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At least 19 recordsLinked to original sources
PubMed2026

Systematic mapping of insertion-tolerant regions enables capsid engineering of an infectious RNA phage.

RNA phages are attractive platforms for the design of programmable bioparticles, but their development has been constrained by limited knowledge of genomic sites that can tolerate sequence insertion. Here, we combined MuA transposase-mediated in vitro insertion mutagenesis with our established reverse genetics systems to systematically identify insertion-tolerant regions (ITRs) in the RNA phages MS2 and PP7. Screening of 4,555 MS2 and 2,228 PP7 random insertion clones identified 29 and 26 non-redundant ITRs, respectively. We further analyzed and compared these ITRs in the context of RNA genome organization and virion architecture. Both phages contained ITRs within the maturation protein, whereas only PP7 tolerated insertions within the coat protein (CP). On the basis of structural location and plaque-forming capacity, an ITR situated between Gly74 and Glu75 (GGC^GAG) in the PP7 CP was selected for further study. Infectious phage particles generated from complementary DNA clones retained the 15-bp insertion at both the RNA and protein levels. Engineered PP7 phages carrying an Arg-Gly-Asp motif inserted into the CP at this ITR displayed enhanced in vivo clearance in a Drosophila model, despite having in vitro stability comparable to that of the wild type. These findings provide the first example of CP engineering in an infectious RNA phage and establish a framework for engineering RNA phages for biological and biotechnological applications.IMPORTANCEA major obstacle to developing RNA phages as synthetic biology platforms is the lack of design principles for genomic insertion. Here, we address this limitation by establishing a mutagenesis-and-recovery workflow that systematically identifies insertion-tolerant regions (ITRs) in the RNA phages MS2 and PP7. The resulting maps reveal distinct structural constraints in the two phages and enable rational engineering of a peptide-display site in the PP7 capsid. Using this approach, we generated an engineered infectious phage with a modified capsid, thereby providing the first demonstration of capsid engineering in an infectious RNA phage, to our knowledge. This study lays the groundwork for the rational design of live RNA phage virions as tractable and engineerable scaffolds for future biological and biotechnological applications.

Animals↗
PubMed1975

The host factor required for RNA phage Qbeta RNA replication in vitro. Intracellular location, quantitation, and purification by polyadenylate-cellulose chromatography.

The Qbeta host factor, a heat-stable protein necessary in concert with Qbeta replicase for phage Qbeta RNA replication in vitro, has been localized in Escherichia coli and found to be associated primarily with ribosomes. This location has been established both by complement fixation assays with highly specific antiserum directed against the host factor, and by in vitro stimulation of Qbeta RNA replication by the Qbeta replicase. The complement fixation assay has provided the estimate that there are approximately 2500 copies of the host factor polypeptide per cell. The host factor is released from the ribosomes by a 1 M NH4Cl wash and concentrated by ammonium sulfate precipitation. It can be purified to apparent homogeneity in one further step by chromatography on poly(A)-cellulose. Ribosomal protein S1 subunit I of Qbeta replicase) also binds to the poly(A)-cellulose column and elutes before the host factor. In agreement with previous reports, we find that the host factor has a monomer molecular weight of 12,000 as judged by sodium dodecyl sulfate-polyacrylamide gels, and a native molecular weight of 72,000 as judged by the stoichiometric interaction of the host factor with Qbeta RNA, by sedimentation in sucrose velocity gradients, and by sodium dodecyl sulfate gel mobility when incompletely disaggregated. The Qbeta host factor is a potent inhibitor of an in vitro poly(A)-directed polylysine protein-synthesizing system, but has less effect on the in vitro translation of poly(U), R17 RNA, late T7 mRNA, or endogenous E. coli mRNA. The amino acid composition and NH2- terminal sequence rule out the host factor as one of the known 30 S or 50 S E. coli ribosomal proteins. The finding that the Qbeta host factor is associated with ribosomes in vivo completes the demonstration that all of the host-supplied proteins required for phage Qbeta RNA replication in vitro are either associated with ribosomes or are involved in the protein-synthetic machinery of the cell.

Animals↗
PubMed1977

The effect of the ribosomal protein S1 from Escherichia coli on the synthesis in vitro of bacterial-, DNA phage- and RNA phage proteins.

Using an in vitro preparation for protein synthesis, we have studied the effect of the ribosomal protein S1 from Escherichia coli on the synthesis of the coat protein of the RNA-containing phages Qbeta and MS2, on that of an "early" and a "late" enzyme encoded by the DNA containing phage T7, and on that of anthranilate synthetase, an enzyme encoded by the bacterial tryptophan operon. Our results indicate that for the synthesis of these five proteins the presence of S1 is required. From these results we conclude that S1 is an essential protein for the translation of bacterial and bacteriophage messenger RNA.

Anthranilate Synthase↗
PubMed2006

Analysis of six new genes encoding lysis proteins and coat proteins in Escherichia coli group A RNA phages.

Group A RNA phages consist of four genes-maturation protein, coat protein, lysis protein and replicase genes. We analyzed six plasmids containing lysis protein genes and coat protein genes of Escherichia coli group A RNA phages and compared their amino acid sequences with the known proteins of E. coli(group A), Pseudomonas aeruginosa(PP7) RNA phages and Rg-lysis protein from Qbeta phage. The size of lysis proteins was different by the groups but the coat proteins were almost the same size among phages. The phylogenetic analysis shows that the sub-groups A-I and A-II of E. coli RNA phages were clearly dispersed into two clusters.

Allolevivirus↗
PubMed1976

Characteristics of phage AP50, an RNA phage containing phospholipids.

A bacteriophage specific for Bacillus anthracis was isolated and designated as AP50. The nucleic acid of phage AP50 is RNA and the virion contains five different phospholipids. Some physical and biological characteristics of the phage, including morphology, were examined. To the best of our knowledge, this RNA bacteriophage containing phospholipids is the first to be isolated for a Gram-positive host.

Bacillus anthracis↗
PubMed1992

Reversion of Q beta RNA phage mutants by homologous RNA recombination.

Q beta phage RNAs with inactivating insertion (8-base) or deletion (17-base) mutations within their replicase genes were prepared from modified Q beta cDNAs and transfected into Escherichia coli spheroplasts containing Q beta replicase provided in trans by a resident plasmid. Replicase-defective (Rep-) Q beta phage produced by these spheroplasts were detected as normal-sized plaques on lawns of cells containing plasmid-derived Q beta replicase, but were unable to form plaques on cells lacking this plasmid. When individual Rep- phage were isolated and grown to high titer in cells containing plasmid-derived Q beta replicase, revertant (Rep+) Q beta phage were obtained at a frequency of ca. 10(-8). To investigate the mechanism of this reversion, a point mutation was placed into the plasmid-derived Q beta replicase gene by site-directed mutagenesis. Q beta mutants amplified on cells containing the resultant plasmid also yielded Rep+ revertants. Genomic RNA was isolated from several of the latter phage revertants and sequenced. Results showed that the original mutation (insertion or deletion) was no longer present in the phage revertants but that the marker mutation placed into the plasmid was now present in the genomic RNAs, indicating that recombination was one mechanism involved in the reversion of the Q beta mutants. Further experiments demonstrated that the 3' noncoding region of the plasmid-derived replicase gene was necessary for the reversion-recombination of the deletion mutant, whereas this region was not required for reversion or recombination of the insertion mutant. Results are discussed in terms of a template-switching model of RNA recombination involving Q beta replicase, the mutant phage genome, and plasmid-derived replicase mRNA.

Amino Acid Sequence↗
PubMed1971

Grouping of RNA phages based on the template specificity of their RNA replicases.

We attempted to apply a new criterion, the template specificity of RNA replicases of Escherichia coli RNA phages, for the grouping of these phages. Based on the template specificity, it was shown that (a) Qbeta, VK, and ST belonged to one group (group III), and SP and FI to another group (group IV), and (b) some similarity existed between groups III and IV. Considering such template specificity in addition to previously reported properties of phage particles, we could separate RNA phages into four major groups (I-IV) with subgroups a, b, and c in group III, and subgroups a and b in group IV.

Coliphages↗
PubMed1996

An oligonucleotide hybridization assay for the identification and enumeration of F-specific RNA phages in surface water.

F-specific RNA phages can be used as model organisms for enteric viruses to monitor the effectiveness of sewage treatment, and to assess the potential contamination of surface water with these viruses. In this paper a method is described which identifies RNA phages quantitatively by a plaque hybridization assay. Oligonucleotide probes were developed that can assign phages to their phylogenetic subgroups. Such a distinction is important, since some subgroups preferentially occur in sewage of human origin, while others tend to be associated with animal wastewater. The method has been tested on a large number of isolates and represents an improvement in time and reliability over the previously used serological classification.

Base Sequence↗