Although the importance of this diversity awaits future verification, the ultimate determinant of the oncogenic property of EBV lies, of course, at the level of viral genes, which code for proteins and their propensity to alter by environmental factors. From the technical point of view, by using a sequential symmetry relaxation and classification workflow in subparticle reconstruction, we have overcome two intrinsic challenges in high-resolution structural studies of EBV: the scarcity of virion particles and the intrinsic structural plasticity of EBV proteins.
Reflecting on the astonishing accomplishment of reconstructing the tomato bushy stunt virus by combining merely six virus particles at the dawn of 3D electron microscopy 37 , the work presented here—resolving 45, amino acid residues from only 2, EBV virus particles—highlights the progress in cryo-EM enabled by direct electron detection and advanced data analysis.
Future efforts towards structure-based inhibitor design 11 , 18 and vaccine development 38 should extend to, and would probably benefit from, the structural plasticity documented in the present study.
EBV is mostly latent in infected cells in vitro and grows to very low titres compared with other herpesviruses, presenting a major challenge in isolating high-concentration virions for structural studies.
We obtained EBV virions by chemical induction of latently infected B cells. Tetradecanoyl phorbol acetate and sodium butyrate can both reactivate EBV from latency to virion-producing lytic replication, and the reduced level of FBS can minimize the secretion of EBV-like vesicles Then viral particles were pelleted by ultracentrifugation 21, g for 1.
Aliquots of 2. These grids were stored in a liquid nitrogen Dewar before cryo-EM imaging. Before imaging, the electron microscope was carefully aligned and the parallel beam was optimized using the coma-free alignment tool in SerialEM We circumvented the scarcity of virion particles by employing a combination of advanced imaging technologies 40 , 41 , 42 to precisely target sparsely distributed EBV virions barely one particle per movie, for example, see Supplementary Fig.
Movies were processed using MotionCor2 ref. The best class contained 2, particles and showed good structural features. Those particles were then subjected to 3D auto-refine with I3 icosahedral symmetry and post-processing, yielding an I3 reconstruction at 6. To improve the resolution of this icosahedral reconstruction, we ran three additional steps to calibrate defocus, astigmatism and beam tilt, a procedure that we refer to as iterative CTF refinement.
The 3D auto-refine, with the defocus-calibrated data STAR file and post-processing, yielded a new icosahedral reconstruction at 5. In the second step, the defocus and the astigmatism of all particles in the new data STAR file were calibrated simultaneously, and the resolution of the icosahedral reconstruction was pushed to 5. In the third step, not only were parameters of defocus and astigmatism of all particles calibrated, but also the beam tilt parameters of the microscopes were estimated.
The resolution of the resulting icosahedral reconstruction was pushed to 5. As the resolution limit Nyquist limit for bin2 images pixel size 2. These main-axis subparticle reconstructions began with the above-described I3 icosahedral reconstruction and the corresponding I3-icosahedral data STAR file. Our workflow see Supplementary Fig. In the first step, we extracted main-axis subparticles. These entries differ in their orientations. As each EBV virus only contains 12 fivefold vertices other than 60, this subparticle data STAR file contains 5 subparticle entries corresponding to each fivefold vertex.
To extract subparticles around twofold and threefold axes with more accurate initial orientation information, virus particles listed in the I3-icosahedral data STAR file obtained from the above section were subjected to another round of 3D auto-refine with icosahedral symmetry in the I2 convention Crowther setting, with icosahedral twofold axes along x , y and z axes , yielding an I2 icosahedral reconstruction identical to the icosahedral I3 reconstruction but oriented in the Crowther setting and a corresponding I2 icosahedral data STAR file.
For the icosahedral I2 reconstruction, a twofold axis is along the z axis and a threefold axis lies in the YZ plane about In the present study, we describe only fivefold subparticles as one example to illustrate this step.
As described, during extraction of fivefold subparticles, we did not adjust the subparticles by considering their locations in the virus, so we did not eliminate effectively the depth-of-focus problem for the enormous virus particles 48 , Instead, we used the iterative CTF refinement strategy described in Micrograph pre-processing and icosahedral reconstruction to alleviate this problem.
With three iterations of CTF refinement, the resolution of the C5 fivefold subparticle reconstruction finally converged at 3. The workflow for twofold subparticles was the same as for the fivefold subparticles, except the symmetry was set to C2 during the focused 3D auto-refine step. The resolution of the final C2 twofold subparticle reconstruction is also 3. For threefold subparticles, the C3 symmetry axis of the cropped map from the I2 icosahedral reconstruction is not along the z axis, so we first aligned the C3 symmetry axis of the cropped map to the z axis manually and resampled the map to one map reference the C2 twofold subparticle reconstruction with Chimera After three iterations of CTF refinement, the resolution of the final threefold subparticle reconstruction was pushed to 3.
Resolutions were based on the 0. Similarly, we extracted hexon subparticles and performed subparticle reconstruction see Supplementary Fig. The nearest main axis of E, C and P hexons is the twofold, threefold and threefold axis, respectively. We used the RELION data STAR file of the main-axis subparticle reconstruction to guide extracting the hexon subparticles nearest to the corresponding main axis; thus, E, C and P hexon subparticle extractions were guided by the STAR files of the twofold, threefold and fivefold subparticle reconstructions, respectively.
As E hexon is at the centre of the C2 twofold subparticle reconstruction, there was no need to expand the final C2 twofold reconstruction-related data STAR file. The initial parameters for orientation, defocus, astigmatism and beam tilt of each hexon subparticle are the same as those of the nearest main-axis subparticle processed above E hexons to twofold, C hexons to threefold and P hexons to fivefold.
Iterative CTF refinement in this step did not improve the final resolution further. The new data STAR file was then used to run a focused 3D classification without orientation search, by requesting three classes and applying a soft mask that covers only the triplex Tf protein area. After removing duplicative particles, the particles belonging to one class were subjected to 3D auto-refine with C1 symmetry and post-processing, yielding a C1 threefold subparticle reconstruction at 4.
To obtain the C5 portal subparticle reconstruction and the C12 portal vertex reconstruction, we used a similar data-processing strategy as descried previously We only had about 2, portal vertex subparticles, which presented difficulties in the initial steps to determine a C12 portal vertex reconstruction if using the same strategy as before 19 , so we included 4, and 2, portal vertex subparticles from HCMV and HSV-1, respectively, to assist our initial data processing.
We expanded the fivefold symmetry of the combined portal vertex dataset, generating a new data STAR file that contains five unique orientation entries for each subparticle.
The new data STAR file was then used to run a focused 3D classification step with C12 symmetry by requesting three classes. The 1, non-duplicative particles were subjected to 3D auto-refine by applying C5 symmetry and post-processing, yielding a C5 whole-virus reconstruction at 7. The penton vertex subparticles obtained were subjected to another 3D auto-refine and post-processing, yielding a C5 penton vertex subparticle reconstruction at 3.
We used a soft mask to mask just the CATC area, thus creating five sub-subparticle entries containing only the CATC region for each penton vertex subparticle. The new data STAR file was used to run one round of focused 3D classification by requesting three classes without orientation search see Supplementary Fig.
About The density of the CATC-absent penton vertex reconstruction 3. To figure out how many CATCs each penton contains, we examined the five sub-subparticle entries from each penton and counted their frequency of appearance in the three resulting 3D classes.
If only one of the five entries was classified into the CATC-binding class, then this penton vertex has only one CATC bound; if two entries were classified into this class, then this penton vertex is bound by two CATCs; likewise, if three, four or five entries were classified into this class, then this vertex is bounded by three, four or five CATCs, respectively. This statistical analysis result was summarized in the plot of Extended Data Fig. Local resolution assessments indicate that density maps at the capsid shell region in our subparticle reconstructions have resolutions uniformly better than 3.
These density maps have clear features of amino-acid side chains see Fig. The C2 twofold subparticle, C3 threefold subparticle and C5 penton vertex subparticle reconstructions described in the "C5 fivefold, C3 threefold and C2 twofold subparticle reconstructions" section have sufficient resolution for us to model 47 unique protein subunits in the icosahedral asymmetrical unit with Coot 52 by following the model-building workflow detailed previously 53 and referencing the atomic models of KSHV capsid PDB accession nos.
For those regions that could not simply be adjusted to match the model, we rebuilt the model de novo by referencing secondary structures predicted with the Phyre2 server 55 and using bulky amino-acid side chains as landmarks. This manual modelling step resulted in initial atomic models for an icosahedral asymmetrical unit. We built a triplex Tf atomic model based on the C1 threefold subparticle reconstruction see Fig.
The resolution for this reconstruction is 4. Nevertheless, in these two density maps, we can identify bumps corresponding to bulky amino-acid side chains to support homology-guided modelling. These subunits and the penton CATC model were fitted into the C1 CATC-binding penton vertex subparticle reconstruction and manually adjusted and, when necessary, modelled de novo with Coot, as described above at the beginning of this section. These subunits and five copies of the penton CATC model were fitted into the portal vertex subparticle reconstruction and manually adjusted and, when necessary, modelled de novo with Coot.
The atomic model of the recombinant dodecameric portal PDB model accession no. The manually built models were then iteratively improved through both Phenix real-space refinement 56 and manual readjustment in Coot The 47 PDB files in each asymmetrical unit were divided into 3 groups: group 1 contained 16 subunits around the twofold axis, group 2 contained 15 subunits around the threefold axis and group 3 contained 16 subunits around the fivefold axis see Supplementary Table 1. The atomic models in groups 1—3 were subjected to multiple iterations of refinement based on C2 twofold subparticle, C3 threefold subparticle and C5 penton vertex subparticle reconstructions, respectively.
Each iteration consisted of two steps. The first step is real-space refinement against subparticle reconstructions. Using group 1 as an example, we combined the 16 group 1 subunits with the atomic models of the 9 neighbouring protein subunits that make direct contact with group 1 subunits into a single concatenated PDB coordinate file.
This PDB file was subjected to real-space refinement against the C2 twofold subparticle reconstruction using Phenix. We obtained a PDB coordinate file for the refined 16 group 1 subunits by discarding the neighbouring subunits in the resulting PDB coordinate file. Likewise, the coordinates for the 15 subunits belonging to group 2 and 16 subunits belonging to group 3 were combined with their corresponding 14 and 10 neighbouring protein subunits, and then refined against the C3 threefold and C5 penton vertex subparticle reconstructions, respectively.
After discarding the neighbouring subunits from the resulting PDB files, we obtained a group 2 and a group 3 PDB coordinate file containing 15 and 16 refined subunits, respectively. The second step is model evaluation and manual fixing. The above refined models were assessed by various programs and, when necessary, manually corrected. For models in each group, wwPDB outputs a list of outliers based on bond length and angle, planarity and chirality.
When using these refinement tools, we turned on the following restraint options: Torsion, Planar Peptide, Trans Peptide and Ramachandran. Utilities such as a Ramachandran plot, geometry analysis, rotamer analysis and probe clashes in the pull-down validation menu of Coot provided various properties of the residues being refined. Occasionally, some refinement steps can cause residues to move away from the cryo-EM densities, leading to misfit.
When this happened, we manually fixed such refinement-introduced anomalies so that all residues fitted the cryo-EM densities. The above two steps were repeated until no further improvements were made and the models converged.
The number of iterations to convergence varied for the three groups and was about On achieving refinement convergence for all three groups, the three refined PDB coordinate files were combined to produce a final PDB coordinate file containing 47 protein subunits in the asymmetrical unit. Similarly, the initial atomic models of triplex Tf, CATC-binding penton vertex and portal vertex were refined against the C1 threefold subparticle reconstruction, the CATC-binding penton vertex subparticle reconstruction and the C5 portal vertex subparticle reconstruction, respectively.
However, no neighbours were used in the real-space refinement step for all these three models. In addition, when the portal vertex model was refined, we excluded the dodecameric portal complex. The number of iterations for this refinement was seven, ten and eight for the model of triplex Tf, the CATC-binding penton vertex and the portal vertex, respectively. Further information on research design is available in the Nature Research Reporting Summary linked to this article.
Epstein, M. Lancet , — Google Scholar. Salahuddin, S. Isolation of a new virus, HBLV, in patients with lymphoproliferative disorders. Science , — Andersson, J. Clinical aspects on Epstein—Barr virus infection. Fugl, A. Epstein—Barr virus and its association with disease—a review of relevance to general practice. BMC Fam. Bochkarev, A. Cell 83 , 39—46 Huang, H. Structural basis underlying viral hijacking of a histone chaperone complex.
Mullen, M. Cell 9 , — Matsuura, H. Natl Acad. USA , — Atomic structure of the Epstein—Barr virus portal. Germi, R. Three-dimensional structure of the Epstein—Barr virus capsid. Dai, X. Structure and mutagenesis reveal essential capsid protein interactions for KSHV replication. Nature , — Structure of the herpes simplex virus 1 capsid with associated tegument protein complexes. Science , eaao Wang, J. The capsid itself has a nucleocapsid with a icosahedral shape, which means it has twenty sides.
The capsid measures about nanometers in diameter. The tegument is the space between the capsid and envelope. This space is filled with proteins, and handles proteins and enzymes needed for replication and prepares them to do so.
To learn more about the disease these structures help cause, click here! Design by Free Web Templates. Home References About Me multipleorganisms. Taxonomy Habitat Interesting Facts Interactions. How does it work? Spikes : The "spikes" that are visible in the picture below are the many glycoproteins that coat the outside surface of the virus envelope.
Envelope : The envelope is a protective outer membrane that surrounds the virus. There is in vitro activity of the above mentioned drugs; however, there is no clinical benefit when administered to patients with uncomplicated IM. That is, the virus is susceptible in vitro but resistant in vivo.
Patients from lower socioeconomic groups are at higher risk of acquiring the infection early in life. In these groups of patients, intrafamiliar propagation is frequent. Endemic infectious mononucleosis is frequent in college students living in close contact, such as those living in dorms and frequenting other educational facilities.
It is an ubiquitous infection. The age at initial primary infection varies in different cultural and socioeconomic settings. In higher socioeconomic groups and industrialized countries the infection takes place later in life.
A higher rate of infection has been noted in white persons than in other ethnic groups, and a modest increase of infection in males than in females. Primary infections during pregnancy, but not reinfection have been associated with fetal involvement.
Transmission via breast milk is unclear. There is scarce evidence of EBV transmission and IM illness with sexual intercourse and closely related behaviors. EBV has been detected in genital ulcers, the uterine cervix, and the male reproductive tract. These findings raise the potential for venereal transmission. EBV is shed regularly in saliva, and, therefore, infection control policies are difficult to develop. In addition, mechanisms for its transmission are not clearly understood.
For the hospitalized patient, standard precautions are recommended. Transmission can also occur through blood products and organ transplantation; thus, persons with a recent EBV infection or IM like-illness are urged to refrain from donating blood or organs. A safe and efficacious vaccine is not available, although efforts continue on its development. Because of concern over the administration of a vaccine with unknown long-term effects, including malignancy, most vaccine development attempts have focused on development of subunit EBV vaccines.
These types of vaccines have shown to be immunogenic and well tolerated; recipients were not protected from EBV infection; however, symptoms of IM were reduced. An effective vaccine is needed for persons at high risk for development of EBV-associated lymphoproliferative diseases LPD and cancer, such as patients with acquired immunodeficiency syndrome AIDS , those with X-linked LPD, and organ transplant recipients.
Adults and children at high risk for developing post-transplant LPD e. EBV is a herpetic lymphotropic microorganism; latently infected B-cells are its primary reservoir in the body, although monocytes may also play a role in its latency. After this T-cell response, the number of EBV infected cells drops significantly during the next 4 to 6 weeks. The histopathology will vary according to the clinical syndrome or disease caused by EBV.
Most cases of acute IM are benign, and the diagnosis is based on clinical and serologic findings, thus, histopathology examination of tissues is rarely required. There is information on tissues, such as lymph nodes, tonsils, and spleen, from patients with unusual disease that requires surgery. Most histopathologic changes due to EBV infection have been attained from patients with LPD and other serious complications. The lymph nodes show active lymphoid follicles with lymphoid proliferation of the sinuses, blood vessels, trabecula, and capsule this latter one usually remains intact.
The response is mediated mainly by T and B immunoblasts with a pleomorphic pattern and mitosis indicative of rapid cell turnover. Small and large atypical lymphocytes can be seen, as well as plasma cells, eosinophils, histiocytes, and micronecrosis.
The tonsils also contain an active lymphoproliferative response with more prominent follicles and extensive necrosis. The spleen is enlarged two to three times its normal size and weight during the acute stage of the infection because of hyperplasia of the red pulp.
As in lymph nodes, the cells are primarily immunoblasts with pleomorphism; subcapsular hemorrhage is commonly encountered. The liver shows minimal disease with infiltration by lymphocytes and monocytes in the portal area and minor degeneration of the hepatocytes. The bone marrow can appear normal during acute IM; however, there have been reports of hypercellularity and small granulomas. The central nervous system CNS shows, when involved, lymphocytic infiltration of the meninges. Less commonly, there can be demyelinization, degenerative changes, focal hemorrhages, congestion, and edema.
In EBV-related LPD syndromes and B-cell lymphomas collectively named post-transplantation lymphoproliferative disorder [PTLD] , the histologic findings range from benign lymphoid hyperplasia with normal tissue architecture and a pleomorphic response polyclonality to well defined malignant lymphomas.
There are diffuse inflammatory cell infiltrates; some of the neoplastic cells have classically been termed Red-Sternberg cells and mononuclear Hodgkin cells collectively termed as H-RS cells. These cells are large, and their multinucleated pattern is evidence derived from germinal centers of B-cells.
Their nucleoli are strongly stained and surrounded by a clear area resembling a halo. The classic HD includes four categories: lymphocyte rich; nodular sclerosis; mixed cellularity; and lymphocyte depleted. The sclerosing variety is most commonly found in children with HD. Epithelial tumors of the nasopharynx: In children, the most common type of epithelial cells is the undifferentiated variety characterized by undifferentiated squamous cells with multiple copies of monoclonal EBV DNA.
Other EBV virus-associated diseases: Tissues of patients with hemophagocytic lymphohistiocytosis HLH , such as bone marrow, spleen, lymph nodes, brain, and skin can be affected. The bone marrow shows hypocellularity, mainly activated macrophages with engulfment of all bone marrow elements, their precursors, or fragments, including erythrocytes, platelets, and leukocytes.
Oral hairy leukoplakia: Tissues of patients usually adults with AIDS with this disorder show keratin or parakeratin projections, acanthosis, ballooning, hyperparakeratosis, and hyperplasia of the prickle cell layer. Mild inflammatory cell infiltrates in the connective tissue can be seen. The incubation period is approximately 4 to 6 weeks, whether acquired through contact with infected secretions or after a blood transfusion.
The prodrome lasts 3 to 5 days and is that of mild headache, malaise, and fatigue. Generalized lymphadenopathy is a hallmark of IM; any group of lymph nodes can be involved; however, those of the anterior and posterior cervical chains are the most frequent.
They are tender, firm, usually single, 2 to 4cm in diameter, and not matted. Massive mediastinal and hilar lymph nodes can cause respiratory distress. Mesenteric lymphadenopathy can mimic acute appendicitis. Lymph node enlargement tends to subside over a period of days to weeks. Pharyngitis characterized by sore throat is the cardinal symptom of IM.
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