Structural atlas of a human gut crassvirus.

CrAssphage and related viruses of the order Crassvirales (hereafter referred to as crassviruses) were originally discovered by cross-assembly of metagenomic sequences. They are the most abundant viruses in the human gut, are found in the majority of individual gut viromes, and account for up to 95% of the viral sequences in some individuals1-4. Crassviruses are likely to have major roles in shaping the composition and functionality of the human microbiome, but the structures and roles of most of the virally encoded proteins are unknown, with only generic predictions resulting from bioinformatic analyses4,5. Here we present a cryo-electron microscopy reconstruction of Bacteroides intestinalis virus ΦcrAss0016, providing the structural basis for the functional assignment of most of its virion proteins. The muzzle protein forms an assembly about 1 MDa in size at the end of the tail and exhibits a previously unknown fold that we designate the 'crass fold', that is likely to serve as a gatekeeper that controls the ejection of cargos. In addition to packing the approximately 103 kb of virus DNA, the ΦcrAss001 virion has extensive storage space for virally encoded cargo proteins in the capsid and, unusually, within the tail. One of the cargo proteins is present in both the capsid and the tail, suggesting a general mechanism for protein ejection, which involves partial unfolding of proteins during their extrusion through the tail. These findings provide a structural basis for understanding the mechanisms of assembly and infection of these highly abundant crassviruses.

RNA-mediated interference and reverse transcription control the persistence of RNA viruses in the insect model Drosophila.

How persistent viral infections are established and maintained is widely debated and remains poorly understood. We found here that the persistence of RNA viruses in Drosophila melanogaster was achieved through the combined action of cellular reverse-transcriptase activity and the RNA-mediated interference (RNAi) pathway. Fragments of diverse RNA viruses were reverse-transcribed early during infection, which resulted in DNA forms embedded in retrotransposon sequences. Those virus-retrotransposon DNA chimeras produced transcripts processed by the RNAi machinery, which in turn inhibited viral replication. Conversely, inhibition of reverse transcription hindered the appearance of chimeric DNA and prevented persistence. Our results identify a cooperative function for retrotransposons and antiviral RNAi in the control of lethal acute infection for the establishment of viral persistence.

Yaravirus: A novel 80-nm virus infecting Acanthamoeba castellanii.

Here we report the discovery of Yaravirus, a lineage of amoebal virus with a puzzling origin and evolution. Yaravirus presents 80-nm-sized particles and a 44,924-bp dsDNA genome encoding for 74 predicted proteins. Yaravirus genome annotation showed that none of its genes matched with sequences of known organisms at the nucleotide level; at the amino acid level, six predicted proteins had distant matches in the nr database. Complimentary prediction of three-dimensional structures indicated possible function of 17 proteins in total. Furthermore, we were not able to retrieve viral genomes closely related to Yaravirus in 8,535 publicly available metagenomes spanning diverse habitats around the globe. The Yaravirus genome also contained six types of tRNAs that did not match commonly used codons. Proteomics revealed that Yaravirus particles contain 26 viral proteins, one of which potentially representing a divergent major capsid protein (MCP) with a predicted double jelly-roll domain. Structure-guided phylogeny of MCP suggests that Yaravirus groups together with the MCPs of Pleurochrysis endemic viruses. Yaravirus expands our knowledge of the diversity of DNA viruses. The phylogenetic distance between Yaravirus and all other viruses highlights our still preliminary assessment of the genomic diversity of eukaryotic viruses, reinforcing the need for the isolation of new viruses of protists.

Structures of filamentous viruses infecting hyperthermophilic archaea explain DNA stabilization in extreme environments.

Living organisms expend metabolic energy to repair and maintain their genomes, while viruses protect their genetic material by completely passive means. We have used cryo-electron microscopy (cryo-EM) to solve the atomic structures of two filamentous double-stranded DNA viruses that infect archaeal hosts living in nearly boiling acid: Saccharolobus solfataricus rod-shaped virus 1 (SSRV1), at 2.8-Å resolution, and Sulfolobus islandicus filamentous virus (SIFV), at 4.0-Å resolution. The SIFV nucleocapsid is formed by a heterodimer of two homologous proteins and is membrane enveloped, while SSRV1 has a nucleocapsid formed by a homodimer and is not enveloped. In both, the capsid proteins wrap around the DNA and maintain it in an A-form. We suggest that the A-form is due to both a nonspecific desolvation of the DNA by the protein, and a specific coordination of the DNA phosphate groups by positively charged residues. We extend these observations by comparisons with four other archaeal filamentous viruses whose structures we have previously determined, and show that all 10 capsid proteins (from four heterodimers and two homodimers) have obvious structural homology while sequence similarity can be nonexistent. This arises from most capsid residues not being under any strong selective pressure. The inability to detect homology at the sequence level arises from the sampling of viruses in this part of the biosphere being extremely sparse. Comparative structural and genomic analyses suggest that nonenveloped archaeal viruses have evolved from enveloped viruses by shedding the membrane, indicating that this trait may be relatively easily lost during virus evolution.

Building the atomic model of a boreal lake virus of unknown fold in a 3.9 Šcryo-EM map.

We report here the protocol adopted to build the atomic model of the newly discovered virus FLiP (Flavobacterium infecting, lipid-containing phage) into 3.9 Šcryo-electron microscopy (cryo-EM) maps. In particular, this report discusses the combination of density modification procedures, automatic model building and bioinformatics tools applied to guide the tracing of the major capsid protein (MCP) of this virus. The protocol outlined here may serve as a reference for future structural determination by cryo-EM of viruses lacking detectable structural homologues.

The genome of the nucleopolyhedrosis-causing virus from Tipula oleracea sheds new light on the Nudiviridae family.

UNLABELLED: A large double-stranded DNA (dsDNA) virus that produces occlusion bodies, typical of baculoviruses, has been described to infect crane fly larvae of the genus Tipula (Diptera, Tipulidae). Because of a lack of genomic data, this virus has remained unclassified. Electron microscopy of an archival virus isolated from Tipula oleracea, T. oleracea nudivirus (ToNV), showed irregularly shaped occlusion bodies measuring from 2 to 5 µm in length and 2 µm in middiameter, filled with rod-shape virions containing single nucleocapsids within a bilayer envelope. Whole-genome amplification and Roche 454 sequencing revealed a complete circular genome sequence of 145.7 kb, containing five direct repeat regions. We predicted 131 open reading frames, including a homolog of the polyhedrin gene encoding the major occlusion body protein of T. paludosa nucleopolyhedrovirus (NPV). BLAST searches demonstrated that ToNV had 21 of the 37 baculovirus core genes but shared 52 genes with nudiviruses (NVs). Phylogenomic analyses indicated that ToNV clearly belongs to the Nudiviridae family but should probably be assigned to a new genus. Among nudiviruses, ToNV was most closely related to the Penaeus monodon NV and Heliothis zea NV clade but distantly related to Drosophila innubia NV, the other nudivirus infecting a Diptera. Lastly, ToNV was found to be most closely related to the nuvidirus ancestor of bracoviruses. This was also reflected in terms of gene content, as ToNV was the only known exogenous virus harboring homologs of the Cc50C22.6 and 27b (Cc50C22.7) genes found in the nudiviral genomic cluster involved in bracovirus particle production. IMPORTANCE: The Nudiviridae is a family of arthropod dsDNA viruses from which striking cases of endogenization have been reported (i.e., symbiotic bracoviruses deriving from a nudivirus and the endogenous nudivirus of the brown planthopper). Although related to baculoviruses, relatively little is known about the genomic diversity of exogenous nudiviruses. Here, we characterized, morphologically and genetically, an archival sample of the Tipula oleracea nudivirus (ToNV), which has the particularity of forming occlusion bodies. Comparative genomic and phylogenomic analyses showed ToNV to be to date the closest known relative of the exogenous ancestor of bracoviruses and that ToNV should be assigned to a new genus. Moreover, we revised the homology relationships of nudiviral genes and identified a new set of 32 core genes for the Nudiviridae, of which 21 were also baculovirus core genes. These findings provide important insights into the evolutionary history of large arthropod dsDNA viruses.

Structure of viruses: a short history.

This review is a partially personal account of the discovery of virus structure and its implication for virus function. Although I have endeavored to cover all aspects of structural virology and to acknowledge relevant individuals, I know that I have favored taking examples from my own experience in telling this story. I am anxious to apologize to all those who I might have unintentionally offended by omitting their work. The first knowledge of virus structure was a result of Stanley's studies of tobacco mosaic virus (TMV) and the subsequent X-ray fiber diffraction analysis by Bernal and Fankuchen in the 1930s. At about the same time it became apparent that crystals of small RNA plant and animal viruses could diffract X-rays, demonstrating that viruses must have distinct and unique structures. More advances were made in the 1950s with the realization by Watson and Crick that viruses might have icosahedral symmetry. With the improvement of experimental and computational techniques in the 1970s, it became possible to determine the three-dimensional, near-atomic resolution structures of some small icosahedral plant and animal RNA viruses. It was a great surprise that the protecting capsids of the first virus structures to be determined had the same architecture. The capsid proteins of these viruses all had a 'jelly-roll' fold and, furthermore, the organization of the capsid protein in the virus were similar, suggesting a common ancestral virus from which many of today's viruses have evolved. By this time a more detailed structure of TMV had also been established, but both the architecture and capsid protein fold were quite different to that of the icosahedral viruses. The small icosahedral RNA virus structures were also informative of how and where cellular receptors, anti-viral compounds, and neutralizing antibodies bound to these viruses. However, larger lipid membrane enveloped viruses did not form sufficiently ordered crystals to obtain good X-ray diffraction. Starting in the 1990s, these enveloped viruses were studied by combining cryo-electron microscopy of the whole virus with X-ray crystallography of their protein components. These structures gave information on virus assembly, virus neutralization by antibodies, and virus fusion with and entry into the host cell. The same techniques were also employed in the study of complex bacteriophages that were too large to crystallize. Nevertheless, there still remained many pleomorphic, highly pathogenic viruses that lacked the icosahedral symmetry and homogeneity that had made the earlier structural investigations possible. Currently some of these viruses are starting to be studied by combining X-ray crystallography with cryo-electron tomography.

Coupling viruses to dynein and kinesin-1.

It is now clear that transport on microtubules by dynein and kinesin family motors has an important if not critical role in the replication and spread of many different viruses. Understanding how viruses hijack dynein and kinesin motors using a limited repertoire of proteins offers a great opportunity to determine the molecular basis of motor recruitment. In this review, we discuss the interactions of dynein and kinesin-1 with adenovirus, the α herpes viruses: herpes simplex virus (HSV1) and pseudorabies virus (PrV), human immunodeficiency virus type 1 (HIV-1) and vaccinia virus. We highlight where the molecular links to these opposite polarity motors have been defined and discuss the difficulties associated with identifying viral binding partners where the basis of motor recruitment remains to be established. Ultimately, studying microtubule-based motility of viruses promises to answer fundamental questions as to how the activity and recruitment of the dynein and kinesin-1 motors are coordinated and regulated during bi-directional transport.

Structure of Sputnik, a virophage, at 3.5-Å resolution.

"Sputnik" is a dsDNA virus, referred to as a virophage, that is coassembled with Mimivirus in the host amoeba. We have used cryo-EM to produce an electron density map of the icosahedral Sputnik virus at 3.5-Å resolution, sufficient to verify the identity of most amino acids in the capsid proteins and to establish the identity of the pentameric protein forming the fivefold vertices. It was also shown that the virus lacks an internal membrane. The capsid is organized into a T = 27 lattice in which there are 260 trimeric capsomers and 12 pentameric capsomers. The trimeric capsomers consist of three double "jelly-roll" major capsid proteins creating pseudohexameric capsomer symmetry. The pentameric capsomers consist of five single jelly-roll proteins. The release of the genome by displacing one or more of the pentameric capsomers may be the result of a low-pH environment. These results suggest a mechanism of Sputnik DNA ejection that probably also occurs in other big icosahedral double jelly-roll viruses such as Adenovirus. In this study, the near-atomic resolution structure of a virus has been established where crystallization for X-ray crystallography was not feasible.

Structure of large dsDNA viruses.

Nucleocytoplasmic large dsDNA viruses (NCLDVs) encompass an ever-increasing group of large eukaryotic viruses, infecting a wide variety of organisms. The set of core genes shared by all these viruses includes a major capsid protein with a double jelly-roll fold forming an icosahedral capsid, which surrounds a double layer membrane that contains the viral genome. Furthermore, some of these viruses, such as the members of the Mimiviridae and Phycodnaviridae have a unique vertex that is used during infection to transport DNA into the host.

Crystal structure of a poxvirus-like zalpha domain from cyprinid herpesvirus 3.

Zalpha domains are a subfamily of the winged helix-turn-helix domains sharing the unique ability to recognize CpG repeats in the left-handed Z-DNA conformation. In vertebrates, domains of this family are found exclusively in proteins that detect foreign nucleic acids and activate components of the antiviral interferon response. Moreover, poxviruses encode the Zalpha domain-containing protein E3L, a well-studied and potent inhibitor of interferon response. Here we describe a herpesvirus Zalpha-domain-containing protein (ORF112) from cyprinid herpesvirus 3. We demonstrate that ORF112 also binds CpG repeats in the left-handed conformation, and moreover, its structure at 1.75 Å reveals the Zalpha fold found in ADAR1, DAI, PKZ, and E3L. Unlike other Zalpha domains, however, ORF112 forms a dimer through a unique domain-swapping mechanism. Thus, ORF112 may be considered a new member of the Z-domain family having DNA binding properties similar to those of the poxvirus E3L inhibitor of interferon response.

Backbone structure of the infectious epsilon15 virus capsid revealed by electron cryomicroscopy.

A half-century after the determination of the first three-dimensional crystal structure of a protein, more than 40,000 structures ranging from single polypeptides to large assemblies have been reported. The challenge for crystallographers, however, remains the growing of a diffracting crystal. Here we report the 4.5-A resolution structure of a 22-MDa macromolecular assembly, the capsid of the infectious epsilon15 (epsilon15) particle, by single-particle electron cryomicroscopy. From this density map we constructed a complete backbone trace of its major capsid protein, gene product 7 (gp7). The structure reveals a similar protein architecture to that of other tailed double-stranded DNA viruses, even in the absence of detectable sequence similarity. However, the connectivity of the secondary structure elements (topology) in gp7 is unique. Protruding densities are observed around the two-fold axes that cannot be accounted for by gp7. A subsequent proteomic analysis of the whole virus identifies these densities as gp10, a 12-kDa protein. Its structure, location and high binding affinity to the capsid indicate that the gp10 dimer functions as a molecular staple between neighbouring capsomeres to ensure the particle's stability. Beyond epsilon15, this method potentially offers a new approach for modelling the backbone conformations of the protein subunits in other macromolecular assemblies at near-native solution states.

Characterization of a virulent bacteriophage LK1 specific for Citrobacter freundii isolated from sewage water.

Citrobacter freundii is a worldwide emerging nosocomial pathogen with escalating incidence of multidrug resistance. Citrobacter freundii exists in natural environment, especially in health care settings and is difficult to eradicate. Phage therapy is considered as an alternative way of controlling bacterial infections and contaminations. In this study, we have described isolation and characterization of a virulent bacteriophage LK1 capable of specifically infecting Citrobacter freundii. A virulent bacteriophage LK1, specific for Citrobacter freundii was isolated from sewage water sample. TEM showed that phage Lk1 has an icosahedral head 70 nm in diameter and short tail of 17 nm, and can be classified as a member of the Podoviridae family. Restriction analysis indicated that phage LK1 was a dsDNA virus with an approximate genome size of 20-23 kb. Proteomic pattern generated by SDS PAGE using purified LK1 phage particles, revealed three major and six minor protein bands with molecular weight ranging from 25 to 80 kDa. Adsorption rate of LK1 relative to the host bacterium was also determined which showed significant improvement in adsorption with the addition of CaCl2 . In a single step growth experiment, LK1 exhibited a latent period of 24 min and burst size of 801 particle/cell. Moreover, pH and thermal stability of phage LK1 demonstrated a pH range of 5.0-6.0 and phage viability decreased to 0% at 65 °C. When LK1 was used to infect six other clinically isolated pathogenic strains, it showed relatively narrow host range. LK1 was capable of eliciting efficient lysis of Citrobacter freundii, revealing its potential as a non-toxic sanitizer for controlling Citrobacter freundii infection and contamination in both hospital and other public environments.

Liquid-liquid Phase Separation in Viral Function.

An emerging set of results suggests that liquid-liquid phase separation (LLPS) is the basis for the formation of membrane-less compartments in cells. Evidence is now mounting that various types of virus-induced membrane-less compartments and organelles are also assembled via LLPS. Specifically, viruses appear to use intracellular phase transitions to form subcellular microenvironments known as viral factories, inclusion bodies, or viroplasms. These compartments - collectively referred to as viral biomolecular condensates - can be used to concentrate replicase proteins, viral genomes, and host proteins that are required for virus replication. They can also be used to subvert or avoid the intracellular immune response. This review examines how certain DNA or RNA viruses drive the formation of viral condensates, the possible biological functions of those condensates, and the biophysical and biochemical basis for their assembly.

Vital for Viruses: Intrinsically Disordered Proteins.

Viruses infect all kingdoms of life; their genomes vary from DNA to RNA and in size from 2kB to 1 MB or more. Viruses frequently employ disordered proteins, that is, protein products of virus genes that do not themselves fold into independent three-dimensional structures, but rather, constitute a versatile molecular toolkit to accomplish a range of functions necessary for viral infection, assembly, and proliferation. Interestingly, disordered proteins have been discovered in almost all viruses so far studied, whether the viral genome consists of DNA or RNA, and whatever the configuration of the viral capsid or other outer covering. In this review, I present a wide-ranging set of stories illustrating the range of functions of IDPs in viruses. The field is rapidly expanding, and I have not tried to include everything. What is included is meant to be a survey of the variety of tasks that viruses accomplish using disordered proteins.

Role of channel lysines and the "push through a one-way valve" mechanism of the viral DNA packaging motor.

Linear double-stranded DNA (dsDNA) viruses package their genomes into preformed protein shells via nanomotors using ATP as an energy source. The central hub of the bacteriophage φ29 DNA-packaging motor contains a 3.6-nm channel for dsDNA to enter during packaging and to exit during infection. The negatively charged interior channel wall is decorated with a total of 48 positively charged lysine residues displayed as four 12-lysine rings from the 12 gp10 subunits that enclose the channel. The standard notion derived from many models is that these uniquely arranged, positively charged rings play active roles in DNA translocation through the channel. In this study, we tested this prevailing view by examining the effect of mutating these basic lysines to alanines, and assessing the impact of altering the pH environment. Unexpectedly, mutating these basic lysine residues or changing the pH to 4 or 10, which could alter the charge of lysines, did not measurably impair DNA translocation or affect the one-way traffic property of the channel. The results support our recent findings regarding the dsDNA packaging mechanism known as the "push through a one-way valve".

RNA silencing pathways of plants: silencing and its suppression by plant DNA viruses.

RNA silencing refers to processes that depend on small (s)RNAs to regulate the expression of eukaryotic genomes. In plants, these processes play critical roles in development, in responses to a wide array of stresses, in maintaining genome integrity and in defense against viral and bacterial pathogens. We provide here an updated view on the array of endogenous sRNA pathways, including microRNAs (miRNAs), discovered in the model plant Arabidopsis, which are also the basis for antiviral silencing. We emphasize the current knowledge as well as the recent advances made on understanding the defense and counter-defense strategies evolved in the arms race between plants and DNA viruses on both the nuclear and the cytoplasmic front. This article is part of a Special Issue entitled: MicroRNA's in viral gene regulation.

Structural studies of the giant mimivirus.

Mimivirus is the largest known virus whose genome and physical size are comparable to some small bacteria, blurring the boundary between a virus and a cell. Structural studies of Mimivirus have been difficult because of its size and long surface fibers. Here we report the use of enzymatic digestions to remove the surface fibers of Mimivirus in order to expose the surface of the viral capsid. Cryo-electron microscopy (cryoEM) and atomic force microscopy were able to show that the 20 icosahedral faces of Mimivirus capsids have hexagonal arrays of depressions. Each depression is surrounded by six trimeric capsomers that are similar in structure to those in many other large, icosahedral double-stranded DNA viruses. Whereas in most viruses these capsomers are hexagonally close-packed with the same orientation in each face, in Mimivirus there are vacancies at the systematic depressions with neighboring capsomers differing in orientation by 60 degrees . The previously observed starfish-shaped feature is well-resolved and found to be on each virus particle and is associated with a special pentameric vertex. The arms of the starfish fit into the gaps between the five faces surrounding the unique vertex, acting as a seal. Furthermore, the enveloped nucleocapsid is accurately positioned and oriented within the capsid with a concave surface facing the unique vertex. Thus, the starfish-shaped feature and the organization of the nucleocapsid might regulate the delivery of the genome to the host. The structure of Mimivirus, as well as the various fiber components observed in the virus, suggests that the Mimivirus genome includes genes derived from both eukaryotic and prokaryotic organisms. The three-dimensional cryoEM reconstruction reported here is of a virus with a volume that is one order of magnitude larger than any previously reported molecular assembly studied at a resolution of equal to or better than 65 Angstroms.

Numerous variants of leucine rich repeats in proteins from nucleo-cytoplasmic large DNA viruses.

Leucine rich repeats (LRRs) occurring in tandem are 20-29 amino acids long. Eleven LRR types have been recognized. Sequence features of LRRs from viruses were investigated using over 600 LRR proteins from 89 species. Directly before, metagenome data of nucleo-cytoplasmic large dsDNA viruses (NCLDVs) have been published; the 2,074 NCLDVs encode 199,021 proteins. From the NCLDVs 547 LRR proteins were identified and 502 were used for analysis. Various variants of known LRR types were identified in viral LRRs. A comprehensive analysis of TpLRR and FNIP that belong to an LRR type was first performed. The repeating unit lengths (RULs) in five types are 19 residues which is the shortest among all LRRs. The RULs of eight LRR types including FNIP are one to five residues shorter than those of the known, corresponding LRR types. The conserved hydrophobic residues such as Leu, Val or Ile in the consensus sequences are frequently substituted by cysteine at one or two positions. Four unique LRR motifs that are different from those identified previously are observed. The present study enhances the previous result. An evolutionary scenario of short or unique LRR was discussed.

The Role of Tape Measure Protein in Nucleocytoplasmic Large DNA Virus Capsid Assembly.

Nucleocytoplasmic large DNA viruses (NCLDVs) are a group of large viruses that infect a wide range of hosts, from animals to protists. These viruses are grouped together in NCLDV based on genomic sequence analyses. They share a set of essential genes for virion morphogenesis and replication. Most NCLDVs generally have large physical sizes while their morphologies vary in different families, such as icosahedral, brick, or oval shape, raising the question of the possible regulatory factor on their morphogenesis. The capsids of icosahedral NCLDVs are assembled from small building blocks, named capsomers, which are the trimeric form of the major capsid proteins. Note that the capsids of immature poxvirus are spherical even though they are assembled from capsomers that share high structural conservation with those icosahedral NCLDVs. The recently published high resolution structure of NCLDVs, Paramecium bursaria Chlorella virus 1 and African swine fever virus, described the intensive network of minor capsid proteins that are located underneath the capsomers. Among these minor proteins is the elongated tape measure protein (TmP) that spans from one icosahedral fivefold vertex to another. In this study, we focused on the critical roles that TmP plays in the assembly of icosahedral NCLDV capsids, answering a question raised in a previously proposed spiral mechanism. Interestingly, basic local alignment search on the TmPs showed no significant hits in poxviruses, which might be the factor that differentiates poxviruses and icosahedral NCLDVs in their morphogenesis.