Component tree analysis of cystovirus φ6 nucleocapsid Cryo-EM single particle reconstructions.

The 3-dimensional structure of the nucleocapsid (NC) of bacteriophage φ6 is described utilizing component tree analysis, a topological and geometric image descriptor. The component trees are derived from density maps of cryo-electron microscopy single particle reconstructions. Analysis determines position and occupancy of structure elements responsible for RNA packaging and transcription. Occupancy of the hexameric nucleotide triphosphorylase (P4) and RNA polymerase (P2) are found to be essentially complete in the NC. The P8 protein lattice likely fixes P4 and P2 in place during maturation. We propose that the viral procapsid (PC) is a dynamic structural intermediate where the P4 and P2 can attach and detach until held in place in mature NCs. During packaging, the PC expands to accommodate the RNA, and P2 translates from its original site near the inner 3-fold axis (20 sites) to the inner 5-fold axis (12 sites) with excess P2 positioned inside the central region of the NC.

Disassembly of the cystovirus ϕ6 envelope by montmorillonite clay.

Prior studies of clay-virus interactions have focused on the stability and infectivity of nonenveloped viruses, yielding contradictory results. We hypothesize that the surface charge distribution of the clay and virus envelope dictates how the components react and affect aggregation, viral stability, and infectivity. The bacteriophage Cystoviridae species φ6 used in this study is a good model for enveloped pathogens. The interaction between φ6 and montmorillonite (MMT) clay (the primary component of bentonite) is explored by transmission electron microscopy. The analyses show that MMT-φ6 mixtures undergo heteroaggregation, forming structures in which virtually all the virions are either sequestered between MMT platelet layers or attached to platelet edges. The virions swell and undergo disassembly resulting in partial or total envelope loss. Edge-attached viral envelopes distort to increase contact area with the positively charged platelet edges indicating that the virion surface is negatively charged. The nucleocapsid (NCs) remaining after envelope removal also exhibit distortion, in contrast to detergent-produced NCs which exhibit no distortion. This visually discernible disassembly is a mechanism for loss of infectivity previously unreported by studies of nonenveloped viruses. The MMT-mediated sequestration and disassembly result in reduced infectivity, suggesting that clays may reduce infectivity of enveloped pathogenic viruses in soils and sediments.

Cystovirus maturation at atomic resolution.

Cystoviruses are dsRNA viruses that infect bacteria and include bacteriophages Φ8 and Φ6. In this issue of Structure, El Omari and colleagues and Nemecek and colleagues report crystal structures of capsid protein P1 pentamers found in procapsid cores of Φ8 and Φ6. The two structures show a striking resemblance in the absence of sequence similarity and offer new mechanistic and evolutionary insights.

Plate tectonics of virus shell assembly and reorganization in phage φ8, a distant relative of mammalian reoviruses.

The hallmark of a virus is its capsid, which harbors the viral genome and is formed from protein subunits, which assemble following precise geometric rules. dsRNA viruses use an unusual protein multiplicity (120 copies) to form their closed capsids. We have determined the atomic structure of the capsid protein (P1) from the dsRNA cystovirus Φ8. In the crystal P1 forms pentamers, very similar in shape to facets of empty procapsids, suggesting an unexpected assembly pathway that proceeds via a pentameric intermediate. Unlike the elongated proteins used by dsRNA mammalian reoviruses, P1 has a compact trapezoid-like shape and a distinct arrangement in the shell, with two near-identical conformers in nonequivalent structural environments. Nevertheless, structural similarity with the analogous protein from the mammalian viruses suggests a common ancestor. The unusual shape of the molecule may facilitate dramatic capsid expansion during phage maturation, allowing P1 to switch interaction interfaces to provide capsid plasticity.

Assembly of large icosahedral double-stranded RNA viruses.

Double-stranded RNA (dsRNA) viruses are a diverse group of viruses infecting hosts from bacteria to higher eukaryotes. Among the hosts are humans, domestic animals, and economically important plant species. Fine details of high-resolution virion structures have revealed common structural characteristics unique to these viruses including an internal icosahedral capsid built from 60 asymmetric dimers (120 monomers!) of the major coat protein. Here we focus mainly on the structures and assembly principles of large icosahedral dsRNA viruses belonging to the families of Cystoviridae and Reoviridae. It is obvious that there are a variety of assembly pathways utilized by different viruses starting from similar building blocks and reaching in all cases a similar capsid architecture. This is true even with closely related viruses indicating that the assembly pathway per se is not an indicator of relatedness and is achieved with minor changes in the interacting components.

Electron cryo-tomographic structure of cystovirus phi 12.

Bacteriophage phi 12 is a member of the Cystoviridae virus family and contains a genome consisting of three segments of double-stranded RNA (dsRNA). This virus family contains eight identified members, of which four have been classified in regard to their complete genomic sequence and encoded viral proteins. A phospholipid envelope that contains the integral proteins P6, P9, P10, and P13 surrounds the viral particles. In species phi 6, host infection requires binding of a multimeric P3 complex to type IV pili. In species varphi8, phi 12, and phi 13, the attachment apparatus is a heteromeric protein assembly that utilizes the rough lipopolysaccharide (rlps) as a receptor. In phi 8 the protein components are designated P3a and P3b while in species phi 12 proteins P3a and P3c have been identified in the complex. The phospholipid envelope of the cystoviruses surrounds a nucleocapsid (NC) composed of two shells. The outer shell is composed of protein P8 with a T=13 icosahedral lattice while the primary component of the inner shell is a dodecahedral frame composed of dimeric protein P1. For the current study, the 3D architecture of the intact phi 12 virus was obtained by electron cryo-tomography. The nucleocapsid appears to be centered within the membrane envelope and possibly attached to it by bridging structures. Two types of densities were observed protruding from the membrane envelope. The densities of the first type were elongated, running parallel, and closely associated to the envelope outer surface. In contrast, the second density was positioned about 12 nm above the envelope connected to it by a flexible low-density stem. This second structure formed a torroidal structure termed "the donut" and appears to inhibit BHT-induced viral envelope fusion.

Electron cryomicroscopy comparison of the architectures of the enveloped bacteriophages phi6 and phi8.

The enveloped dsRNA bacteriophages phi6 and phi8 are the two most distantly related members of the Cystoviridae family. Their structure and function are similar to that of the Reoviridae but their assembly can be conveniently studied in vitro. Electron cryomicroscopy and three-dimensional icosahedral reconstruction were used to determine the structures of the phi6 virion (14 A resolution), phi8 virion (18 A resolution), and phi8 core (8.5 A resolution). Spikes protrude 2 nm from the membrane bilayer in phi6 and 7 nm in phi8. In the phi6 nucleocapsid, 600 copies of P8 and 72 copies of P4 interact with the membrane, whereas in phi8 it is only P4 and 60 copies of a minor protein. The major polymerase complex protein P1 forms a dodecahedral shell from 60 asymmetric dimers in both viruses, but the alpha-helical fold has apparently diverged. These structural differences reflect the different host ranges and entry and assembly mechanisms of the two viruses.

Virus particles monitored by fluorescence spectroscopy: a potential detection assay for macromolecular assembly.

Native fluorescence spectroscopy was used for in situ investigations of two lipid-containing bacteriophages from the cystovirus family as well as their Pseudomonad host cells. Both the viruses phi6 and phi12 and their bacterial host proteins contain the amino acid tryptophan (trp), which is the predominant fluorophore in UV. Within proteins, trp's structural environment differs, and the differences are reflected in their spectroscopic signatures. It was observed that the peak of the trp emission from both viruses was at 330 nm, a significantly shorter wavelength than trp in either the Pseudomonad host cells or the amino acid's chemical form. This allowed us to monitor the viral attachment process and subsequent lytic release of progeny virus particles by measurement of the trp emission spectra during the infection process. This work demonstrates that fluorescence may offer a novel tool to detect viruses and monitor viral infection of cells and may be part of a biodefense application.

Packaging, replication and recombination of the segmented genome of bacteriophage Phi6 and its relatives.

The genomes of bacteriophage Phi6 and its relatives are packaged through a mechanism that involves the recognition and translocation of the three different plus strand transcripts of the segmented dsRNA genomes into preformed polyhedral structures called procapsids or inner cores. The packaging requires hydrolysis of NTPs and takes place in the order S:M:L. Minus strand synthesis begins after the completion of the plus strand packaging. The packaging and replication reactions can be studied in vitro with purified components. A model has been presented that proposes that the program of serially dependent packaging is determined by the conformational changes at the surface of the procapsid due to the amount of RNA packaged at each step. The in vitro packaging and replication system has facilitated the application of reverse genetics and the study of recombination in the family of Cystoviridae.

Bacteriophage observations and evolution.

Bacteriophages are classified into one order and 13 families. Over 5100 phages have been examined in the electron microscope since 1959. At least 4950 phages (96%) are tailed. They constitute the order Caudovirales and three families. Siphoviridae or phages with long, noncontractile tails predominate (61% of tailed phages). Polyhedral, filamentous, and pleomorphic phages comprise less than 4% of bacterial viruses. Bacteriophages occur in over 140 bacterial or archaeal genera. Their distribution reflects their origin and bacterial phylogeny. Bacteriophages are polyphyletic, arose repeatedly in different hosts, and constitute 11 lines of descent. Tailed phages appear as monophyletic and as the oldest known virus group.