In this issue of Structure, Lerch and colleagues present a 4.5 Å cryo electron microscopy (cryoEM) structure of a variant of an adeno-associated virus that has been genetically engineered for liver gene therapy. The identification of two structurally distinct loops flanking the highly conserved jellyroll β barrel highlights the potentials of high-resolution cryoEM.
To avoid the challenges of crystallization and the size limitations of NMR, it has long been hoped that single-particle cryo-electron microscopy (cryo-EM) would eventually yield atomically interpretable reconstructions. For the most favorable class of specimens (large icosahedral viruses), one of the key obstacles is curvature of the Ewald sphere, which leads to a breakdown of the Projection Theorem used by conventional three-dimensional (3D) reconstruction programs. Here, we review the basic problem and our implementation of the “paraboloid” reconstruction method, which overcomes the limitation by averaging information from images recorded from different points of view.
To bring cryo electron microscopy (cryoEM) of large biological complexes to atomic resolution, several factors – in both cryoEM image acquisition and 3D reconstruction – that may be neglected at low resolution become significantly limiting. Here we present thorough analyses of four limiting factors: (a) electron-beam tilt, (b) inaccurate determination of defocus values, (c) focus gradient through particles, and (d) particularly for large particles, dynamic (multiple) scattering of electrons. We also propose strategies to cope with these factors: (a) the divergence and direction tilt components of electron-beam tilt could be reduced by maintaining parallel illumination and by using a coma-free alignment procedure, respectively. Moreover, the effect of all beam tilt components, including spiral tilt, could be eliminated by use of a spherical aberration corrector. (b) More accurate measurement of defocus value could be obtained by imaging areas adjacent to the target area at high electron dose and by measuring the image shift induced by tilting the electron beam. (c) Each known Fourier coefficient in the Fourier transform of a cryoEM image is the sum of two Fourier coefficients of the 3D structure, one on each of two curved ‘characteristic surfaces’ in 3D Fourier space. We describe a simple model-based iterative method that could recover these two Fourier coefficients on the two characteristic surfaces. (d) The effect of dynamic scattering could be corrected by deconvolution of a transfer function. These analyses and our proposed strategies offer useful guidance for future experimental designs targeting atomic resolution cryoEM reconstruction.
cryoEM; Atomic resolution; Beam tilt; Dynamic scattering; Defocus gradient
Storage granules are an important component of metabolism in many organisms spanning the bacterial, eukaryal and archaeal domains, but systematic analysis of their organization inside cells is lacking. In this study, we identify and characterize granulelike inclusion bodies in a methanogenic archaeon, Methanospirillum hungatei, an anaerobic microorganism that plays an important role in nutrient recycling in the ecosystem. Using cryo electron microscopy, we show that granules in mature M. hungatei are amorphous in structure with a uniform size. Energy dispersive X-ray spectroscopy analysis establishes that each granule is a polyphosphate body (PPB) that consists of high concentrations of phosphorous and oxygen, and increased levels of iron and magnesium. By scanning transmission electron tomography, we further estimate that the mass density within a PPB is a little less than metal titanium at room temperature and is about four times higher than that of the surrounding cytoplasm. Finally, three-dimensional cryo electron tomography reveals that PPBs are positioned off-centre in their radial locations relative to the cylindrical axis of the cell, and almost uniformly placed near cell ends. This positioning ability points to a genetic program that spatially and temporally directs the accumulation of polyphosphate into a storage granule, perhaps for energy-consuming activities, such as cell maintenance, division or motility.
Single-particle cryo electron microscopy (cryoEM) is a technique for determining three-dimensional (3D) structures from projection images of molecular complexes preserved in their “native,” noncrystalline state. Recently, atomic or near-atomic resolution structures of several viruses and protein assemblies have been determined by single-particle cryoEM, allowing ab initio atomic model building by following the amino acid side chains or nucleic acid bases identifiable in their cryoEM density maps. In particular, these cryoEM structures have revealed extended arms contributing to molecular interactions that are otherwise not resolved by the conventional structural method of X-ray crystallography at similar resolutions. High-resolution cryoEM requires careful consideration of a number of factors, including proper sample preparation to ensure structural homogeneity, optimal configuration of electron imaging conditions to record high-resolution cryoEM images, accurate determination of image parameters to correct image distortions, efficient refinement and computation to reconstruct a 3D density map, and finally appropriate choice of modeling tools to construct atomic models for functional interpretation. This progress illustrates the power of cryoEM and ushers it into the arsenal of structural biology, alongside conventional techniques of X-ray crystallography and NMR, as a major tool (and sometimes the preferred one) for the studies of molecular interactions in supramolecular assemblies or machines.
Regulated by pH, membrane-anchored proteins E and M play a series of roles during dengue virus maturation and membrane fusion. Our atomic model of the whole virion from cryo electron microscopy at 3.5Å resolution reveals that in the mature virus at neutral extracellular pH, the N-terminal 20-amino acid segment of M (involving three pH-sensing histidines) latches and thereby prevents spring-loaded E fusion protein from prematurely exposing its fusion peptide. This M latch was fastened at an earlier stage, during maturation at acid pH in the trans-Golgi network. At a later stage, to initiate infection in response to acid pH in the late endosome, M releases the latch and exposes the fusion peptide. Thus, M serves as a multistep chaperone of E to control the conformational changes accompanying maturation and infection. These pH-sensitive interactions could serve as targets for drug discovery.
cryo electron microscopy; flavivirus; bio-threat agent; enveloped viruses; chaperone; viral maturation
Recent advances in cryo-electron microscopy and single-particle reconstruction (collectively referred to as “cryoEM”) have made it possible to determine the three-dimensional (3D) structures of several macromolecular complexes at near-atomic resolution (~3.8 – 4.5 Å). These achievements were accomplished by overcoming challenges in sample handling, instrumentation, image processing, and model building. At near-atomic resolution, many detailed structural features can be resolved, such as the turns and deep grooves of helices, strand separation in β sheets, and densities for loops and bulky amino acid side chains. Such structural data of the cytoplasmic polyhedrosis virus (CPV), the Epsilon 15 bacteriophage and the GroEL complex have provided valuable constraints for atomic model building using integrative tools, thus significantly enhancing the value of the cryoEM structures. The CPV structure revealed a drastic conformational change from a helix to a β hairpin associated with RNA packaging and replication, coupling of RNA processing and release, and the long sought-after polyhedrin-binding domain. These latest advances in single-particle cryoEM provide exciting opportunities for the 3D structural determination of viruses and macromolecular complexes that are either too large or too heterogeneous to be investigated by conventional X-ray crystallography or nuclear magnetic resonance (NMR) methods.
Human cytomegalovirus (HCMV) is the most genetically and structurally complex human herpesvirus and is composed of an envelope, a tegument, and a dsDNA-containing capsid. HCMV tegument plays essential roles in HCMV infection and assembly. Using cryo-electron tomography (cryoET), here we show that HCMV tegument compartment can be divided into two sub-compartments: an inner and an outer tegument. The inner tegument consists of densely-packed proteins surrounding the capsid. The outer tegument contains those components that are loosely packed in the space between the inner tegument and the pleomorphic glycoprotein-containing envelope. To systematically characterize the inner tegument proteins interacting with the capsid, we used chemical treatment to strip off the entire envelope and most tegument proteins to obtain a tegumented capsid with inner tegument proteins. SDS-polyacrylamide gel electrophoresis analyses show that only two tegument proteins, UL32-encoded pp150 and UL48-encoded high molecular weight protein (HMWP), remains unchanged in their abundance in the tegumented capsids as compared to their abundance in the intact particles. 3D reconstructions by single particle cryo-electron microscopy (cryoEM) reveal that the net-like layer of icosahedrally-ordered tegument densities are also the same in the tegumented capsid and in the intact particles. CryoET reconstruction of the tegumented capsid labeled with an anti-pp150 antibody is consistent with the biochemical and cryoEM data in localizing pp150 within the ordered tegument. Taken together, these results suggest that pp150, a betaherpesvirus-specific tegument protein, is a constituent of the net-like layer of icosahedrally-ordered capsid-bound tegument densities, a structure lacking similarities in alpha and gammaherpesviruses.
human cytomegalovirus; tegument proteins; single-particle analysis; cryo-electron microscopy; cryo-electron tomography; chemical treatment; pp150
Deregulation of mini-chromosome maintenance (MCM) proteins is associated with genomic instability and cancer. MCM complexes are recruited to replication origins for genome duplication. Paradoxically, MCM proteins are in excess than the number of origins and are associated with chromatin regions away from the origins during G1 and S phases. Here, we report an unusually wide left-handed filament structure for an archaeal MCM, as determined by X-ray and electron microscopy. The crystal structure reveals that an α-helix bundle formed between two neighboring subunits plays a critical role in filament formation. The filament has a remarkably strong electro-positive surface spiraling along the inner filament channel for DNA binding. We show that this MCM filament binding to DNA causes dramatic DNA topology change. This newly identified function of MCM to change DNA topology may imply a wider functional role for MCM in DNA metabolisms beyond helicase function. Finally, using yeast genetics, we show that the inter-subunit interactions, important for MCM filament formation, play a role for cell growth and survival.
To achieve cell entry, many nonenveloped viruses must transform from a dormant to a primed state. In contrast to the membrane fusion mechanism of enveloped viruses (e.g., influenza virus), this membrane penetration mechanism is poorly understood. Here, using single-particle cryo-electron microscopy, we report a 3.3 Å structure of the primed, infectious subvirion particle of aquareovirus. The density map reveals side-chain densities of all types of amino acids (except glycine), enabling construction of a full-atom model of the viral particle. Our structure and biochemical results show that priming involves autocleavage of the membrane penetration protein and suggest that Lys84 and Glu76 may facilitate this autocleavage in a nucleophilic attack. We observe a myristoyl group, covalently linked to the N terminus of the penetration protein and embedded in a hydrophobic pocket. These results suggest a well-orchestrated process of nonenveloped virus entry involving autocleavage of the penetration protein prior to exposure of its membrane-insertion finger.
Construction of a complex virus may involve a hierarchy of assembly elements. Here, we report the structure of the whole human adenovirus virion at 3.6Å resolution by cryo-electron microscopy, revealing in situ atomic models of three minor capsid proteins (IIIa, VIII and IX), extensions of the major (penton base and hexon) capsid proteins, and interactions within three protein-protein networks. One network is mediated by protein IIIa within Group-of-Six (GOS) tiles – a penton base and its five surrounding hexons – at vertices. Another is mediated by ropes (protein IX) that lash hexons together to form Group-of-Nine (GON) tiles and bind GONs to GONs. The third, mediated by IIIa and VIII, binds each GOS to five surrounding GONs. Optimization of adenovirus for cancer and gene therapy could target these networks.
3-methylcrotonyl-CoA carboxylase (MCC), a member of the biotin-dependent carboxylase superfamily, is essential for the metabolism of leucine, and deficient mutations in this enzyme are linked to methylcrotonylglycinuria (MCG) and other serious diseases in humans 1–8. MCC has strong sequence conservation with propionyl-CoA carboxylase (PCC), and their holoenzymes are both 750 kD α6β6 dodecamers. Therefore the architecture of the MCC holoenzyme is expected to be highly similar to that of PCC 9. Here we report the crystal structures of the Pseudomonas aeruginosa MCC (PaMCC) holoenzyme, alone and in complex with coenzyme A. Surprisingly, the structures show that the architecture and overall shape of PaMCC are strikingly different when compared to PCC. The α subunits display trimeric association in the PaMCC holoenzyme while they have no contacts with each other in PCC. Moreover, the positions of the two domains in the β subunit in PaMCC are swapped relative to those in PCC. The structural information establishes a foundation for understanding the disease-causing mutations of MCC and provides new insights into the catalytic mechanism and evolution of biotin-dependent carboxylases. The large structural differences between MCC and PCC also have general implications for the relationship between sequence conservation and structural similarity.
fatty acid metabolism; amino acid metabolism; propionic acidemia; 3-methylcrotonylglycinuria; protein complex; biotin-dependent carboxylase; acetyl-CoA carboxylase; propionyl-CoA carboxylase
Adenovirus invades host cells by first binding to host receptors through a trimeric fiber, which contains three domains: a receptor-binding knob domain, a long flexible shaft domain and a penton base-attachment tail domain. Although the structure of the knob domain associated with a portion of the shaft has been solved by X-ray crystallography, the in situ structure of the fiber in the virion is not known; thus it remains a mystery how the trimeric fiber attaches to its underlying pentameric penton base. By high-resolution cryo-electron microscopy (cryoEM), we have determined the structure of the human adenovirus type 5 (Ad5) to 3.6Å resolution and have reported the full atomic models for its capsid proteins, but not for the fiber whose density cannot be directly interpreted due to symmetry mismatch with the penton base. Here we report the determination of the Ad5 fiber structure and its mode of attachment to the pentameric penton base by using an integrative approach of multi-resolution filtering, homology modeling, computational simulation of mismatched symmetries and fitting of atomic models into cryoEM density maps. Our structure reveals that the interactions between the trimeric fiber and the pentameric penton base are mediated by a hydrophobic ring on the top surface of the penton base and three flexible tails inserted into three of the five available grooves formed by neighboring subunits of penton base. These interaction sites provide the molecular basis for the symmetry mismatch and can be targeted for optimizing adenovirus for gene therapy applications.
adenovirus; fiber; penton base; symmetry mismatch; cryoEM
Type IV pili (TFP) are membrane-anchored filaments with a number of important biological functions. In the model organism Myxococcus xanthus, TFP act as molecular engines that power social (S) motility through cycles of extension and retraction. TFP filaments consist of several thousand copies of a protein called PilA or pilin. PilA contains an N-terminal α-helix essential for TFP assembly and a C-terminal globular domain important for its activity. The role of the PilA sequence and its structure–function relationship in TFP-dependent S motility remain active areas of research. In this study, we identified an M. xanthus PilA mutant carrying an alanine to valine substitution at position 32 in the α-helix, which produced structurally intact but retraction-defective TFP. Characterization of this mutant and additional single-residue variants at this position in PilA demonstrated the critical role of alanine 32 in PilA stability, TFP assembly and retraction.
Although the aspect ratio (AR) of engineered nanomaterials (ENMs) is one of the key physicochemical parameters that could determine biological outcome, not much is understood about how AR contributes to shaping biological outcome. By using a mesoporous silica nanoparticle (MSNP) library that has been constructed to cover a range of different lengths, we could demonstrate that the AR of rod-shaped particles determine the rate and abundance of MSNP uptake by a macropinocytosis process in HeLa and A549 cancer cell lines. MSNPs with an AR of 2.1–2.5 were taken up in larger quantities compared to shorter or longer length rods by a process that is sensitive to amiloride, cytochalasin D, azide and 4 °C inhibition. The rods with intermediary AR also induced the maximal number of filopodia, actin polymerization and activation of small GTP-binding proteins (e.g. Rac1, CDC42) that involve assembly of the actin cytoskeleton and filopodia formation. When assessing the role of AR in the delivery of paclitaxel or camptothecin, the rods with AR 2.1–2.5 were clearly more efficient for drug delivery and generation of cytotoxic killing in HeLa cells. All considered, our data suggest an active sensoring mechanism by which HeLa and A549 cells are capable of detecting AR differences in MSNP to the extent that accelerated macropinocytosis can be used to achieve more efficient drug delivery.
Aspect ratio; Macropinocytosis; Cell uptake; Mesoporous silica nanoparticles; Drug delivery; Anticancer drug
Recent advancements in cryo-electron microscopy (cryoEM) have made it technically possible to determine the three-dimensional (3D) structures of macromolecular complexes at atomic resolution. However, processing the large amount of data needed for atomic resolution reconstructions requires either accessing to very expensive computer clusters or waiting for weeks of continuous computation in a personal computer (PC). In this paper, we present a practical computational solution to this 3D reconstruction problem through the optimal utilization of the processing capabilities of both commodity graphics hardware [i.e., general purpose graphics processing unit (GPGPU)]. Our solution, which is implemented in a new program, called eLite3D, has a number of advanced features of general interests. We construct interleaved schemes to prevent the data race condition intrinsic in merging of 2D data into a 3D volume. The speedup of eLite3D is up to 100 times over other commonly used 3D reconstruction programs with the same accuracy, thus allowing completion of atomic resolution 3D reconstructions of large complexes in a PC in 1–2 hours other than days or weeks. Our result provides a practical solution to atomic resolution cryoEM (asymmetric or symmetric) reconstruction and offers useful guidelines for developing GPGPU applications in general.
atomic resolution; cryoEM; 3D reconstruction; GPGPU; parallel processing; CUDA
Flagellum motility is critical for normal human development and for transmission of pathogenic protozoa that cause tremendous human suffering worldwide. Biophysical principles underlying motility of eukaryotic flagella are conserved from protists to vertebrates. However, individual cells exhibit diverse waveforms that depend on cell-specific elaborations on basic flagellum architecture. Trypanosoma brucei is a uniflagellated protozoan parasite that causes African sleeping sickness. The T. brucei flagellum is comprised of a 9+2 axoneme and an extra-axonemal paraflagellar rod (PFR), but the three-dimensional (3D) arrangement of the underlying structural units is poorly defined. Here, we use dual-axis electron tomography to determine the 3D architecture of the T. brucei flagellum. We define the T. brucei axonemal repeating unit. We observe direct connections between the PFR and axonemal dyneins, suggesting a mechanism by which mechanochemical signals may be transmitted from the PFR to axonemal dyneins. We find that the PFR itself is comprised of overlapping laths organized into distinct zones that are connected through twisting elements at the zonal interfaces. The overall structure has an underlying 57nm repeating unit. Biomechanical properties inferred from PFR structure lead us to propose that the PFR functions as a biomechanical spring that may store and transmit energy derived from axonemal beating. These findings provide insight into the structural foundations that underlie the distinctive flagellar waveform that is a hallmark of T. brucei cell motility.
Previous studies have described the structure of purified cytoplasmic polyhedrosis virus (CPV) and that of polyhedrin protein. However, how polyhedrin molecules embed CPV particles inside infectious polyhedra is not known. By using electron tomography, we show that CPV particles are occluded within the polyhedrin crystalline lattice with a random spatial distribution and interact with the polyhedrin protein through the A-spike rather than as previously thought through the B-spike. Furthermore, both full (with RNA) and empty (no RNA) capsids were found inside polyhedra, suggesting a spontaneous RNA encapsidating process for CPV assembly in vivo.
High resolution cryo-electron tomography (cryo-ET) was utilized to visualize Treponema pallidum, the causative agent of syphilis, at the molecular level. Three-dimensional (3-D) reconstructions from 304 infectious organisms revealed unprecedented cellular structures of this unusual member in the spirochetal family. High resolution cryo-ET reconstructions provided the detailed structures of the cell envelope, which is significantly different from that of gram-negative bacteria. The 4 nm lipid bilayer of both outer and cytoplasmic membranes resolved in 3-D reconstructions, providing an important marker for interpreting membrane-associated structures. Abundant lipoproteins cover the outer leaflet of the cytoplasmic membrane, in contrast to the rare outer membrane proteins visible by scanning probe microscopy. High resolution cryo-ET images also provided the first observation of T. pallidum chemoreceptor arrays, as well as structural details of the periplasmically located, cone-shaped structure at both ends of bacterium. Furthermore, 3-D subvolume averages of the periplasmic flagellar motors and filaments from living organisms revealed the novel flagellar architectures that may facilitate their rotation within the confining periplasmic space. Together, our findings provide the most detailed structural understanding of the periplasmic flagella and the surrounding cell envelope, which enable this enigmatic bacterium to efficiently penetrate tissue and escape host immune responses.
The genomic RNA of negative-strand RNA viruses, such as vesicular stomatitis virus (VSV), is completely enwrapped by the nucleocapsid protein (N) in every stage of virus infection. During viral transcription/replication, however, the genomic RNA in the nucleocapsid must be accessible by the virus-encoded RNA-dependent RNA polymerase in order to serve as the template for RNA synthesis. With the VSV nucleocapsid and a nucleocapsid-like particle (NLP) produced in Escherichia coli, we have found that the RNA in the VSV nucleocapsid can be removed by RNase A, in contrast to what was previously reported. Removal of the RNA did not disrupt the assembly of the N protein, resulting in an empty capsid. Polyribonucleotides were reencapsidated into the empty NLP, and the crystal structures were determined. The crystal structures revealed variable degrees of association of the N protein with a specific RNA sequence.
The Fab fragment of NC-1, a murine antibody that specifically recognizes the six-helix bundle core of HIV-1 gp41, has been crystallized in space group P3221. An X-ray diffraction data set was collected at 3.2 Å resolution and a clear molecular-replacement solution was obtained for solution of the structure.
NC-1 is a murine monoclonal antibody that specifically recognizes the six-helix bundle core of the human immunodeficiency virus type 1 (HIV-1) gp41. As such, it is a useful tool for probing gp41 conformations in HIV-1 membrane fusion. To establish the structural basis underlying the NC-1 specificity, X-ray crystallography was employed to solve its three-dimensional structure. To accomplish this, hybridoma-produced NC-1 antibody was first purified and digested with papain. Its Fab fragment was then purified using size-exclusion chromatography following Fc depletion using a Protein A affinity column. Finally, crystallization of NC-1 Fab was performed by the hanging-drop vapour-diffusion method and the protein was crystallized at pH 8.0 using PEG 6000 as precipitant. The results showed that the NC-1 Fab crystals belonged to the trigonal space group P3221, with unit-cell parameters a = b = 118.7, c = 106.0 Å. There is one Fab molecule in the asymmetric unit, with 67.5% solvent content. An X-ray diffraction data set was collected at 3.2 Å resolution and a clear molecular-replacement solution was obtained for solution of the structure.
NC-1 antibody; HIV-1 gp41; Fab fragments
Propionyl-coenzyme A carboxylase (PCC), a mitochondrial biotin-dependent enzyme, is essential for the catabolism of the amino acids Thr, Val, Ile and Met, cholesterol, and fatty acids with an odd number of carbon atoms. Deficiencies of PCC activity in humans are linked to the disease propionic acidemia (PA), an autosomal recessive disorder that can be fatal in infants 1–4. The holoenzyme of PCC is an α6β6 dodecamer, with a molecular weight of 750 kD. The α subunit contains the biotin carboxylase (BC) and biotin carboxyl carrier protein (BCCP) domains, while the β subunit supplies the carboxyltransferase (CT) activity. Here we report the crystal structure at 3.2 Å resolution of a bacterial PCC α6β6 holoenzyme as well as cryo-electron microscopy (cryo-EM) reconstructionat 15 Å resolution demonstrating a similar structure for human PCC. The structure defines the overall architecture of PCC and reveals unexpectedly that the α subunits are arranged as monomers in the holoenzyme, decorating a central β6 hexamer. A hitherto unrecognized domain in the α subunit, formed by residues between the BC and BCCP domains, is crucial for interactions with the β subunit. We have named it the BT domain. The structure reveals for the first time the relative positions of the BC and CT active sites in the holoenzyme. They are separated by approximately 55 Å, indicating that the entire BCCP domain must translocate during catalysis. The BCCP domain is located in the active site of the β subunit in the current structure, providing insight for its involvement in the CT reaction. The structural information establishes a molecular basis for understanding the large collection of disease-causing mutations in PCC, and also has important relevance for the holoenzymes of other biotin-dependent carboxylases, including 3-methylcrotonyl-CoA carboxylase (MCC) 5–7 and eukaryotic acetyl-CoA carboxylase (ACC) 8,9.
fatty acid metabolism; amino acid metabolism; propionic acidemia; 3-methylcrotonylglycinuria; protein complex; biotin-dependent carboxylase; acetyl-CoA carboxylase; 3-methylcrotonyl-CoA carboxylase
Gammaherpesviruses are etiologically associated with human tumors. A three-dimensional (3D) examination of their life cycle in the host is lacking, significantly limiting our understanding of the structural and molecular basis of virus-host interactions. Here, we report the first 3D visualization of key stages of the murine gammaherpesvirus 68 life cycle in NIH3T3 cells, including viral attachment, entry, assembly and egress, by dual-axis electron tomography. In particular, we revealed the transient processes of incoming capsids injecting viral DNA through nuclear pore complexes and nascent DNA being packaged into progeny capsids in vivo as a spool coaxial with the putative portal vertex. We discovered that intranuclear invagination of both nuclear membranes is involved in nuclear egress of herpesvirus capsids. Taken together, our results provide the structural basis for a detailed mechanistic description of gammaherpesvirus life cycle and also demonstrate the advantage of electron tomography in dissecting complex cellular processes of viral infection.
Gammaherpesvirus; Life cycle; Assembly; Electron Tomography
Influenza viruses are enveloped, negative stranded, segmented RNA viruses belonging to Orthomyxoviridae family. Each virion consists of three major subviral components, namely (i) a viral envelope decorated with three transmembrane proteins hemagglutinin (HA), neuraminidase (NA) and M2, (ii) an intermediate layer of matrix protein (M1), and (iii) an innermost helical viral ribonucleocapsid [vRNP] core formed by nucleoprotein (NP) and negative strand viral RNA (vRNA). Since complete virus particles are not found inside the cell, the processes of assembly, morphogenesis, budding and release of progeny virus particles at the plasma membrane of the infected cells are critically important for the production of infectious virions and pathogenesis of influenza viruses as well. Morphogenesis and budding require that all virus components must be brought to the budding site which is the apical plasma membrane in polarized epithelial cells whether in vitro cultured cells or in vivo infected animals. HA and NA forming the outer spikes on the viral envelope possess apical sorting signals and use exocytic pathways and lipid rafts for cell surface transport and apical sorting. NP also has apical determinant(s) and is probably transported to the apical budding site similarly via lipid rafts and/or through cortical actin microfilaments. M1 binds the NP and the exposed RNAs of vRNPs, as well as to the cytoplasmic tails (CT) and transmembrane (TM) domains of HA, NA and M2, and is likely brought to the budding site on the piggy-back of vRNP and transmembrane proteins.
Budding processes involve bud initiation, bud growth and bud release. Presence of lipid rafts and assembly of viral components at the budding site can cause asymmetry of lipid bilayers and outward membrane bending leading to bud initiation and bud growth. Bud release requires fusion of the apposing viral and cellular membranes and scission of the virus buds from the infected cellular membrane. The processes involved in bud initiation, bud growth and bud scission/release require involvement both viral and host components and can affect bud closing and virus release in both positive and negative ways. Among the viral components, M1, M2 and NA play important roles in bud release and M1, M2 and NA mutations all affect the morphology of buds and released viruses. Disassembly of host cortical actin microfilaments at the pinching-off site appears to facilitate bud fission and release. Bud scission is energy dependent and only a small fraction of virus buds present on the cell surface is released. Discontinuity of M1 layer underneath the lipid bilayer, absence of outer membrane spikes, absence of lipid rafts in the lipid bilayer, as well as possible presence of M2 and disassembly of cortical actin microfilaments at the pinching off site appear to facilitate bud fission and bud release. We provide our current understanding of these important processes leading to the production of infectious influenza virus particles.
Grass carp reovirus (GCRV) is a member of the Aquareovirus genus of the family Reoviridae, a large family of dsRNA viruses infecting plants, insects, fishes and mammals. We report the first subnanometer-resolution three-dimensional (3D) structures of both GCRV core and virion by cryo-electron microscopy (cryoEM). These structures have allowed the delineation of interactions among the over 1000 molecules in this enormous macromolecular machine, and a detail comparison with other dsRNA viruses at the secondary structure level. The GCRV core structure shows that the inner proteins have strong structural similarities even at the level of secondary structure elements with those of orthoreoviruses, indicating that the structures involved in viral dsRNA interaction and transcription are highly conserved. In contrast, the level of similarity in structures decreases in the proteins situated in the outer layers of the virion. The proteins involved in host recognition and attachment exhibit the least similarities to other members of Reoviridae. Furthermore, in GCRV, the RNA-translocating turrets are in an open state and lack a counterpart for the σ1 protein situated on top of the close turrets observed in mammalian orthoreovirus (MRV). Interestingly, the distribution and organization of GCRV core proteins resembles those of the cytoplasmic polyhedrosis virus (CPV), a cypovirus and the structurally simplest member of the Reoviridae family. Our results suggest that GCRV occupies a unique structure niche between the simpler cypoviruses and the considerably more complex MRV, thus providing an important model for understanding the structural and functional conservation and diversity of this enormous family of dsRNA viruses.
Grass carp reovirus; aquareovirus; dsRNA virus; 3D structure; Reoviridae; subnanometer-resolution; cryo-electron microscopy; evolution; divergence