Dissecting the Components of Sindbis Virus from Arthropod and Vertebrate Hosts: Implications for Infectivity Differences.

Sindbis virus (SINV) is an enveloped, single-stranded RNA virus, which is transmitted via mosquitos to a wide range of vertebrate hosts. SINV produced by vertebrate, baby hamster kidney (BHK) cells is more than an order of magnitude less infectious than SINV produced from mosquito (C6/36) cells. The cause of this difference is poorly understood. In this study, charge detection mass spectrometry was used to determine the masses of intact SINV particles isolated from BHK and C6/36 cells. The measured masses are substantially different: 52.88 MDa for BHK derived SINV and 50.69 MDa for C6/36 derived. Further analysis using several mass spectrometry-based methods and biophysical approaches indicates that BHK derived SINV has a substantially higher mass than C6/36 derived because in the lipid bilayer, there is a higher portion of lipids containing long chain fatty acids. The difference in lipid composition could influence the organization of the lipid bilayer. As a result, multiple stages of the viral lifecycle may be affected including assembly and budding, particle stability during transmission, and fusion events, all of which could contribute to the differences in infectivity.

Domains of the TF protein important in regulating its own palmitoylation.

Sindbis virus particles contain the viral proteins capsid, E1 and E2, and low levels of a small membrane protein called TF. TF is produced during a (-1) programmed ribosomal frameshifting event during the translation of the structural polyprotein. TF from Sindbis virus-infected cells is present in two palmitoylated states, basal and maximal; unpalmitoylated TF is not detectable. Mutagenesis studies demonstrated that without palmitoylation, TF is not incorporated into released virions, suggesting palmitoylation of TF is a regulated step in virus assembly. In this work, we identified Domains within the TF protein that regulate its palmitoylation state. Mutations and insertions in Domain III, a region proposed to be in the cytoplasmic loop of TF, increase levels of unpalmitoylated TF found during an infection but still unpalmitoylated TF was not incorporated into virions. Mutations in Domain IV, the TF unique region, are likely to impact the balance between basal and maximal palmitoylation.

A simple strategy for retargeting lentiviral vectors to desired cell types via a disulfide-bond-forming protein-peptide pair.

Despite recent improvements in the engineering of viral envelope proteins, it remains a significant challenge to create lentiviral vectors that allow targeted transduction to specific cell populations of interest. In this study, we developed a simple 'plug and play' strategy to retarget lentiviral vectors to any desired cell types through in vitro covalent modification of the virions with specific cell-targeting proteins (CTPs). This strategy exploits a disulfide bond-forming protein-peptide pair PDZ1 and its pentapeptide ligand (ThrGluPheCysAla, TEFCA). PDZ1 was incorporated into an engineered Sindbis virus envelope protein (Sind-PDZ1) and displayed on lentiviral particles while the TEFCA pentapeptide ligand was genetically linked to the CTP. Her2/neu-binding designed ankyrin repeat proteins (DARPin) were used as our model CTPs. DARPin-functionalized unconcentrated lentiviral vectors harboring Sind-PDZ1 envelope protein (Sind-PDZ1-pp) exhibited >800-fold higher infectious titer in HER2+ cells than the unfunctionalized virions (8.5 × 106 vs. <104 IU/mL). Moreover, by virtue of the covalent disulfide bond interaction between PDZ1 and TEFCA, the association of the CTP with the virions is nonreversible under non-reducing conditions (e.g. serum), making these functionalized virions potentially stable in an in vivo setting.

Why Enveloped Viruses Need Cores-The Contribution of a Nucleocapsid Core to Viral Budding.

During the lifecycle of many enveloped viruses, a nucleocapsid core buds through the cell membrane to acquire an outer envelope of lipid membrane and viral glycoproteins. However, the presence of a nucleocapsid core is not required for assembly of infectious particles. To determine the role of the nucleocapsid core, we develop a coarse-grained computational model with which we investigate budding dynamics as a function of glycoprotein and nucleocapsid interactions, as well as budding in the absence of a nucleocapsid. We find that there is a transition between glycoprotein-directed budding and nucleocapsid-directed budding that occurs above a threshold strength of nucleocapsid interactions. The simulations predict that glycoprotein-directed budding leads to significantly increased size polydispersity and particle polymorphism. This polydispersity can be explained by a theoretical model accounting for the competition between bending energy of the membrane and the glycoprotein shell. The simulations also show that the geometry of a budding particle leads to a barrier to subunit diffusion, which can result in a stalled, partially budded state. We present a phase diagram for this and other morphologies of budded particles. Comparison of these structures against experiments could establish bounds on whether budding is directed by glycoprotein or nucleocapsid interactions. Although our model is motivated by alphaviruses, we discuss implications of our results for other enveloped viruses.

Structural divergence creates new functional features in alphavirus genomes.

Alphaviruses are mosquito-borne pathogens that cause human diseases ranging from debilitating arthritis to lethal encephalitis. Studies with Sindbis virus (SINV), which causes fever, rash, and arthralgia in humans, and Venezuelan equine encephalitis virus (VEEV), which causes encephalitis, have identified RNA structural elements that play key roles in replication and pathogenesis. However, a complete genomic structural profile has not been established for these viruses. We used the structural probing technique SHAPE-MaP to identify structured elements within the SINV and VEEV genomes. Our SHAPE-directed structural models recapitulate known RNA structures, while also identifying novel structural elements, including a new functional element in the nsP1 region of SINV whose disruption causes a defect in infectivity. Although RNA structural elements are important for multiple aspects of alphavirus biology, we found the majority of RNA structures were not conserved between SINV and VEEV. Our data suggest that alphavirus RNA genomes are highly divergent structurally despite similar genomic architecture and sequence conservation; still, RNA structural elements are critical to the viral life cycle. These findings reframe traditional assumptions about RNA structure and evolution: rather than structures being conserved, alphaviruses frequently evolve new structures that may shape interactions with host immune systems or co-evolve with viral proteins.

An icosahedral virus as a fluorescent calibration standard: a method for counting protein molecules in cells by fluorescence microscopy.

The ability to replace genes coding for cellular proteins with DNA that codes for fluorescent protein-tagged versions opens the way to counting the number of molecules of each protein component of macromolecular assemblies in vivo by measuring fluorescence microscopically. Converting fluorescence to absolute numbers of molecules requires a fluorescent standard whose molecular composition is known precisely. In this report, the construction, properties and mode of using a set of fluorescence calibration standards are described. The standards are based on an icosahedral virus engineered to contain exactly 240 copies of one of seven different fluorescent proteins. Two applications of the fluorescent standards to counting molecules in the human parasite Toxoplasma gondii are described. Methods for improving the preciseness of the measurements and minimizing potential inaccuracies are emphasized.

Full inactivation of alphaviruses in single particle and crystallized forms.

Inherent in the study of viruses is the risk of pathogenic exposure, which necessitates appropriate levels of biosafety containment. Unfortunately, this also limits the availability of useful research instruments that are located at facilities not equipped to handle infectious pathogens. Abrogation of viral infectivity can be accomplished without severely disrupting the physical structure of the virus particle. Virus samples that are verifiably intact but not infectious may be enabled for study at research facilities where they would otherwise not be allowed. Inactivated viruses are also used in the development of vaccines, where immunogenicity is sought in the absence of active infection. We demonstrate the inactivation of Sindbis alphavirus particles in solution, as well as in crystallized form. Inactivation was accomplished by two different approaches: crosslinking of proteins by glutaraldehyde treatment, and crosslinking of nucleic acids by UV irradiation. Biophysical characterization methods, including dynamic light scattering and transmission electron microscopy, were used to demonstrate that the glutaraldehyde and UV inactivated Sindbis virus particles remain intact structurally. SDS-PAGE was also used to show evidence of the protein crosslinking that was expected with glutaraldehyde treatment, but also observed with UV irradiation.

Enveloped virus flocculation and removal in osmolyte solutions.

Our ability to reduce infectious disease burden throughout the world has been greatly improved by the creation of vaccines. However, worldwide immunization rates are low. The two most likely reasons are the lack of sufficient distribution in underdeveloped countries and the high cost of vaccine products. The high costs are due to the difficulties of manufacturing individual vaccine products with specialized purification trains. In this study, we propose to use virus flocculation in osmolytes, followed by microfiltration, as an alternative vaccine purification operation. In our previous work, we demonstrated that osmolytes preferentially flocculate a non-enveloped virus, porcine parvovirus (PPV). In this work we show that osmolytes flocculate the enveloped virus, Sindbis virus heat resistant strain (SVHR), and demonstrate a >80% removal with a 0.2 µm microfilter membrane while leaving proteins in solution. The best osmolytes were tested for their ability to flocculate SVHR at different concentrations, pH and ionic strengths. Our best removal was 98% of SVHR in 0.3M mannitol at a pH of 5. We propose that osmolytes are able to flocculate hydrophobic non-enveloped and enveloped virus particles by the reduction of the hydration layer around the particles, which stimulates virus aggregation. Now that we have demonstrated that protecting osmolytes flocculate viruses, this method has the potential to be a future platform purification process for vaccines.

Imaging the alphavirus exit pathway.

UNLABELLED: Alphaviruses are small enveloped RNA viruses with highly organized structures that exclude host cell proteins. They contain an internal nucleocapsid and an external lattice of the viral E2 and E1 transmembrane proteins. Alphaviruses bud from the plasma membrane (PM), but the process and dynamics of alphavirus assembly and budding are poorly understood. Here we generated Sindbis viruses (SINVs) with fluorescent protein labels on the E2 envelope protein and exploited them to characterize virus assembly and budding in living cells. During virus infection, E2 became enriched in localized patches on the PM and in filopodium-like extensions. These E2-labeled patches and extensions contained all of the viral structural proteins. Correlative light and electron microscopy studies established that the patches and extensions colocalized with virus budding structures, while light microscopy showed that they excluded a freely diffusing PM marker protein. Exclusion required the interaction of the E2 protein with the capsid protein, a critical step in virus budding, and was associated with the immobilization of the envelope proteins on the cell surface. Virus infection induced two distinct types of extensions: tubulin-negative extensions that were ∼2 to 4 µm in length and excluded the PM marker, and tubulin-positive extensions that were >10 µm long, contained the PM marker, and could transfer virus particles to noninfected cells. Tubulin-positive extensions were selectively reduced in cells infected with a nonbudding SINV mutant. Together, our data support a model in which alphavirus infection induces reorganization of the PM and cytoskeleton, leading to virus budding from specialized sites. IMPORTANCE: Alphaviruses are important and widely distributed human pathogens for which vaccines and antiviral therapies are urgently needed. These small highly organized viruses bud from the host cell PM. Virus assembly and budding are critical but little understood steps in the alphavirus life cycle. We developed alphaviruses with fluorescent protein tags on one of the viral membrane (envelope) proteins and used a variety of microscopy techniques to follow the envelope protein and a host cell PM protein during budding. We showed that alphavirus infection induced the formation of patches and extensions on the PM where the envelope proteins accumulate. These sites excluded other PM proteins and correlated with virus budding structures. Exclusion of PM proteins required specific interactions of the viral envelope proteins with the internal capsid protein. Together, our data indicate that alphaviruses extensively reorganize the cell surface and cytoskeleton to promote their assembly and budding.

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.

Mutating conserved cysteines in the alphavirus e2 glycoprotein causes virus-specific assembly defects.

There are 80 trimeric, glycoprotein spikes that cover the surface of an alphavirus particle. The spikes, which are composed of three E2 and E1 glycoprotein heterodimers, are responsible for receptor binding and mediating fusion between the viral and host-cell membranes during entry. In addition, the cytoplasmic domain of E2 interacts with the nucleocapsid core during the last stages of particle assembly, possibly to aid in particle stability. During assembly, the spikes are nonfusogenic until the E3 glycoprotein is cleaved from E2 in the trans-Golgi network. Thus, a mutation in E2 potentially has effects on virus entry, spike assembly, or spike maturation. E2 is a highly conserved, cysteine-rich transmembrane glycoprotein. We made single cysteine-to-serine mutations within two distinct regions of the E2 ectodomain in both Sindbis virus and Ross River virus. Each of the E2 Cys mutants produced fewer infectious particles than wild-type virus. Further characterization of the mutant viruses revealed differences in particle morphology, fusion activity, and polyprotein cleavage between Sindbis and Ross River virus mutants, despite the mutations being made at corresponding positions in E2. The nonconserved assembly defects suggest that E2 folding and function is species dependent, possibly due to interactions with a virus-specific chaperone.

Interactions of the cytoplasmic domain of Sindbis virus E2 with nucleocapsid cores promote alphavirus budding.

Alphavirus budding from the plasma membrane occurs through the specific interaction of the nucleocapsid core with the cytoplasmic domain of the E2 glycoprotein (cdE2). Structural studies of the Sindbis virus capsid protein (CP) have suggested that these critical interactions are mediated by the binding of cdE2 into a hydrophobic pocket in the CP. Several molecular genetic studies have implicated amino acids Y400 and L402 in cdE2 as important for the budding of alphaviruses. In this study, we characterized the role of cdE2 residues in structural polyprotein processing, glycoprotein transport, and capsid interactions. Along with hydrophobic residues, charged residues in the N terminus of cdE2 were critical for the effective interaction of cores with cdE2, a process required for virus budding. Mutations in the C-terminal signal sequence region of cdE2 affected E2 protein transport to the plasma membrane, while nonbudding mutants that were defective in cdE2-CP interaction accumulated E2 on the plasma membrane. The interaction of cdE2 with cytoplasmic cores purified from infected cells and in vitro-assembled core-like particles suggests that cdE2 interacts with assembled cores to mediate budding. We hypothesize that these cdE2 interactions induce a change in the organization of the nucleocapsid core upon binding leading to particle budding and priming of the nucleocapsid cores for disassembly that is required for virus infection.

Conformational changes in Sindbis virus induced by decreased pH are revealed by small-angle neutron scattering.

Alphaviruses, such as Sindbis virus, undergo dramatic changes in three-dimensional structure upon exposure to low pH, and such exposure can establish conditions allowing fusion of the virus membrane with a cell plasma membrane upon return to neutral pH. While exposure to low pH is not required for entry of Sindbis virus into vertebrate or invertebrate cells, the conformational changes occurring at low pH may mimic those occurring upon virus-receptor interaction. Here, we employed small-angle neutron scattering with contrast variation to probe how the structure of a mammalian-grown Sindbis virus responds to moderately acidic pH. Several changes took place throughout the virion structure when the pH decreased from 7.2 to 6.4. Specifically, the RNA in the virion core underwent a conformational change. Additionally, the protein was redistributed. A significant amount of protein moved from the layer containing the lipid bilayer to the exterior of the virion. The results improve our understanding of the pH-driven alteration of Sindbis virus structure.

Structural changes of envelope proteins during alphavirus fusion.

Alphaviruses are enveloped RNA viruses that have a diameter of about 700 Å and can be lethal human pathogens. Entry of virus into host cells by endocytosis is controlled by two envelope glycoproteins, E1 and E2. The E2-E1 heterodimers form 80 trimeric spikes on the icosahedral virus surface, 60 with quasi-three-fold symmetry and 20 coincident with the icosahedral three-fold axes arranged with T = 4 quasi-symmetry. The E1 glycoprotein has a hydrophobic fusion loop at one end and is responsible for membrane fusion. The E2 protein is responsible for receptor binding and protects the fusion loop at neutral pH. The lower pH in the endosome induces the virions to undergo an irreversible conformational change in which E2 and E1 dissociate and E1 forms homotrimers, triggering fusion of the viral membrane with the endosomal membrane and then releasing the viral genome into the cytoplasm. Here we report the structure of an alphavirus spike, crystallized at low pH, representing an intermediate in the fusion process and clarifying the maturation process. The trimer of E2-E1 in the crystal structure is similar to the spikes in the neutral pH virus except that the E2 middle region is disordered, exposing the fusion loop. The amino- and carboxy-terminal domains of E2 each form immunoglobulin-like folds, consistent with the receptor attachment properties of E2.

The structure of Sindbis virus produced from vertebrate and invertebrate hosts as determined by small-angle neutron scattering.

The complex natural cycle of vectored viruses that transition between host species, such as between insects and mammals, makes understanding the full life cycle of the virus an incredibly complex problem. Sindbis virus, an arbovirus and prototypic alphavirus having an inner protein shell and an outer glycoprotein coat separated by a lipid membrane, is one example of a vectored virus that transitions between vertebrate and insect hosts. While evidence of host-specific differences in Sindbis virus has been observed, no work has been performed to characterize the impact of the host species on the structure of the virus. Here, we report the first study of the structural differences between Sindbis viruses grown in mammalian and insect cells, which were determined by small-angle neutron scattering (SANS), a nondestructive technique that did not decrease the infectivity of the Sindbis virus particles studied. The scattering data and modeling showed that, while the radial position of the lipid bilayer did not change significantly, it was possible to conclude that it did have significantly more cholesterol when the virus was grown in mammalian cells. Additionally, the outer protein coat was found to be more extended in the mammalian Sindbis virus. The SANS data also demonstrated that the RNA and nucleocapsid protein share a closer interaction in the mammalian-cell-grown virus than in the virus from insect cells.

Interaction of Sindbis virus non-structural protein 3 with poly(ADP-ribose) polymerase 1 in neuronal cells.

The alphavirus non-structural protein 3 (nsP3) has a conserved N-terminal macro domain and a variable highly phosphorylated C-terminal domain. nsP3 forms complexes with cellular proteins, but its role in virus replication is poorly understood and protein interaction domains have not been defined. As the N-terminal macro domain can bind poly(ADP-ribose) (PAR), and PAR polymerase-1 (PARP-1) is activated and autoribosylated during Sindbis virus (SINV) infection, it was hypothesized that PARP-1 and nsP3 may interact. Co-immunoprecipitation studies showed that PARP-1 interacted with nsP3 during SINV infection of NSC34 neuronal cells and was most abundantly present in replication complexes that contained plus- and minus-strand SINV RNAs 10-14 h after infection, prior to PARP-1 activation or automodification with PAR. Treatment with an inhibitor of PARP enzymic activity did not affect the interaction between nsP3 and PARP-1 or SINV replication. Co-expression of individual domains of nsP3 with PARP-1 showed that nsP3 interacted with PARP-1 through the C-terminal domain, not the N-terminal macro domain, and that phosphorylation was not required. It was concluded that PARP-1 interacts with the C-terminal domain of nsP3, is present in replication complexes during virus amplification and may play a role in regulating virus RNA synthesis in neuronal cells.

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.

Structural and functional analyses of stem-loop 1 of the Sindbis virus genome.

Alphavirus genome function is controlled by elements at both the 5' and 3' ends. The 5' 220 nt of the Sindbis virus genome is predicted to consist of four stem-loop structures the first of which has been demonstrated to be required for efficient minus-strand RNA synthesis. To understand the role of the structure of the first stem-loop (SL1) in regulating genome function, we performed enzymatic and chemical probing analyses. There were significant differences between the computer-predicted structures and our experimental data. In the 5' terminus, two loop regions appear to be interacting in a complex and interdependent fashion with non-Watson-Crick interactions involving multiple adenosine residues playing a critical role in determining the overall structure. Some of the mutations that disrupted these interactions had significant affects, both positive and negative, on minus-strand synthesis, and translational efficiency was generally increased. In the context of full-length virus, these structural changes resulted in reduced virus growth kinetics particularly in mosquito cells suggesting host-specific effects of mutations in this region of the viral genome. Possible SL1 structures based on our experimental data are discussed.