Structure of the Borrelia Bacteriophage φBB1 Procapsid.

Bacteriophages of Borrelia burgdorferi are a biologically important but under-investigated feature of the Lyme disease-causing spirochete. No virulent borrelial viruses have been identified, but all B. burgdorferi isolates carry a prophage φBB1 as resident circular plasmids. Like its host, the φBB1 phage is quite distinctive and shares little sequence similarity with other known bacteriophages. We expressed φBB1 head morphogenesis proteins in Escherichia coli which resulted in assembly of homogeneous prolate procapsid structures and used cryo-electron microscopy to determine the three-dimensional structure of these particles. The φBB1 procapsids consist of 415 copies of the major capsid protein and an equal combined number of three homologous capsid decoration proteins that form trimeric knobs on the outside of the particle. One of the end vertices of the particle is occupied by a portal assembled from twelve copies of the portal protein. The φBB1 scaffolding protein is entirely α-helical and has an elongated shape with a small globular domain in the middle. Within the tubular section of the procapsid, the internal scaffold is built of stacked rings, each composed of 32 scaffolding protein molecules, which run in opposite directions from both caps with a heterogeneous part in the middle. Inside the portal-containing cap, the scaffold is organized asymmetrically with ten scaffolding protein molecules bound to the portal. The φBB1 procapsid structure provides better insight into the vast structural diversity of bacteriophages and presents clues of how elongated bacteriophage particles might be assembled.

Abolishment of morphology-based taxa and change to binomial species names: 2022 taxonomy update of the ICTV bacterial viruses subcommittee.

This article summarises the activities of the Bacterial Viruses Subcommittee of the International Committee on Taxonomy of Viruses for the period of March 2021-March 2022. We provide an overview of the new taxa proposed in 2021, approved by the Executive Committee, and ratified by vote in 2022. Significant changes to the taxonomy of bacterial viruses were introduced: the paraphyletic morphological families Podoviridae, Siphoviridae, and Myoviridae as well as the order Caudovirales were abolished, and a binomial system of nomenclature for species was established. In addition, one order, 22 families, 30 subfamilies, 321 genera, and 862 species were newly created, promoted, or moved.

Diversity of the lysozyme fold: structure of the catalytic domain from an unusual endolysin encoded by phage Enc34.

Endolysins are bacteriophage-encoded peptidoglycan-degrading enzymes with potential applications for treatment of multidrug-resistant bacterial infections. Hafnia phage Enc34 encodes an unusual endolysin with an N-terminal enzymatically active domain and a C-terminal transmembrane domain. The catalytic domain of the endolysin belongs to the conserved protein family PHA02564 which has no recognizable sequence similarity to other known endolysin types. Turbidity reduction assays indicate that the Enc34 enzyme is active against peptidoglycan from a variety of Gram-negative bacteria including the opportunistic pathogen Pseudomonas aeruginosa PAO1. The crystal structure of the catalytic domain of the Enc34 endolysin shows a distinctive all-helical architecture that distantly resembles the α-lobe of the lysozyme fold. Conserved catalytically important residues suggest a shared evolutionary history between the Enc34 endolysin and GH73 and GH23 family glycoside hydrolases and propose a molecular signature for substrate cleavage for a large group of peptidoglycan-degrading enzymes.

Guidance for creating individual and batch latinized binomial virus species names.

The International Committee on Taxonomy of Viruses recently adopted, and is gradually implementing, a binomial naming format for virus species. Although full Latinization of these names remains optional, a standardized nomenclature based on Latinized binomials has the advantage of comparability with all other biological taxonomies. As a language without living native speakers, Latin is more culturally neutral than many contemporary languages, and words built from Latin roots are already widely used in the language of science across the world. Conversion of established species names to Latinized binomials or creation of Latinized binomials de novo may seem daunting, but the rules for name creation are straightforward and can be implemented in a formulaic manner. Here, we describe approaches, strategies and steps for creating Latinized binomials for virus species without prior knowledge of Latin. We also discuss a novel approach to the automated generation of large batches of novel genus and species names. Importantly, conversion to a binomial format does not affect virus names, many of which are created from local languages.

Leviviricetes: expanding and restructuring the taxonomy of bacteria-infecting single-stranded RNA viruses.

The vast majority of described prokaryotic viruses have double-stranded or single-stranded DNA or double-stranded RNA genomes. Until 2020, a mere four prokaryotic single-stranded, positive-sense RNA viruses have been classified in two genera (Riboviria; Lenarviricota; Allassoviricetes; Leviviridae). Several recent metagenomic and metatranscriptomic studies revealed a vastly greater diversity of these viruses in prokaryotic soil communities than ever anticipated. Phylogenetic analysis of these newly discovered viruses prompted the reorganization of class Allassoviricetes, now renamed Leviviricetes, to include two orders, Norzivirales and Timlovirales, and a total of six families, 428 genera and 882 species. Here we outline the new taxonomy of Leviviricetes, approved and ratified in 2021 by the International Committee on Taxonomy of Viruses, and describe open-access hidden Markov models to accommodate the anticipated identification and future classification of hundreds, if not thousands, of additional class members into this new taxonomic framework.

Bacterial Viruses Subcommittee and Archaeal Viruses Subcommittee of the ICTV: update of taxonomy changes in 2021.

In this article, we - the Bacterial Viruses Subcommittee and the Archaeal Viruses Subcommittee of the International Committee on Taxonomy of Viruses (ICTV) - summarise the results of our activities for the period March 2020 - March 2021. We report the division of the former Bacterial and Archaeal Viruses Subcommittee in two separate Subcommittees, welcome new members, a new Subcommittee Chair and Vice Chair, and give an overview of the new taxa that were proposed in 2020, approved by the Executive Committee and ratified by vote in 2021. In particular, a new realm, three orders, 15 families, 31 subfamilies, 734 genera and 1845 species were newly created or redefined (moved/promoted).

Structural Characterization of a Single-Stranded DNA-Binding Protein: A Case Study of the ORF6 Protein from Bacteriophage Enc34.

In the quest to understand how single-stranded DNA-binding proteins function and evolve at a molecular level, determination of their high-resolution three-dimensional structure using methods such as X-ray crystallography is indispensable. Here we present a collection of methods used in crystallographic studies of the single-stranded DNA-binding protein from the bacteriophage Enc34, from designing expression constructs through to protein production, purification, and crystallization, to determination and analysis of the protein's three-dimensional structure. The chapter aims to shed light on all the essential stages in a structural study of a single-stranded DNA-binding protein, with a spotlight on procedures specific to X-ray crystallography to aid those interested in venturing into structural biology.

Three-dimensional structure of 22 uncultured ssRNA bacteriophages: Flexibility of the coat protein fold and variations in particle shapes.

The single-stranded RNA (ssRNA) bacteriophages are among the simplest known viruses with small genomes and exceptionally high mutation rates. The number of ssRNA phage isolates has remained very low, but recent metagenomic studies have uncovered an immense variety of distinct uncultured ssRNA phages. The coat proteins (CPs) in these genomes are particularly diverse, with notable variation in length and often no recognizable similarity to previously known viruses. We recombinantly expressed metagenome-derived ssRNA phage CPs to produce virus-like particles and determined the three-dimensional structure of 22 previously uncharacterized ssRNA phage capsids covering nine distinct CP types. The structures revealed substantial deviations from the previously known ssRNA phage CP fold, uncovered an unusual prolate particle shape, and revealed a previously unseen dsRNA binding mode. These data expand our knowledge of the evolution of viral structural proteins and are of relevance for applications such as ssRNA phage-based vaccine design.

Novel ssRNA phage VLP platform for displaying foreign epitopes by genetic fusion.

Virus-like particles (VLPs) can be used as efficient carriers of various antigens and therefore serve as attractive tools in vaccine development. Although VLPs of different viruses can be used, VLPs of ssRNA phages have convincing advantages due to their unique properties, including efficient protein production in bacterial and yeast expression systems, low production cost and easy and fast purification. Currently, the range of ssRNA phage VLPs is limited. In particular, this is true for VLPs that tolerate insertions at the N- and C-termini of the coat protein. It is therefore necessary to find new alternatives within the known ssRNA phage VLP range. From previous studies, we found approximately 80 new VLPs forming ssRNA phage coat proteins. In the current study, we attached a model peptide to the N- and C-termini of coat proteins. As a model peptide, we used a triple repeat of 23 N-terminal residues of the ectodomain of the influenza M2 protein, used previously in the development of the flu vaccine. Examining 43 novel phage coat proteins for the ability to form chimeric VLPs, we found ten new promising candidates for further vaccine design, five of which were tolerant to insertions at both the N- and C-termini. Furthermore, we demonstrate that most of the chimeric VLPs have good antigenic properties as judged from their reactivity with anti-M2 antibodies.

Taxonomy of prokaryotic viruses: 2018-2019 update from the ICTV Bacterial and Archaeal Viruses Subcommittee.

This article is a summary of the activities of the ICTV's Bacterial and Archaeal Viruses Subcommittee for the years 2018 and 2019. Highlights include the creation of a new order, 10 families, 22 subfamilies, 424 genera and 964 species. Some of our concerns about the ICTV's ability to adjust to and incorporate new DNA- and protein-based taxonomic tools are discussed.

Production and characterization of novel ssRNA bacteriophage virus-like particles from metagenomic sequencing data.

BACKGROUND: Protein shells assembled from viral coat proteins are an attractive platform for development of new vaccines and other tools such as targeted bioimaging and drug delivery agents. Virus-like particles (VLPs) derived from the single-stranded RNA (ssRNA) bacteriophage coat proteins (CPs) have been important and successful contenders in the area due to their simplicity and robustness. However, only a few different VLP types are available that put certain limitations on continued developments and expanded adaptation of ssRNA phage VLP technology. Metagenomic studies have been a rich source for discovering novel viral sequences, and in recent years have unraveled numerous ssRNA phage genomes significantly different from those known before. Here, we describe the use of ssRNA CP sequences found in metagenomic data to experimentally produce and characterize novel VLPs. RESULTS: Approximately 150 ssRNA phage CP sequences were sourced from metagenomic sequence data and grouped into 14 different clusters based on CP sequence similarity analysis. 110 CP-encoding sequences were obtained by gene synthesis and expressed in bacteria which in 80 cases resulted in VLP assembly. Production and purification of the VLPs was straightforward and compatible with established protocols, with the only exception that a considerable proportion of the CPs had to be produced at a lower temperature to ensure VLP assembly. The VLP morphology was similar to that of the previously studied phages, although a few deviations such as elongated or smaller particles were noted in certain cases. In addition, stabilizing inter-subunit disulfide bonds were detected in six VLPs and several possible candidate RNA structures in the phage genomes were identified that might bind to the coat protein and ensure specific RNA packaging. CONCLUSIONS: Compared to the few types of ssRNA phage VLPs that were used before, several dozens of new particles representing ten distinct similarity groups are now available with a notable potential for biotechnological applications. It is believed that the novel VLPs described in this paper will provide the groundwork for future development of new vaccines and other applications based on ssRNA bacteriophage VLPs.

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.

Structural Basis for DNA Recognition of a Single-stranded DNA-binding Protein from Enterobacter Phage Enc34.

Modern DNA sequencing capabilities have led to the discovery of a large number of new bacteriophage genomes, which are a rich source of novel proteins with an unidentified biological role. The genome of Enterobacter cancerogenus bacteriophage Enc34 contains several proteins of unknown function that are nevertheless conserved among distantly related phages. Here, we report the crystal structure of a conserved Enc34 replication protein ORF6 which contains a domain of unknown function DUF2815. Despite the low (~15%) sequence identity, the Enc34 ORF6 structurally resembles the gene 2.5 protein from bacteriophage T7, and likewise is a single-stranded DNA (ssDNA)-binding protein (SSB) that consists of a variation of the oligosaccharide/oligonucleotide-binding (OB)-fold and an unstructured C-terminal segment. We further report the crystal structure of a C-terminally truncated ORF6 in complex with an ssDNA oligonucleotide that reveals a DNA-binding mode involving two aromatic stacks and multiple electrostatic interactions, with implications for a common ssDNA recognition mechanism for all T7-type SSBs.

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.

Structure of AP205 Coat Protein Reveals Circular Permutation in ssRNA Bacteriophages.

AP205 is a single-stranded RNA bacteriophage that has a coat protein sequence not similar to any other known single-stranded RNA phage. Here, we report an atomic-resolution model of the AP205 virus-like particle based on a crystal structure of an unassembled coat protein dimer and a cryo-electron microscopy reconstruction of the assembled particle, together with secondary structure information from site-specific solid-state NMR data. The AP205 coat protein dimer adopts the conserved Leviviridae coat protein fold except for the N-terminal region, which forms a beta-hairpin in the other known single-stranded RNA phages. AP205 has a similar structure at the same location formed by N- and C-terminal beta-strands, making it a circular permutant compared to the other coat proteins. The permutation moves the coat protein termini to the most surface-exposed part of the assembled particle, which explains its increased tolerance to long N- and C-terminal fusions.

Crystal structure of the bacteriophage Qß coat protein in complex with the RNA operator of the replicase gene.

The coat proteins of single-stranded RNA bacteriophages specifically recognize and bind to a hairpin structure in their genome at the beginning of the replicase gene. The interaction serves to repress the synthesis of the replicase enzyme late in infection and contributes to the specific encapsidation of phage RNA. While this mechanism is conserved throughout the Leviviridae family, the coat protein and operator sequences from different phages show remarkable variation, serving as prime examples for the co-evolution of protein and RNA structure. To better understand the protein-RNA interactions in this virus family, we have determined the three-dimensional structure of the coat protein from bacteriophage Qß bound to its cognate translational operator. The RNA binding mode of Qß coat protein shares several features with that of the widely studied phage MS2, but only one nucleotide base in the hairpin loop makes sequence-specific contacts with the protein. Unlike in other RNA phages, the Qß coat protein does not utilize an adenine-recognition pocket for binding a bulged adenine base in the hairpin stem but instead uses a stacking interaction with a tyrosine side chain to accommodate the base. The extended loop between ß strands E and F of Qß coat protein makes contacts with the lower part of the RNA stem, explaining the greater length dependence of the RNA helix for optimal binding to the protein. Consequently, the complex structure allows the proposal of a mechanism by which the Qß coat protein recognizes and discriminates in favor of its cognate RNA.

Diversity of pili-specific bacteriophages: genome sequence of IncM plasmid-dependent RNA phage M.

BACKGROUND: Bacteriophages of the Leviviridae family are small RNA viruses with linear, positive-sense, single-stranded RNA genomes that encode only four proteins. All phages of this family require bacterial pili to attach to and infect cells. Leviviridae phages utilizing F-pili for this purpose have been extensively studied. RNA phages specific for conjugative plasmid-encoded pili other than that of plasmid F have been isolated, but are much less understood and their relation to the F-pili-specific phages in many cases is not known. RESULTS: Phage M has the smallest known Leviviridae genome to date and has the typical genome organization with maturation, coat and replicase genes in the 5' to 3' direction. The lysis gene is located in a different position than in other known Leviviridae phages and completely overlaps with the replicase gene in a different reading frame. It encodes a 37 residue long polypeptide that contains a transmembrane helix like the other known lysis proteins of leviviruses. Sequence identities of M proteins to those of other phages do not exceed 25% for maturation protein, 51% for coat protein and 41% for replicase. Similarities in protein sequences and RNA secondary structures at the 3' untranslated region place phage M together with phages specific for IncP, IncC and IncH, but not IncF plasmid-encoded pili. Phylogenetic analysis using the complete genome sequences and replicase proteins suggests that phage M represents a lineage that branched off early in the course of RNA phage specialization on different conjugative plasmids. CONCLUSIONS: The genome sequence of phage M shows that it is clearly related to other conjugative pili-specific leviviruses but has an atypical location of the lysis gene. It provides a better view on the remarkable diversification of the plasmid-specific RNA phages.