RNA and Sugars, Unique Properties of Bacteriophages Infecting Multidrug Resistant Acinetobacter radioresistens Strain LH6.

Bacteriophages (phages) are predicted to be the most ubiquitous biological entity on earth, and yet, there are still vast knowledge gaps in our understanding of phage diversity and phage-host interactions. Approximately one hundred Acinetobacter-infecting DNA viruses have been identified, and in this report, we describe eight more. We isolated two typical dsDNA lytic podoviruses (CAP1-2), five unique dsRNA lytic cystoviruses (CAP3-7), and one dsDNA lysogenic siphovirus (SLAP1), all capable of infecting the multidrug resistant isolate Acinetobacter radioresistens LH6. Using transmission electron microscopy, bacterial mutagenesis, phage infectivity assays, carbohydrate staining, mass-spectrometry, genomic sequencing, and comparative studies, we further characterized these phages. Mutation of the LH6 initiating glycosyltransferase homolog, PglC, necessary for both O-linked glycoprotein and capsular polysaccharide (CPS) biosynthesis, prevented infection by the lytic podovirus CAP1, while mutation of the pilin protein, PilA, prevented infection by CAP3, representing the lytic cystoviruses. Genome sequencing of the three dsRNA segments of the isolated cystoviruses revealed low levels of homology, but conserved synteny with the only other reported cystoviruses that infect Pseudomonas species. In Pseudomonas, the cystoviruses are known to be enveloped phages surrounding their capsids with the inner membrane from the infected host. To characterize any membrane-associated glycoconjugates in the CAP3 cystovirus, carbohydrate staining was used to identify a low molecular weight lipid-linked glycoconjugate subsequently identified by mutagenesis and mass-spectrometry as bacterial lipooligosaccharide. Together, this study demonstrates the isolation of new Acinetobacter-infecting phages and the determination of their cell receptors. Further, we describe the genomes of a new genus of Cystoviruses and perform an initial characterization of membrane-associated glycoconjugates.

Recognition of six additional cystoviruses: Pseudomonas virus phi6 is no longer the sole species of the family Cystoviridae.

Cystoviridae is a family of bacterial viruses (bacteriophages) with a tri-segmented dsRNA genome. It includes a single genus Cystovirus, which has presently only one recognised virus species, Pseudomonas virus phi6. However, a large number of additional dsRNA phages have been isolated from various environmental samples, indicating that such viruses are more widespread and abundant than previously recognised. Six of the additional dsRNA phage isolates (Pseudomonas phages phi8, phi12, phi13, phi2954, phiNN and phiYY) have been fully sequenced. They all infect Pseudomonas species, primarily plant pathogenic Pseudomonas syringae strains. Due to the notable genetic and structural similarities with Pseudomonas phage phi6, we propose that these viruses should be included into the Cystovirus genus (and consequently into the Cystoviridae family). Here, we present an updated taxonomy of the family Cystoviridae and give a short overview of the properties of the type member phi6 as well as the putative new members of the family.

ICTV Virus Taxonomy Profile: Cystoviridae.

The family Cystoviridae includes enveloped viruses with a tri-segmented dsRNA genome and a double-layered protein capsid. The innermost protein shell is a polymerase complex responsible for genome packaging, replication and transcription. Cystoviruses infect Gram-negative bacteria, primarily plant-pathogenic Pseudomonas syringae strains. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the Cystoviridae, which is available at http://www.ictv.global/report/cystoviridae.

The ϕ6 cystovirus protein P7 becomes accessible to antibodies in the transcribing nucleocapsid: a probe for viral structural elements.

Protein P7 is a component of the cystovirus viral polymerase complex. In the unpackaged procapsid, the protein is situated in close proximity to the viral directed RNA polymerase, P2. Cryo-electron microscopy difference maps from the species ϕ6 procapsid have demonstrated that P7 and P2 likely interact prior to viral RNA packaging. The location of P7 in the post-packaged nucleocapsid (NC) remains unknown. P7 may translocate closer to the five-fold axis of a filled procapsid but this has not been directly visualized. We propose that monoclonal antibodies (Mabs) can be selected that serve as probe- reagents for viral assembly and structure. A set of Mabs have been isolated that recognize and bind to the ϕ6 P7. The antibody set contains five unique Mabs, four of which recognize a linear epitope and one which recognizes a conformational epitope. The four unique Mabs that recognize a linear epitope display restricted utilization of Vκ and VH genes. The restricted genetic range among 4 of the 5 antibodies implies that the antibody repertoire is limited. The limitation could be the consequence of a paucity of exposed antigenic sites on the ϕ6 P7 surface. It is further demonstrated that within ϕ6 nucleocapsids that are primed for early-phase transcription, P7 is partially accessible to the Mabs, indicating that the nucleocapsid shell (protein P8) has undergone partial disassembly exposing the protein's antigenic sites.

New enveloped dsRNA phage from freshwater habitat.

Cystoviridae is a family of bacteriophages with a tri-segmented dsRNA genome enclosed in a tri-layered virion structure. Here, we present a new putative member of the Cystoviridae family, bacteriophage ϕNN. ϕNN was isolated from a Finnish lake in contrast to the previously identified cystoviruses, which originate from various legume samples collected in the USA. The nucleotide sequence of the virus reveals a strong genetic similarity (~80 % for the L-segments, ~55 % for the M-segments and ~84 % for the S-segments) to Pseudomonas phage ϕ6, the type member of the virus family. However, the relationship between ϕNN and other cystoviruses is more distant. In general, proteins located in the internal parts of the virion were more conserved than those exposed on the virion surface, a phenomenon previously reported among eukaryotic dsRNA viruses. Structural models of several putative ϕNN proteins propose that cystoviral structures are highly conserved.

Electrophoretic mobility confirms reassortment bias among geographic isolates of segmented RNA phages.

BACKGROUND: Sex presents evolutionary costs and benefits, leading to the expectation that the amount of genetic exchange should vary in conditions with contrasting cost-benefit equations. Like eukaryotes, viruses also engage in sex, but the rate of genetic exchange is often assumed to be a relatively invariant property of a particular virus. However, the rates of genetic exchange can vary within one type of virus according to geography, as highlighted by phylogeographic studies of cystoviruses. Here we merge environmental microbiology with experimental evolution to examine sex in a diverse set of cystoviruses, consisting of the bacteriophage ϕ6 and its relatives. To quantify reassortment we manipulated - by experimental evolution - electrophoretic mobility of intact virus particles for use as a phenotypic marker to estimate genetic exchange. RESULTS: We generated descendants of ϕ6 that exhibited fast and slow mobility during gel electrophoresis. We identified mutations associated with slow and fast phenotypes using whole genome sequencing and used crosses to establish the production of hybrids of intermediate mobility. We documented natural variation in electrophoretic mobility among environmental isolates of cystoviruses and used crosses against a common fast mobility ϕ6 strain to monitor the production of hybrids with intermediate mobility, thus estimating the amount of genetic exchange. Cystoviruses from different geographic locations have very different reassortment rates when measured against ϕ6, with viruses isolated from California showing higher reassortment rates than those from the Northeastern US. CONCLUSIONS: The results confirm that cystoviruses from different geographic locations have remarkably different reassortment rates -despite similar genome structure and replication mechanisms- and that these differences are in large part due to sexual reproduction. This suggests that particular viruses may indeed exhibit diverse sexual behavior, but wide geographic sampling, across varying environmental conditions may be necessary to characterize the full repertoire. Variation in reassortment rates can assist in the delineation of viral populations and is likely to provide insight into important viral evolutionary dynamics including the rate of coinfection, virulence, and host range shifts. Electrophoretic mobility may be an indicator of important determinants of fitness and the techniques herein can be applied to the study of other viruses.