells and non-FRNA coliphages.

F-specific RNA (FRNA) coliphages, which infect Escherichia coli by attachment to F pili, might serve as indicators of human enteric viruses in groundwater, provided these phages do not replicate in groundwater and replicate only to a limited extent in wastewater. Several factors that could influence phage replication in either of these environments were examined. Q beta did not replicate when host cells were fewer than 10(4) cfu ml-1. Replication selected for insusceptible cells when Q beta was incubated with its E. coli host. Loss of Q beta, presumably by inactivation, occurred in autoclaved on-site and urban wastewater, autoclaved groundwater, and in filter-sterilized spent LB broth. Replication did not occur in LB broth diluted with sterile saline to 1% of its original strength, which indicates that replication of FRNA coliphages cannot occur in such nutrient-poor environments as wastewater and groundwater. Competition from non-FRNA coliphages and insusceptible cells tended to reduce Q beta replication, as predicted, but phage yields unexpectedly increased significantly when Enterococcus faecalis was added to cultures.

Photo-induced inactivation of viruses: adsorption of methylene blue, thionine, and thiopyronine on Qbeta bacteriophage.

The adsorption of cationic organic dyes (methylene blue, thionine, and thiopyronine) on Qbeta bacteriophage was studied by UV-visible and fluorescence spectroscopy. The dyes have shown a strong affinity to the virus and some have been used as sensitizers for photo-induced inactivation of virus. In the methylene blue concentration range of 0.1-5 microM and at high ratios of dye to virus (greater than 1000 dye molecules per virion), the dyes bind as aggregates on the virus. Aggregation lowers the efficiency of photoinactivation because of self-quenching of the dye. At lower ratios of dye to virus (lower than 500 dye molecules per virion), the dye binds to the virus as a monomer. Fluorescence polarization and time-resolved studies of the fluorescence support the conclusions based on fluorescence quenching. Increasing the ionic strength (adding NaCl) dissociates bound dye aggregates on the virus and releases monomeric dye into the bulk solution.

The RNA-binding protein HF-I, known as a host factor for phage Qbeta RNA replication, is essential for rpoS translation in Escherichia coli.

The rpoS-encoded sigma(S) subunit of RNA polymerase in Escherichia coli is a global regulatory factor involved in several stress responses. Mainly because of increased rpoS translation and stabilization of sigma(S), which in nonstressed cells is a highly unstable protein, the cellular sigma(S) content increases during entry into stationary phase and in response to hyperosmolarity. Here, we identify the hfq-encoded RNA-binding protein HF-I, which has been known previously only as a host factor for the replication of phage Qbeta RNA, as an essential factor for rpoS translation. An hfq null mutant exhibits strongly reduced sigma(S) levels under all conditions tested and is deficient for growth phase-related and osmotic induction of sigma(S). Using a combination of gene fusion analysis and pulse-chase experiments, we demonstrate that the hfq mutant is specifically impaired in rpoS translation. We also present evidence that the H-NS protein, which has been shown to affect rpoS translation, acts in the same regulatory pathway as HF-I at a position upstream of HF-I or in conjunction with HF-I. In addition, we show that expression and heat induction of the heat shock sigma factor sigma(32) (encoded by rpoH) is not dependent on HF-I, although rpoH and rpoS are both subject to translational regulation probably mediated by changes in mRNA secondary structure. HF-I is the first factor known to be specifically involved in rpoS translation, and this role is the first cellular function to be identified for this abundant ribosome-associated RNA-binding protein in E. coli.

Stability of replicative form and fitness among RNA variants transcribed by Qbeta replicase.

Stability in the replicative form of RNA molecules transcribed by Qbeta replicase was demonstrated to provide a sequence-dependent indicator of their fitness. This follows from the finding that replication rates reported for 17 RNA species (genome length, 77-370 nucleotides) correlate with the self-interaction free energy of these self-annealed strands. Formation of double-stranded molecules during replication conversely decreased with self-interaction free energy. H-bond formation between self-complementary segments of folded RNA molecules plainly produces a potential energy barrier opposing the transition to a double-stranded, non-replicating form. Melting point temperature and resistance of RNA synthesis to elevated salt levels among three variants also increased with strand configuration free energy. Genome-based estimates of fitness in other self-replicating RNA species were therefore possible. Once a link between the kinetic parameters of replication and base sequence of these RNA species is adequately established, estimates of fitness can be dissociated from survival states following evolution, and Darwin's fundamental precept, 'survival of the fittest,' could be appraised as an experimentally testable hypothesis.

The lysis function of RNA bacteriophage Qbeta is mediated by the maturation (A2) protein.

Complete or partial cDNA sequences of the RNA bacteriophage Qbeta were cloned in plasmids under the control of the lambdaP(L) promoter to allow regulated expression in Escherichia coli harbouring the gene for the temperature-sensitive lambdaCI857 repressor. Induction of the complete Qbeta sequence leads to a 100-fold increase in phage production, accompanied by cell lysis. Induction of the 5'-terminal sequence containing the intact maturation protein (A2) cistron also causes cell lysis. Alterations of the A2 cistron, leading to proteins either devoid of approximately 20% of the C-terminal region or of six internal amino acids, abolish the lysis function. Expression of other cistrons in addition to the A2 cistron does not enhance host lysis. Thus, in Qbeta, the A2 protein, in addition to its functions as maturation protein, appears to trigger cell lysis. This contrasts with the situation in the distantly related group I RNA phages such as f2 and MS2 where a small lysis polypeptide is coded for by a region overlapping the end of the coat gene and the beginning of the replicase gene.