The SD1 Subdomain of Venezuelan Equine Encephalitis Virus Capsid Protein Plays a Critical Role in Nucleocapsid and Particle Assembly.

UNLABELLED: Venezuelan equine encephalitis virus (VEEV) is an important human and animal pathogen, for which no safe and efficient vaccines or therapeutic means have been developed. Viral particle assembly and budding processes represent potential targets for therapeutic intervention. However, our understanding of the mechanistic process of VEEV assembly, RNA encapsidation, and the roles of different capsid-specific domains in these events remain to be described. The results of this new study demonstrate that the very amino-terminal VEEV capsid-specific subdomain SD1 is a critical player in the particle assembly process. It functions in a virus-specific mode, and its deletion, mutation, or replacement by the same subdomain derived from other alphaviruses has strong negative effects on infectious virus release. VEEV variants with mutated SD1 accumulate adaptive mutations in both SD1 and SD2, which result in a more efficiently replicating phenotype. Moreover, efficient nucleocapsid and particle assembly proceeds only when the two subdomains, SD1 and SD2, are derived from the same alphavirus. These two subdomains together appear to form the central core of VEEV nucleocapsids, and their interaction is one of the driving forces of virion assembly and budding. The similar domain structures of alphavirus capsid proteins suggest that this new knowledge can be applied to other alphaviruses. IMPORTANCE: Alphaviruses are a group of human and animal pathogens which cause periodic outbreaks of highly debilitating diseases. Despite significant progress made in understanding the overall structure of alphavirus and VEEV virions, and glycoprotein spikes in particular, the mechanistic process of nucleocapsid assembly, RNA encapsidation, and the roles of different capsid-specific domains in these processes remain to be described. Our new data demonstrate that the very amino-terminal subdomain of Venezuelan equine encephalitis virus capsid protein, SD1, plays a critical role in the nucleocapsid assembly. It functions synergistically with the following SD2 (helix I) and appears to form a core in the center of nucleocapsid. The core formation is one of the driving forces of alphavirus particle assembly.

4.4 Å cryo-EM structure of an enveloped alphavirus Venezuelan equine encephalitis virus.

Venezuelan equine encephalitis virus (VEEV), a member of the membrane-containing Alphavirus genus, is a human and equine pathogen, and has been developed as a biological weapon. Using electron cryo-microscopy (cryo-EM), we determined the structure of an attenuated vaccine strain, TC-83, of VEEV to 4.4 Å resolution. Our density map clearly resolves regions (including E1, E2 transmembrane helices and cytoplasmic tails) that were missing in the crystal structures of domains of alphavirus subunits. These new features are implicated in the fusion, assembly and budding processes of alphaviruses. Furthermore, our map reveals the unexpected E3 protein, which is cleaved and generally thought to be absent in the mature VEEV. Our structural results suggest a mechanism for the initial stage of nucleocapsid core formation, and shed light on the virulence attenuation, host recognition and neutralizing activities of VEEV and other alphavirus pathogens.

Structure of a Venezuelan equine encephalitis virus assembly intermediate isolated from infected cells.

Venezuelan equine encephalitis virus (VEEV) is a prototypical enveloped ssRNA virus of the family Togaviridae. To better understand alphavirus assembly, we analyzed newly formed nucleocapsid particles (termed pre-viral nucleocapsids) isolated from infected cells. These particles were intermediates along the virus assembly pathway, and ultimately bind membrane-associated viral glycoproteins to bud as mature infectious virus. Purified pre-viral nucleocapsids were spherical with a unimodal diameter distribution. The structure of one class of pre-viral nucleocapsids was determined with single particle reconstruction of cryo-electron microscopy images. These studies showed that pre-viral nucleocapsids assembled into an icosahedral structure with a capsid stoichiometry similar to the mature nucleocapsid. However, the individual capsomers were organized significantly differently within the pre-viral and mature nucleocapsids. The pre-viral nucleocapsid structure implies that nucleocapsids are highly plastic and undergo glycoprotein and/or lipid-driven rearrangements during virus self-assembly. This mechanism of self-assembly may be general for other enveloped viruses.

Structure of the recombinant alphavirus Western equine encephalitis virus revealed by cryoelectron microscopy.

Western equine encephalitis virus (WEEV; Togaviridae, Alphavirus) is an enveloped RNA virus that is typically transmitted to vertebrate hosts by infected mosquitoes. WEEV is an important cause of viral encephalitis in humans and horses in the Americas, and infection results in a range of disease, from mild flu-like illnesses to encephalitis, coma, and death. In addition to spreading via mosquito vectors, human WEEV infections can potentially occur directly via aerosol transmission. Due to its aerosol infectivity and virulence, WEEV is thus classified as a biological safety level 3 (BSL-3) agent. Because of its highly infectious nature and containment requirements, it has not been possible to investigate WEEV's structure or assembly mechanism using standard structural biology techniques. Thus, to image WEEV and other BSL-3 agents, we have constructed a first-of-its-kind BSL-3 cryoelectron microscopy (cryoEM) containment facility. cryoEM images of WEEV were used to determine the first three-dimensional structure of this important human pathogen. The overall organization of WEEV is similar to those of other alphaviruses, consistent with the high sequence similarity among alphavirus structural proteins. Surprisingly, the nucleocapsid of WEEV, a New World virus, is more similar to the Old World alphavirus Sindbis virus than to other New World alphaviruses.

Complete inactivation of Venezuelan equine encephalitis virus by 1,5-iodonaphthylazide.

Hydrophobic alkylating compounds like 1,5-iodonaphthylazide (INA) partitions into biological membranes and accumulates selectively into the hydrophobic domain of the lipid bilayer. Upon irradiation with far UV light, INA binds selectively to transmembrane proteins in the viral envelope and renders them inactive. Such inactivation does not alter the ectodomains of the membrane proteins thus preserving the structural and conformational integrity of immunogens on the surface of the virus. In this study, we have used INA to inactivate Venezuelan equine encephalitis virus (VEEV). Treatment of VEEV with INA followed by irradiation with UV light resulted in complete inactivation of the virus. Immuno-fluorescence for VEEV and virus titration showed no virus replication in-vitro. Complete loss of infectivity was also achieved in mice infected with INA treated plus irradiated preparations of VEEV. No change in the structural integrity of VEEV particles were observed after treatment with INA plus irradiation as assessed by electron microscopy. This data suggest that such inactivation strategies can be used for developing vaccine candidates for VEEV and other enveloped viruses.

Venezuelan equine encephalomyelitis virus structure and its divergence from old world alphaviruses.

Although alphaviruses have been extensively studied as model systems for the structural organization of enveloped viruses, no structures exist for the phylogenetically distinct eastern equine encephalomyelitis (EEE)-Venezuelan equine encephalomyelitis (VEE) lineage of New World alphaviruses. Here we report the 25-A structure of VEE virus, obtained from electron cryomicroscopy and image reconstruction. The envelope spike glycoproteins of VEE virus have a T=4 icosahedral arrangement, similar to that observed in Old World Sindbis, Semliki Forest, and Ross River alphaviruses. However, VEE virus has pronounced differences in its nucleocapsid structure relative to nucleocapsid structures repeatedly observed in Old World alphaviruses.

[Importance of transmission electron microscopy in the diagnosis of a Venezuelan equine encephalitis outbreak in 1995 in the Venezuelan Guajira]. / Importancia de la microscopía electrónica de transmisión en el diagnóstico de la epidemia de encefalitis equina venezolana de 1995 en la Guajira Venezolana.

Transmission electron microscopy of the brain in 25 newborn mice was performed. Mice were intracerebrally inoculated with cultured VERO cells infected with VEE to be used as positive control, with samples of serum of cerebrospinal fluid from patients with clinical symptoms and signs of encephalitis, with serum from healthy patients or with serum from sick equines. Borate bovine albumin serum was also inoculated in some mice to be used as negative control. All samples were obtained during the epizootic and epidemic outbreak in the Venezuelan Guajira area, northern of Zulia State during October 1995. The ultrastructural study was blindly performed, however the presence of Togavirus particles were detected in 100% of the virologically positive cases. The usefulness, accuracy and speed of the employed methodology is stressed.

[The use of atomic-force microscopy for the analysis of microbiological objects]. / Ispol'zovanie atomno-silovoi mikroskopii dlia analiza mikrobiologicheskikh ob''ektov.

Atomic force microscopy (AFM) was used for the analysis of rickettsiae and viruses. The specificity of interaction was evaluated on the basis of the adsorption of the analyzed antigen on the polymer-antibody film. The film was formed and transferred onto highly oriented pyrolytic graphite (HOPG) by the method of Langmuir-Schaefer with the use of amphiphilic polymers, alkylated polyethyleneimines. According to the data of AFM, polymer-antibody film was 10-30 nm thick and had ruptures, uneven surface. AFM images of Coxiella burnetii, rotavirus and Venezuelan equine encephalitis virus, immunoadsorbed on the antibody film, were obtained. C.burnetii, due to their size equal to (700-900) x (300-500) x Z = 200 nm, were clearly visible on the underlying surface and could be directly counted. Individual virus particles (60-80 nm) cold not be identified on such surface. To analyze such preparations, the program of image analysis was developed. The program classified the registered image with a certain standard. This program determined the presence of virus antigen on the underlying affinity surface with a high degree of precision.

[Course of infection in white rats, infected with Venezuelan equine encephalomyelitis virus by a respiratory route]. / Techenie infektsii u belykh drys, respiratorno zarazhennykh virusom venesuél'skogo éntsefalomielita loshadei.

Time course of accumulation of Venezuelan equine encephalomyelitis virus (strain Trinidad) in the organism of white rats after respiratory infection was studied. Electronmicroscopic examination of the organs was carried out over the course of the disease. The virus was for the first time detected in the lungs of animals after aerogenous infection and in the olfactory area of the nasal cavity of animals infected by applying the virus into the nasal mucosa. Disease development in aerosol-infected rats was characterized by viremia and infection of the hemopoietic and lymphomyeloid systems, particularly of the thymus and bifurcation lymph nodes, as well as of the olfactory system and brain. At the late stage of infection the concentration of the agent in all the examined organs was below the detection threshold, and neutralizing antibodies were detected in the blood serum.

Spread of Venezuelan equine encephalitis virus in mice olfactory tract.

Spread of Venezuelan equine encephalitis (VEE) virus and damage of the central nervous system (CNS) in mice infected by respiratory route was studied. Virus concentration in organs and blood, "dose-effect" relationships, and ultrastructural lesions in various tissues were examined in immune and normal mice. We showed, via three independent methods--characteristic curve investigations, tissue virus concentration dynamics, and ultrastructural methods--the spread of VEE virus through the olfactory tract into the brain of immune mice. From these experiments it was concluded that in case of respiratory challenge VEE virus can enter the CNS of normal mice by both vascular and olfactory pathways, while in immune mice the main route is olfactory.

[Interactions between Mycoplasma and human and animal viruses. Ultrastructural analysis]. / Vzaimodeistvie mikoplazm s virusami cheloveka i zhivotnykh. Ul'trastrukturnyi analiz.

Presented are the data on the ultrastructural analysis of interaction between mycoplasma and certain cancerogenic and infectious viruses in humans and animals. Revealed are spontaneous associations of mycoplasma with viruses of cattle leukemia, T-cell human leukemia and with a representative of Bunyaviruses. Immediate interaction of these agents is found possible. Simulated complexes of mycoplasma with infectious viruses are developed. Electron microscopy on supramolecular levels revealed immediate interaction of different agents in membranes. Some methodological procedures help to reveal that the interaction of M. pneumoniae and A. laidlawii with orthomyxo-, paramyxo and togavirus is of specific character and is realized as receptor ligand form due to the affinity in the receptor requirements of these pathogens. This property as well as a bequeath distribution and frequent association in respiratory infections enable one to suggest the possibility of their immediate interactions in a host body.

[Covalent-bound aggregates of equine venezuelan encephalomyelitis virus induced by UV-irradiation]. / Kovalentno-sviazannye agregaty virusa venesuél'skogo éntsefalomielita loshadei, indutsirovannye UF-oblucheniem.

Formation of Venecuelan equine encephalomyelitis virus (VEE) aggregates induced by UV-light has been studied. The high doses of UV-irradiation induced the protein-protein cross-links resulting in formation of fast sedimenting viral structures. The latter structures are supposed to be presented by the aggregates of several virions linked by the UV-light induced RNA-protein and protein-protein covalent bonds. The lesions in the fine structure of virion envelope was registered by the electron microscopy technique.

[Effect of remantadine on the morphogenesis of the Venezuelan equine encephalomyelitis virus in a Vero cell culture]. / Vliianie remantadina na morfogenez virusa venesuél'skogo éntsefalomielita loshadei v kul'ture kletok Vero.

Morphogenesis of Venezuelan equine encephalomyelitis (VEE) virus was studied in Vero cell cultures with or without remantadine treatment. Remantadine was found to inhibit late stages of VEE virus morphogenesis, especially formation of virus cores in the cytoplasm of the infected cells.

[Concentration of the Venezuelan equine encephalomyelitis virus in a 2-phase system of water-soluble polymers]. / Kontsentratsiia virusa venesuél'skogo éntsefalomielita loshadei v dvukhfaznoi sisteme vodorastvorimykh polimerov.

A three-step concentration of Venezuelan equine encephalomyelitis (VEE) virus from tissue culture fluid was carried out in a two-phase system of polyethyleneglycol (PEG)--sodium dextran sulphate (SDS). The concentration method was based on the dependence of virus distribution coefficient upon NaCl content in the system which allowed alternating transfer of the virus from one phase of the system into the other. The infectious activity of the virus increased approximately 100-fold after the first step, 190-fold after the second, and 300-fold after the third step. The process of concentration was accompanied by purification of virus preparations. This was indicated by the results of immune electron microscopy which allowed visual observation of accumulation of the virus material in the preparations and decrease of the content of cell membrane elements. SDS was removed from virus preparations by precipitation with 3 M KCl solution in the cold, the procedure having no effect on the infectious activity of the preparations.

Electron microscopic analysis of infection patterns for Venezuelan equine encephalomyelitis virus in the vector mosquito, Culex (Melanoconion) taeniopus.

The infectious process for Venezuelan equine encephalomyelitis virus in an enzootic vector mosquito, Culex (Melanoconion) taeniopus, was examined by transmission electron microscopy. Following large dose oral infection, virus was seen in the abdominal fat body and epidermis within 1 hr of engorgement. Replicated virus appeared to leave the mesenteron 3-4 hr post-infection. Dissemination to other organs occurred between days 1 and 2 of extrinsic incubation, when the hindgut and abdominal nerve ganglia were found to be infected. Virus reached the thoracic nerve ganglia, brain and salivary glands by 4 days post-infection, and flight muscles contained virus by day 6. Virus was never detected in the malphigian tubules or ovaries of infected mosquitoes within 21 days of extrinsic incubation. These results suggest that virus particles penetrate the mesenteron and accumulate in the abdominal fat body prior to replication within the vector. This pattern differs from that reported for other arbovirus-vector pairs.

[Ultrastructural organization of liposomes and isolated glycoproteins of enveloped viruses obtained by using the nonionic detergent MESK]. / Ul'trastrukturnaia organizatsiia liposom i izolirovannykh glikoproteidov obolochechnykh virusov, poluchennykh s ispol'zovaniem neionnogo detergenta MESK.

Materials on comparative ultrastructural analysis of isolated glycoproteins (GP) of influenza, parainfluenza, and Venezuelan equine encephalomyelitis viruses as well as liposomes reconstructed on their basis prepared with the use of MECK, a nonionic detergent, are presented. Original virions, subvirion particles, and isolated GP were characterized by typical morphology and remained intact in highly purified and concentrated suspensions owing to the use of the sparing and effective method of treatment with MECK. After removal of the detergent typical micellar aggregates were shown to form from GP monomers for all three viruses under study. The forming rosettes morphologically and biologically are similar to those isolated from ortho-, paramyxo-, and togaviruses using the Triton X-100 detergent. Stages of liposome formation and interaction with cell membrane were studied electron microscopically. A common feature for liposomes from GP of all the three viruses consisted in the formation of two morphological variants of structural complexes: filaments of various lengths and composition in them of GP and vesicles, the arrays of GP in which were similar or identical to those of viral membrane. Liposomes from Sendai virus GP possessed hemolytic activity, however, their interaction with the cell studied by means of the immunoferritine label occurred not by fusion but apparently as a result of destruction of the cell membrane at the sites of protein F contact with plasmalemma and formation in the latter of defects leading to erythrocyte lysis.

[Morphological study of a mixed alphavirus infection]. / Morfologicheskoe izuchenie smeshannoi infektsii al'favirusami.

In combined paired cultivation of 8 Venezuelan equine encephalomyelitis virus, multiploid virions formed in 17.8% of cases. Five clones with this effect were used in mixed infections with clones of Semliki Forest and Sindbis viruses. In these infections changes in virions of the virus progeny were observed in 20% and 16.6%, respectively. Mixed cultivation of pairs of Sindbis and Semliki Forest viruses resulted in formation of multiploid virions in 42.8%. Besides, in two mixed populations formed upon combined multiplication of the latter viruses virions of unusual shapes were found: rounded, oval, elongated, and triangular designated as polymorphic.

The surface structure of Venezuelan equine encephalomyelitis virus.

An electronmicroscopic study of a suspension of Venezuelan equine encephalomyelitis (VEE) virus by freeze-drying and freeze-etching methods showed that glycoprotein peplomers are located on the surface of the lipoprotein shell. These peplomers are organized with trimer clustering in a T = 4 icosahedral surface lattice. The mode of glycoprotein clustering for the two clones of VEE are different.

[Action of cycloheximide on RNA synthesis and the formation of structures in the Venezuelan equine encephalomelitis virus]. / Deistvie tsiklogeksimida na sintez RNK i formirovanie struktur virusa venesuel'skogo éntsefalomielita loshadei

The studies showed that the effect of cycloheximide on reproduction of the virus of Venezuelan equine encephalitis (VEE) depended on the time of the antibiotic administration. When the antibiotic was added 3--5 hours after the inoculation (after formation of the virus RNA-polymerase), accumulation of virus RNA and 60--100 S virus structures was observed in the cells, while virus nucleotides and infection virus did not practically form. The 60--100 S structures included the cell proteins. They were resistant to EDTA and their swimming density in cesium chloride was 1.3--1.4 gm/cm3, it was characteristic of RNA of a non-ribosomic nature. According to these properties 60--100 S structures may be classified as belonging to the "hybrid" RNA analogous to the RNA complexes of VEE virus with the cell proteins. Probably addition of cycloheximide provided conditions in the infected cells for increase of the "hybrid", structural pool since the synthesis of the structural virus proteins was suppressed.