The True Story and Advantages of RNA Phage Capsids as Nanotools.

RNA phages are often used as prototypes for modern recombinant virus-like particle (VLP) technologies. Icosahedral RNA phage VLPs can be formed from coat proteins (CPs) and are efficiently produced in bacteria and yeast. Both genetic fusion and chemical coupling have been successfully used for the production of numerous chimeras based on RNA phage VLPs. In this review, we describe advances in RNA phage VLP technology along with the history of the Leviviridae family, including its taxonomical organization, genomic structure, and important role in the development of molecular biology. Comparative 3D structures of different RNA phage VLPs are used to explain the level of VLP tolerance to foreign elements displayed on VLP surfaces. We also summarize data that demonstrate the ability of CPs to tolerate different organic (peptides, oligonucleotides, and carbohydrates) and inorganic (metal ions) compounds either chemically coupled or noncovalently added to the outer and/or inner surfaces of VLPs. Finally, we present lists of nanotechnological RNA phage VLP applications, such as experimental vaccines constructed by genetic fusion and chemical coupling methodologies, nanocontainers for targeted drug delivery, and bioimaging tools.

Gene mapping and phylogenetic analysis of the complete genome from 30 single-stranded RNA male-specific coliphages (family Leviviridae).

Male-specific single-stranded RNA (FRNA) coliphages belong to the family Leviviridae. They are classified into two genera (Levivirus and Allolevivirus), which can be subdivided into four genogroups (genogroups I and II in Levivirus and genogroups III and IV in Allolevivirus). Relatively few strains have been completely characterized, and hence, a detailed knowledge of this virus family is lacking. In this study, we sequenced and characterized the complete genomes of 19 FRNA strains (10 Levivirus strains and 9 Allolevivirus strains) and compared them to the 11 complete genome sequences available in GenBank. Nucleotide similarities among strains of Levivirus genogroups I and II were 75% to 99% and 83 to 94%, respectively, whereas similarities among strains of Allolevivirus genogroups III and IV ranged from 70 to 96% and 75 to 95%, respectively. Although genogroup I strain fr and genogroup III strains MX1 and M11 share only 70 to 78% sequence identity with strains in their respective genogroups, phylogenetic analyses of the complete genome and the individual genes suggest that strain fr should be grouped in Levivirus genogroup I and that the MX1 and M11 strains belong in Allolevivirus genogroup III. Strains within each genus share >50% sequence identity, whereas between the two genera, strains have <40% nucleotide sequence identity. Overall, amino acid composition, nucleotide similarities, and replicase catalytic domain location contributed to phylogenetic assignments. A conserved eight-nucleotide signature at the 3' end of the genome distinguishes leviviruses (5' ACCACCCA 3') from alloleviviruses (5' TCCTCCCA 3').

Application of real-time PCR assays to genotyping of F-specific phages in river water and sediments in Japan.

Genotyping of F-specific RNA phages is currently one of the most promising approaches to differentiate between human and animal fecal contamination in aquatic environments. In this study, a total of 18 river water and sediment samples were collected from the Tonegawa River basin, Japan, in order to describe the genogroup distribution of F-specific RNA and DNA phages using genogroup-specific real-time PCR assays. F-specific phages were detected in nine (100%) river water and six (67%) sediment samples. Eighty-five phage plaques were isolated from these samples and subjected to real-time PCR assays specific for the phages. F-specific RNA phages of human genogroups (II and III) were detected in 32 (38%) plaques, whereas those of animal genogroups (I and IV) were detected in 17 (20%) plaques. No correlation was observed between the genogroup distribution of F-specific RNA phages and the occurrence of human adenovirus genomes, suggesting that genotyping of the phages alone is inadequate for the evaluation of the occurrence of viruses in aquatic environments. SYBR Green-based real-time PCR assay revealed the presence of F-specific DNA phages in four (5%) plaques, which were further classified into two genogroups (fd- and f1-like phages) by sequence analysis. Thirty-two (38%) plaques were not classified as the F-specific phage genogroups, indicating the limited applicability of these real-time PCR assays to a wide range of aquatic environmental samples worldwide.

Salmonella phages examined in the electron microscope.

Out of 177 surveyed bacteriophages, 161 (91%) are tailed and belong to the Myoviridae, Siphoviridae, and Podoviridae families (43, 55, and 59 viruses, respectively). Sixteen filamentous or isometric phages are members of the Inoviridae, Leviviridae, Microviridae, and Tectiviridae families (9%). Many tailed phages belong to established phage genera (P22, T1, T5, and T7), which are widespread in enterobacteria and other Gram-negatives of the Proteobacteria phylum.

Molecular detection and genotyping of male-specific coliphages by reverse transcription-PCR and reverse line blot hybridization.

In recent years, there has been increased interest in the use of male-specific or F+ coliphages as indicators of microbial inputs to source waters. Sero- or genotyping of these coliphages can also be used for microbial source tracking (MST). Among the male-specific coliphages, the F+ RNA (FRNA) viruses are well studied, while little is known about the F+ DNA (FDNA) viruses. We have developed a reverse line blot hybridization (RLB) assay which allows for the simultaneous detection and genotyping of both FRNA as well as FDNA coliphages. These assays included a novel generic duplex reverse transcription-PCR (RT-PCR) assay for FRNA viruses as well as a generic PCR for FDNA viruses. The RT-PCR assays were validated by using 190 field and prototype strains. Subsequent DNA sequencing and phylogenetic analyses of RT-PCR products revealed the classification of six different FRNA clusters, including the well-established subgroups I through IV, and three different FDNA clusters, including one (CH) not previously described. Within the leviviruses, a potentially new subgroup (called JS) including strains having more than 40% nucleotide sequence diversity with the known levivirus subgroups (MS2 and GA) was identified. We designed subgroup-specific oligonucleotides that were able to genotype all nine (six FRNA, three FDNA) different clusters. Application of the method to a panel of 351 enriched phage samples from animal feces and wastewater, including known prototype strains (MS2, GA, Q beta, M11, FI, and SP for FRNA and M13, f1, and fd for FDNA), resulted in successful genotyping of 348 (99%) of the samples. In summary, we developed a novel method for standardized genotyping of F+ coliphages as a useful tool for large-scale MST studies.

Nucleotide sequence of a ssRNA phage from Acinetobacter: kinship to coliphages.

The complete nucleotide sequence of ssRNA phage AP205 propagating in Acinetobacter species is reported. The RNA has three large ORFs, which code for the following homologues of the RNA coliphage proteins: the maturation, coat and replicase proteins. Their gene order is the same as that in coliphages. RNA coliphages or Leviviridae fall into two genera: the alloleviviruses, like Q(beta), which have a coat read-through protein, and the leviviruses, like MS2, which do not have this coat protein extension. AP205 has no read-through protein and may therefore be classified as a levivirus. A major digression from the known leviviruses is the apparent absence of a lysis gene in AP205 at the usual position, overlapping the coat and replicase proteins. Instead, two small ORFs are present at the 5' terminus, preceding the maturation gene. One of these might encode a lysis protein. The other is of unknown function. Other new features concern the 3'-terminal sequence. In all ssRNA coliphages, there are always three cytosine residues at the 3' end, but in AP205, there is only a single terminal cytosine. Distantly related viruses, like AP205 and the coliphages, do not have significant sequence identity; yet, important secondary structural features of the RNA are conserved. This is shown here for the 3' UTR and the replicase-operator hairpin. Interestingly, although AP205 has the genetic map of a levivirus, its 3' UTR has the length and RNA secondary structure of an allolevivirus. Sharing features with both MS2 and Q(beta) suggests that, in an evolutionary sense, AP205 should be placed between Q(beta) and MS2. A phylogenetic tree for the ssRNA phages is presented.

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.