Implication for alphavirus host-cell entry and assembly indicated by a 3.5Å resolution cryo-EM structure.

Alphaviruses are enveloped RNA viruses that contain several human pathogens. Due to intrinsic heterogeneity of alphavirus particles, a high resolution structure of the virion is currently lacking. Here we provide a 3.5 Å cryo-EM structure of Sindbis virus, using block based reconstruction method that overcomes the heterogeneity problem. Our structural analysis identifies a number of conserved residues that play pivotal roles in the virus life cycle. We identify a hydrophobic pocket in the subdomain D of E2 protein that is stabilized by an unknown pocket factor near the viral membrane. Residues in the pocket are conserved in different alphaviruses. The pocket strengthens the interactions of the E1/E2 heterodimer and may facilitate virus assembly. Our study provides structural insights into alphaviruses that may inform the design of drugs and vaccines.

Palmitoylation of Sindbis Virus TF Protein Regulates Its Plasma Membrane Localization and Subsequent Incorporation into Virions.

Palmitoylation is a reversible, posttranslational modification that helps target proteins to cellular membranes. The alphavirus small membrane proteins 6K and TF have been reported to be palmitoylated and to positively regulate budding. 6K and TF are isoforms that are identical in their N termini but unique in their C termini due to a -1 ribosomal frameshift during translation. In this study, we used cysteine (Cys) mutants to test differential palmitoylation of the Sindbis virus 6K and TF proteins. We modularly mutated the five Cys residues in the identical N termini of 6K and TF, the four additional Cys residues in TF's unique C terminus, or all nine Cys residues in TF. Using these mutants, we determined that TF palmitoylation occurs primarily in the N terminus. In contrast, 6K is not palmitoylated, even on these shared residues. In the C-terminal Cys mutant, TF protein levels increase both in the cell and in the released virion compared to the wild type. In viruses with the N-terminal Cys residues mutated, TF is much less efficiently localized to the plasma membrane, and it is not incorporated into the virion. The three Cys mutants have minor defects in cell culture growth but a high incidence of abnormal particle morphologies compared to the wild-type virus as determined by transmission electron microscopy. We propose a model where the C terminus of TF modulates the palmitoylation of TF at the N terminus, and palmitoylated TF is preferentially trafficked to the plasma membrane for virus budding. IMPORTANCE: Alphaviruses are a reemerging viral cause of arthritogenic disease. Recently, the small 6K and TF proteins of alphaviruses were shown to contribute to virulence in vivo Nevertheless, a clear understanding of the molecular mechanisms by which either protein acts to promote virus infection is missing. The TF protein is a component of budded virions, and optimal levels of TF correlate positively with wild-type-like particle morphology. In this study, we show that the palmitoylation of TF regulates its localization to the plasma membrane, which is the site of alphavirus budding. Mutants in which TF is not palmitoylated display drastically reduced plasma membrane localization, which effectively prevents TF from participating in budding or being incorporated into virus particles. Investigation of the regulation of TF will aid current efforts in the alphavirus field searching for approaches to mitigate alphaviral disease in humans.

Single-Site Glycoprotein Mutants Inhibit a Late Event in Sindbis Virus Assembly.

UNLABELLED: A panel of Sindbis virus mutants that were suspected to have deficiencies in one or more aspects of their replication cycles was examined in baby hamster kidney (BHK) cells. These included an amino acid deletion (ΔH230) and substitution (H230A) in the Sindbis glycoprotein E1_H230 and similar mutants in E2_G209 (G209A, G209D, and ΔG209). Neither H230 mutation produced a measurable titer, but repeated passaging of the H230A mutant in BHK cells produced a second-site compensatory mutant (V231I) that partially rescued both H230 mutants. Electron micrograph (EM) images of these mutants showed assembled viral nucleocapsids but no completed, mature virions. EM of the compensatory mutant strains showed complete virus particles, but these now formed paracrystalline arrays. None of the E2_G209 substitution mutants had any effect on virus production; however, the deletion mutant (ΔG209) showed a very low titer when grown at 37°C and no titer when grown at 28°C. When the deletion mutant grown at 28°C was examined by EM, partially budded virions were observed at the cell surface. (35)S labeling of this mutant confirmed the presence of mutant virus protein in the transfected BHK cell lysate. We conclude that H230 is essential for the assembly of complete infectious Sindbis virus virions and that the presence of an amino acid at E2 position 209 is required for complete budding of Sindbis virus particles although several different amino acids can be at this location without affecting the titer. IMPORTANCE: Our data show the importance of single-site mutations at E1_H230 and E2_G209 in Sindbis virus glycoproteins. These sites have been shown to affect assembly and antibody binding in previous studies. Our data indicate that mutation of one histidine residue in E1 is detrimental to the assembly of Sindbis virus particles in baby hamster kidney cells. Repeated passaging leads to a second-site substitution that partially restores the titer although EM still shows an altered phenotype. Substitutions at position G209 in E2 have no effect on titer, but deletion of this residue greatly reduces titer and again prevents assembly. When this mutant is grown at a lower temperature, virus particles bud from the host cell, but budding arrests before the progeny virus escapes. These results allow us to conclude that these sites have essential roles in assembly, and E2_G209 shows us a new viral egress phenotype.

Single-step antibody-based affinity cryo-electron microscopy for imaging and structural analysis of macromolecular assemblies.

Single particle cryo-electron microscopy (cryo-EM) is an emerging powerful tool for structural studies of macromolecular assemblies (i.e., protein complexes and viruses). Although single particle cryo-EM requires less concentrated and smaller amounts of samples than X-ray crystallography, it remains challenging to study specimens that are low-abundance, low-yield, or short-lived. The recent development of affinity grid techniques can potentially further extend single particle cryo-EM to these challenging samples by combining sample purification and cryo-EM grid preparation into a single step. Here we report a new design of affinity cryo-EM approach, cryo-SPIEM, that applies a traditional pathogen diagnosis tool Solid Phase Immune Electron Microscopy (SPIEM) to the single particle cryo-EM method. This approach provides an alternative, largely simplified and easier to use affinity grid that directly works with most native macromolecular complexes with established antibodies, and enables cryo-EM studies of native samples directly from cell cultures. In the present work, we extensively tested the feasibility of cryo-SPIEM with multiple samples including those of high or low molecular weight, macromolecules with low or high symmetry, His-tagged or native particles, and high- or low-yield macromolecules. Results for all these samples (non-purified His-tagged bacteriophage T7, His-tagged Escherichiacoli ribosomes, native Sindbis virus, and purified but low-concentration native Tulane virus) demonstrated the capability of cryo-SPIEM approach in specifically trapping and concentrating target particles on TEM grids with minimal view constraints for cryo-EM imaging and determination of 3D structures.

Isolation of sindbis virus from a hooded crow in Germany.

Sindbis virus (SINV) is an arbovirus that causes clinical symptoms, including arthritis, rash, and fever during acute human infections. In Europe, SINV outbreaks are largely restricted to northern Europe. Intrigued by the isolation of SINV from mosquitoes in southwestern Germany in 2009, we initiated a passive arbovirus-monitoring program in birds and analyzed a total of 685 samples. By this approach, we were able to detect a SINV in a Hooded Crow in Germany for the first time. It was possible to isolate SINV virus in cell cultures and even to visualize virus particles by electron microscopy. After the determination of the complete SINV genome sequence, the phylogenetic analysis revealed its close relationship to SINV genotype I sequences previously obtained from mosquitoes in Germany and Scandinavia. This first report on the isolation of viable SINV indicates the potential involvement of crows in an enzootic circulation of SINV in Germany and Central Europe.

Directed evolution of a virus exclusively utilizing human epidermal growth factor receptor as the entry receptor.

Rational design and directed evolution are powerful tools to generate and improve protein function; however, their uses are mostly limited to enzyme and antibody engineering. Here we describe a directed-evolution strategy, named the tandem selection and enrichment system (TSES), and its use in generating virus with exclusive specificity for a particular cellular receptor. In TSES, evolving viruses are sequentially and iteratively transferred between two different host cells, one for selection of receptor specificity and the other for enrichment of the fittest virus. By combining rational design and TSES, we generated human epidermal growth factor receptor (EGFR)-specific virus 1 (ESV1). ESV1 has the backbone of Sindbis virus (SINV) and displays an EGF domain engrafted onto structural protein E2 after residue Pro192, together with eight amino acid changes stabilizing the E2-EGF chimera. ESV1 uses EGFR to initiate infection and has lost the capacity to interact with all known SINV receptors. A 12.2-Å cryoelectron microscopic (cryoEM) reconstruction of ESV1 reveals that the E2-EGF fusion adopts a fixed conformation, with EGF sitting at the top of the E2 spike; The EGFR binding interface faces outward, and the EGF domain completely masks SINV receptor binding. The cryoEM structure of ESV1 explains the desirable properties of ESV1 and provides insights for its further modification. TSES expands the scope of directed evolution and can be easily extended to other targeting molecules and viral systems.

Characterization of an early-stage fusion intermediate of Sindbis virus using cryoelectron microscopy.

The sequential steps in the alphavirus membrane fusion pathway have been postulated based on the prefusion and postfusion crystal structures of the viral fusion protein E1 in conjunction with biochemical studies. However, the molecular structures of the hypothesized fusion intermediates have remained obscure due to difficulties inherent in the dynamic nature of the process. We developed an experimental system that uses liposomes as the target membrane to capture Sindbis virus, a prototypical alphavirus, in its membrane-binding form at pH 6.4. Cryoelectron micrograph analyses and 3D reconstructions showed that the virus retains its overall icosahedral structure at this mildly acidic pH, except in the membrane-binding region, where monomeric E1 associates with the target membrane and the E2 glycoprotein retains its original trimeric organization. The remaining E2 trimers may hinder E1 homotrimerization and are a potential target for antiviral drugs.

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.

Helical virus particles formed from morphological subunits of a membrane containing icosahedral virus.

The classic publication by Caspar and Klug in 1962 [Physical principles in the construction of regular viruses. Cold Spring Harbor Symp. Quant. Biol. 27:1-24.] has formed the basis of much research on virus assembly. Caspar and Klug predicted that a single virus morphological unit could form a two dimensional lattice composed of 6-fold arrays (primitive plane), a family of icosahedra of increasing triangulation numbers (T) and helical arrays of varying length. We have shown that icosahedral viruses of varying T numbers can be produced using Sindbis virus [Ferreira, D. F. et al. 2003. Morphological variants of Sindbis virus produced by a mutation in the capsid protein. Virology 307:54-66]. Other studies have shown that Sindbis glycoproteins can also form a 2-dimensional lattice confirming Caspar and Klug's prediction of the primitive plane as a biologically relevant structure [VonBonsdorff, C. H., and S. C. Harrison. 1978. Sindbis virus glycoproteins form a regular icosahedral surface lattice. J. Virol. 28:578]. In this study we have used mutations in the glycoproteins of membrane containing Sindbis virus to create helical-virus-like particles from the morphological subunits of a virus of icosahedral geometry. The resulting virus particles were examined for subunit organization and were determined to be constructed of only 6-fold rotational arrays of the virus glycoproteins. A model of the tubular virus particles created from the 6-fold rotational arrays of Sindbis virus confirmed the observed structure. These experiments show that a common morphological unit (the Sindbis E1-E2 heterodimer) can produce three different morphological entities of varying dimensions in a membrane-containing virus system.

Sindbis virus conformational changes induced by a neutralizing anti-E1 monoclonal antibody.

A rare Sindbis virus anti-E1 neutralizing monoclonal antibody, Sin-33, was investigated to determine the mechanism of in vitro neutralization. A cryoelectron microscopic reconstruction of Sindbis virus (SVHR) neutralized with FAb from Sin-33 (FAb-33) revealed conformational changes on the surface of the virion at a resolution of 24 A. FAb-33 was found to bind E1 in less than 1:1 molar ratios, as shown by the absence of FAb density in the reconstruction and stoichiometric measurements using radiolabeled FAb-33, which determined that about 60 molecules of FAb-33 bound to the 240 possible sites in a single virus particle. FAb-33-neutralized virus particles became sensitive to digestion by endoproteinase Glu-C, providing further evidence of antibody-induced structural changes within the virus particle. The treatment of FAb-33-neutralized or Sin-33-neutralized SVHR with low pH did not induce the conformational rearrangements required for virus membrane-cell membrane fusion. Exposure to low pH, however, increased the amount of Sin-33 or FAb-33 that bound to the virus particles, indicating the exposure of additional epitopes. The neutralization of SVHR infection by FAb-33 or Sin-33 did not prevent the association of virus with host cells. These data are in agreement with the results of previous studies that demonstrated that specific antibodies can inactivate the infectious state of a metastable virus in vitro by the induction of conformational changes to produce an inactive structure. A model is proposed which postulates that the induction of conformational changes in the infectious state of a metastable enveloped virus may be a general mechanism of antibody inactivation of virus infectivity.

Tracking and elucidating alphavirus-host protein interactions.

Viral infections cause profound alterations in host cells. Here, we explore the interactions between proteins of the Alphavirus Sindbis and host factors during the course of mammalian cell infection. Using a mutant virus expressing the viral nsP3 protein tagged with green fluorescent protein (GFP) we directly observed nsP3 localization and isolated nsP3-interacting proteins at various times after infection. These results revealed that host factor recruitment to nsP3-containing complexes was time dependent, with a specific early and persistent recruitment of G3BP and a later recruitment of 14-3-3 proteins. Expression of GFP-tagged G3BP allowed reciprocal isolation of nsP3 in Sindbis infected cells, as well as the identification of novel G3BP-interacting proteins in both uninfected and infected cells. Note-worthy interactions include nuclear pore complex components whose interactions with G3BP were reduced upon Sindbis infection. This suggests that G3BP is a nuclear transport factor, as hypothesized previously, and that viral infection may alter RNA transport. Immunoelectron microscopy showed that a portion of Sindbis nsP3 is localized at the nuclear envelope, suggesting a possible site of G3BP recruitment to nsP3-containing complexes. Our results demonstrate the utility of using a standard GFP tag to both track viral protein localization and elucidate specific viral-host interactions over time in infected mammalian cells.

Mutations in the endodomain of Sindbis virus glycoprotein E2 define sequences critical for virus assembly.

Envelopment of Sindbis virus at the plasma membrane is a multistep process in which an initial step is the association of the E2 protein via a cytoplasmic endodomain with the preassembled nucleocapsid. Sindbis virus is vectored in nature by blood-sucking insects and grows efficiently in a number of avian and mammalian vertebrate hosts. The assembly of Sindbis virus, therefore, must occur in two very different host cell environments. Mammalian cells contain cholesterol which insect membranes lack. This difference in membrane composition may be critical in determining what requirements are placed on the E2 tail for virus assembly. To examine the interaction between the E2 tail and the nucleocapsid in Sindbis virus, we have produced substitutions and deletions in a region of the E2 tail (E2 amino acids 408 to 415) that is initially integrated into the endoplasmic reticulum. This sequence was identified as being critical for nucleocapsid binding in an in vitro peptide protection assay. The effects of these mutations on virus assembly and function were determined in both vertebrate and invertebrate cells. Amino acid substitutions (at positions E2: 408, 410, 411, and 413) reduced infectious virus production in a position-dependent fashion but were not efficient in disrupting assembly in mammalian cells. Deletions in the E2 endodomain (delta406-407, delta409-411, and delta414-417) resulted in the failure to assemble virions in mammalian cells. Electron microscopy of BHK cells transfected with these mutants revealed assembly of nucleocapsids that failed to attach to membranes. However, introduction of these deletion mutants into insect cells resulted in the assembly of virus-like particles but no assayable infectivity. These data help define protein interactions critical for virus assembly and suggest a fundamental difference between Sindbis virus assembly in mammalian and insect cells.

Mapping the structure and function of the E1 and E2 glycoproteins in alphaviruses.

The 9 A resolution cryo-electron microscopy map of Sindbis virus presented here provides structural information on the polypeptide topology of the E2 protein, on the interactions between the E1 and E2 glycoproteins in the formation of a heterodimer, on the difference in conformation of the two types of trimeric spikes, on the interaction between the transmembrane helices of the E1 and E2 proteins, and on the conformational changes that occur when fusing with a host cell. The positions of various markers on the E2 protein established the approximate topology of the E2 structure. The largest conformational differences between the icosahedral surface spikes at icosahedral 3-fold and quasi-3-fold positions are associated with the monomers closest to the 5-fold axes. The long E2 monomers, containing the cell receptor recognition motif at their extremities, are shown to rotate by about 180 degrees and to move away from the center of the spikes during fusion.

Structural characterization of the E2 glycoprotein from Sindbis by lysine biotinylation and LC-MS/MS.

Sindbis is an Alphavirus capable of infecting and replicating in both vertebrate and invertebrate hosts. Mature Sindbis virus particles consist of an inner capsid surrounded by a host-derived lipid bilayer, which in turn is surrounded by a protein shell consisting of the E1 and E2 glycoproteins. While a homolog of the E1 glycoprotein has been structurally characterized, the amount of structural data on the E2 glycoprotein is considerably less. In this study, the organization of the E2 glycoprotein was probed by surface biotinylation of intact virions. The virus remained fully infectious, demonstrating that the biotinylation did not alter the topology of the proteins involved in infection. Seven sites of modification were identified in the E2 glycoprotein (K70, K76, K97, K131, K149, K202, and K235), while one site of modification in the E1 glycoprotein (K16) was identified, confirming that the E1 protein is almost completely buried in the virus structure.

Single amino acid insertions at the junction of the sindbis virus E2 transmembrane domain and endodomain disrupt virus envelopment and alter infectivity.

The final steps in the envelopment of Sindbis virus involve specific interactions of the E2 endodomain with the virus nucleocapsid. Deleting E2 K at position 391 (E2 DeltaK391) resulted in the disruption of virus assembly in mammalian cells but not insect cells (host range mutant). This suggested unique interactions of the E2 DeltaK391 endodomain with the different biochemical environments of the mammalian and insect cell lipid bilayers. To further investigate the role of the amino acid residues located at or around position E2 391 and constraints on the length of the endodomain on virus assembly, amino acid insertions/substitutions at the transmembrane/endodomain junction were constructed. An additional K was inserted at amino acid position 392 (KK391/392), a K-->F substitution at position 391 was constructed (F391), and an additional F was inserted at 392 (FF391/392). These changes should lengthen the endodomain in the KK391/392 insertion mutant or shorten the endodomain in the FF391/392 mutant. The mutant FF391/392 grown in BHK cells formed virus particles containing extruded material not found on wild-type virus. This characteristic was not seen in FF391/392 virus grown in insect cells. The mutant KK391/392 grown in BHK cells was defective in the final membrane fission reaction, producing multicored or conjoined virus particles. The production of these aberrant particles was ameliorated when the KK391/392 mutant was grown in insect cells. These data indicate that there is a critical minimal spanning distance from the E2 membrane proximal amino acid at position 391 and the conserved E2 Y400 residue. The observed phenotypes of these mutants also invoke an important role of the specific host membrane lipid composition on virus architecture and infectivity.

Conformational changes in Sindbis virions resulting from exposure to low pH and interactions with cells suggest that cell penetration may occur at the cell surface in the absence of membrane fusion.

Alphaviruses have the ability to induce cell-cell fusion after exposure to acid pH. This observation has served as an article of proof that these membrane-containing viruses infect cells by fusion of the virus membrane with a host cell membrane upon exposure to acid pH after incorporation into a cell endosome. We have investigated the requirements for the induction of virus-mediated, low pH-induced cell-cell fusion and cell-virus fusion. We have correlated the pH requirements for this process to structural changes they produce in the virus by electron cryo-microscopy. We found that exposure to acid pH was required to establish conditions for membrane fusion but that membrane fusion did not occur until return to neutral pH. Electron cryo-microscopy revealed dramatic changes in the structure of the virion as it was moved to acid pH and then returned to neutral pH. None of these treatments resulted in the disassembly of the virus protein icosahedral shell that is a requisite for the process of virus membrane-cell membrane fusion. The appearance of a prominent protruding structure upon exposure to acid pH and its disappearance upon return to neutral pH suggested that the production of a "pore"-like structure at the fivefold axis may facilitate cell penetration as has been proposed for polio (J. Virol. 74 (2000) 1342) and human rhino virus (Mol. Cell 10 (2002) 317). This transient structural change also provided an explanation for how membrane fusion occurs after return to neutral pH. Examination of virus-cell complexes at neutral pH supported the contention that infection occurs at the cell surface at neutral pH by the production of a virus structure that breaches the plasma membrane bilayer. These data suggest an alternative route of infection for Sindbis virus that occurs by a process that does not involve membrane fusion and does not require disassembly of the virus protein shell.

Assessing the feasibility of using neural precursor cells and peripheral blood mononuclear cells for detection of bioactive Sindbis virus.

Viruses form a significant class of bio-threat agents. Currently, the only method to determine the bioactivity of viruses in vitro is to measure viral and cellular responses after co-incubation of cells with virus. Our goal is to find biomarkers for classification of agents, establishment of bioactivity, and/or prediction of disease outcomes. To begin development of a cell-based biosensor for detection of bioactive Sindbis virus (SV), our model analyte, we surveyed the outcomes of SV interaction with primary rat neural precursor cells (NPC) and human peripheral blood mononuclear cells (PBMC). Confocal fluorescence analysis of NPC treated with recombinant SV carrying green-fluorescent-protein (SV-GFP) showed that most cells were GFP positive by day 1 post inoculation. 4',6-Diamidino-2-phenylindole dihydrochloride (DAPI) staining of the nucleus showed nuclear condensation and fragmentation, and the percentage of TUNEL positive cells were higher in virus-treated cells than in mock-treated control. Also, there were less BrdU positive cells in virus-treated cells compared to control. Thus, SV infects NPC, decreases cellular proliferation, and induces cell death via apoptosis. PBMC were treated with SV- or UV-inactivated SV. By day 5 post infection, there were fewer adherent cells in SV-treated PBMC compared to UV-inactivated SV treated PBMC. However, the percentage of viable cells remained the same, and virus growth curves showed only clearance of virus. Thus, SV induces detachment of a subpopulation of PBMC while not killing most of the cells. Together, these results indicate that NPC and PBMC respond to bioactive SV inoculation, suggesting potential use as detectors of SV in cell-based biosensor paradigm. These studies also provide the rationale, time-scale, and phenotypic correlates for further studies with gene expression arrays.

Structures of immature flavivirus particles.

Structures of prM-containing dengue and yellow fever virus particles were determined to 16 and 25 A resolution, respectively, by cryoelectron microscopy and image reconstruction techniques. The closely similar structures show 60 icosahedrally organized trimeric spikes on the particle surface. Each spike consists of three prM:E heterodimers, where E is an envelope glycoprotein and prM is the precursor to the membrane protein M. The pre-peptide components of the prM proteins in each spike cover the fusion peptides at the distal ends of the E glycoproteins in a manner similar to the organization of the glycoproteins in the alphavirus spikes. Each heterodimer is associated with an E and a prM transmembrane density. These transmembrane densities represent either an EE or prMprM antiparallel coiled coil by which each protein spans the membrane twice, leaving the C-terminus of each protein on the exterior of the viral membrane, consistent with the predicted membrane-spanning domains of the unprocessed polyprotein.

Morphological variants of Sindbis virus produced by a mutation in the capsid protein.

Sindbis virus is a complex aggregate of RNA, protein and lipid. The virus is organized as two nested T = 4 icosahedral protein shells between which is sandwiched a lipid bilayer. The virus RNA resides within the inner protein shell. The inner protein shell is attached to the outer protein shell through contacts to proteins in the outer shell, which penetrate the lipid bilayer. The data presented in the following manuscript show that mutations in the capsid protein can result in the assembly of the virus structural proteins into icosahedra of different triangulation numbers. The triangulation numbers calculated, for these morphological variants, follow the sequence T = 4, 9, 16, 25 and 36. All fall into the class P = 1 of icosadeltahedra as was predicted by. The data support their hypothesis that families of icosahedra would be developed by altering the distance between the points of insertion of the five-fold axis. This capsid protein defect also results in the incorporation of much of the capsid protein, into large cytoplasmic aggregates of protein and RNA. These observations support models suggesting that the geometry of a pre-formed nucleocapsid organizes the assembly of the virus membrane proteins into a structure of identical configuration and argues against models suggesting that assembly of the membrane glycoproteins directs the assembly of the nucleocapsid.

Locations of carbohydrate sites on alphavirus glycoproteins show that E1 forms an icosahedral scaffold.

There are 80 spikes on the surface of Sindbis virus arranged as an icosahedral surface lattice. Each spike consists of three copies of each of the glycoproteins E1 and E2. There are two glycosylation sites on E1 and two on E2. These four sites have been located by removal of the glycosylation recognition motifs using site-specific mutagenesis, followed by cryoelectron microscopy. The positions of these sites have demonstrated that E2 forms the protruding spikes and that E1 must be long and narrow, lying flat on the viral surface, forming an icosahedral scaffold analogous to the arrangement of the E glycoprotein in flaviviruses. This arrangement of E1 leads to both dimeric and trimeric intermolecular contacts, consistent with the observed structural changes that occur on fusion with host cell membranes, suggesting a similar fusion mechanism for alpha- and flaviviruses.