Computational method for designing vaccines applied to virus-like particles (VLPs) as epitope carriers.

Nonenveloped virus-like particles (VLPs) are self-assembled oligomeric structures composed of one or more proteins that originate from diverse viruses. Because these VLPs have similar antigenicity to the parental virus, they are successfully used as vaccines against cognate virus infection. Furthermore, after foreign antigenic sequences are inserted in their protein components (chimVLPs), some VLPs are also amenable to producing vaccines against pathogens other than the virus it originates from (these VLPs are named platform or epitope carrier). Designing chimVLP vaccines is challenging because the immunogenic response must be oriented against a given antigen without altering stimulant properties inherent to the VLP. An important step in this process is choosing the location of the sequence modifications because this must be performed without compromising the assembly and stability of the original VLP. Currently, many immunogenic data and computational tools can help guide the design of chimVLPs, thus reducing experimental costs and work. In this study, we analyze the structure of a novel VLP that originate from an insect virus and describe the putative regions of its three structural proteins amenable to insertion. For this purpose, we employed molecular dynamics (MD) simulations to assess chimVLP stability by comparing mutated and wild-type (WT) VLP protein trajectories. We applied this procedure to design a chimVLP that can serve as a prophylactic vaccine against the SARS-CoV-2 virus. The methodology described in this work is generally applicable for VLP-based vaccine development.

Dimeric Rhodopsin R135L Mutant-Transducin-like Complex Sheds Light on Retinitis Pigmentosa Misfunctions.

Rhodopsin (RHO) is a light-sensitive pigment in the retina and the main prototypical protein of the G-protein-coupled receptor (GCPR) family. After receiving a light stimulus, RHO and its cofactor retinylidene undergo a series of structural changes that initiate an intricate transduction mechanism. Along with RHO, other partner proteins play key roles in the signaling pathway. These include transducin, a GTPase, kinases that phosphorylate RHO, and arrestin (Arr), which ultimately stops the signaling process and promotes RHO regeneration. A large number of RHO genetic mutations may lead to very severe retinal dysfunction and eventually to impaired dark adaptation disease called autosomal dominant retinitis pigmentosa (adRP). In this study, we used molecular dynamics (MD) simulations to evaluate the different behaviors of the dimeric form of wild-type RHO (WT dRHO) and its mutant at position 135 of arginine to leucine (dR135L), both in the free (noncomplexed) and in complex with the transducin-like protein (Gtl). Gtl is a heterotrimeric model composed of a mixture of human and bovine G proteins. Our calculations allow us to explain how the mutation causes structural changes in the RHO dimer and how this can affect the signal that transducin generates when it is bound to RHO. Moreover, the structural modifications induced by the R135L mutation can also account for other misfunctions observed in the up- and downstream signaling pathways. The mechanism of these dysfunctions, together with the transducin activity reduction, provides structure-based explanations of the impairment of some key processes that lead to adRP.

Antibodies response induced by recombinant virus-like particles from Triatoma virus and chimeric antigens from Trypanosoma cruzi.

BACKGROUND: The infection caused by the protozoan Trypanosoma cruzi affects humans and is called as Chagas disease. Currently, the main measures available to reduce the incidence of this disease are drug treatment and vector control. Traditionally, the development of vaccines occurs mainly through the use of antigenic candidates of the etiologic agent in the form of a vaccine preparation. Virus-like particles (VLPs) are structures analogous to viral capsids composed essentially of structural proteins and are widely used in vaccination protocols because of their immunostimulatory properties. In this context, the objective of this study was to use strategies in a murine immunization model to characterize the immunostimulatory capacity of VLPs from Triatoma virus (TrV-VLPs), analysed in the presence or absence of the aluminium vaccine adjuvant. In parallel, to characterize the immunogenic behaviour of four T. cruzi chimeric recombinant proteins (mix-IBMP) associated with TrV-VLPs or aluminium vaccine adjuvant. METHOD: We immunized BALB/c mice once or twice, depending on the strategy, and collected serum samples at 15, 30 and 45 days after the immunization. Subsequently, serum samples from animals immunized with TrV-VLPs were used to determine total IgG, IgG1, IgG2a, IgG2b and IgG3 anti-TrV-VLPs by enzyme-linked immunosorbent assay (ELISA). RESULTS: Data obtained demonstrate the ability of TrV-VLPs to preferably induce IgG2b and IgG3 type antibodies in the absence of aluminium adjuvant. In fact, the use of aluminium did not interfere with the total IgG profile of anti-TrV-VLPs. Interestingly, mix-IBMP had a better profile of total IgG, IgG1 and IgG3 subclasses when mixed with TrV-VLPs. CONCLUSION: In conclusion, these results suggest the potential of TrV-VLPs as a vaccine adjuvant and the use of T. cruzi chimeric antigens as a rational strategy for the development of vaccines against the experimental model of Chagas disease.

VLP-Based Vaccines as a Suitable Technology to Target Trypanosomatid Diseases.

Research on vaccines against trypanosomatids, a family of protozoa that cause neglected tropical diseases, such as Chagas disease, leishmaniasis, and sleeping sickness, is a current need. Today, according to modern vaccinology, virus-like particle (VLP) technology is involved in many vaccines, including those undergoing studies related to COVID-19. The potential use of VLPs as vaccine adjuvants opens an opportunity for the use of protozoan antigens for the development of vaccines against diseases caused by Trypanosoma cruzi, Leishmania spp., and Trypanosoma brucei. In this context, it is important to consider the evasion mechanisms of these protozoa in the host and the antigens involved in the mechanisms of the parasite-host interaction. Thus, the immunostimulatory properties of VLPs can be part of an important strategy for the development and evaluation of new vaccines. This work aims to highlight the potential of VLPs as vaccine adjuvants for the development of immunity in complex diseases, specifically in the context of tropical diseases caused by trypanosomatids.

Virion structure and in vitro genome release mechanism of dicistrovirus Kashmir bee virus.

Infections of Kashmir bee virus (KBV) are lethal for honeybees and have been associated with colony collapse disorder. KBV and closely related viruses contribute to the ongoing decline in the number of honeybee colonies in North America, Europe, Australia, and other parts of the world. Despite the economic and ecological impact of KBV, its structure and infection process remain unknown. Here we present the structure of the virion of KBV determined to a resolution of 2.8 Å. We show that the exposure of KBV to acidic pH induces a reduction in inter-pentamer contacts within capsids and the reorganization of its RNA genome from a uniform distribution to regions of high and low density. Capsids of KBV crack into pieces at acidic pH, resulting in the formation of open particles lacking pentamers of capsid proteins. The large openings of capsids enable the rapid release of genomes and thus limit the probability of their degradation by RNases. The opening of capsids may be a shared mechanism for the genome release of viruses from the family Dicistroviridae ImportanceThe western honeybee (Apis mellifera) is indispensable for maintaining agricultural productivity as well as the abundance and diversity of wild flowering plants. However, bees suffer from environmental pollution, parasites, and pathogens, including viruses. Outbreaks of virus infections cause the deaths of individual honeybees as well as collapses of whole colonies. Kashmir bee virus has been associated with colony collapse disorder in the US, and no cure of the disease is currently available. Here we report the structure of an infectious particle of Kashmir bee virus and show how its protein capsid opens to release the genome. Our structural characterization of the infection process determined that therapeutic compounds stabilizing contacts between pentamers of capsid proteins could prevent the genome release of the virus.

Alkalinization of Icosahedral Non-enveloped Viral Capsid Interior Through Proton Channeling.

Small icosahedral viruses have a compact capsid that apparently lacks holes through which solvents can be exchanged with the external milieu. However, due to the steric hindrance of amino acids, upon folding, capsid proteins form narrow cavities in which water and ions can be trapped. These occluded solvent molecules can form lines of water, called water wires, representing an arrangement with special features for proton conduction. In this chapter, we review the physico-chemical principles that permit proton conduction through protein cavities. We also describe how a combination of these elements found in an insect viral capsid can allow the virus to sense alkaline environments. Through this analysis, we stress the need to combine experimental and theoretical techniques when modeling complex biological systems.

ICTV Virus Taxonomy Profile: Solinviviridae.

Solinviviridae is a family of picorna/calici-like viruses with non-segmented, linear, positive-sense RNA genomes of approximately 10-11 kb. Unusually, their capsid proteins are encoded towards the 3'-end of the genome where they can be expressed both from a subgenomic RNA and as an extension of the replication (picorna-like helicase-protease-polymerase) polyprotein. Members of two species within the family infect ants, but related unclassified virus sequences derive from a large variety of insects and other arthropods. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the Solinviviridae, which is available at www.ictv.global/report/solinviviridae.

ICTV Virus Taxonomy Profile: Polycipiviridae.

Polycipiviridae is a family of picorna-like viruses with non-segmented, linear, positive-sense RNA genomes of approximately 10-12 kb. Unusually for viruses within the order Picornavirales, their genomes are polycistronic, with four (or more) consecutive 5'-proximal open reading frames (ORFs) encoding structural (and possibly other) proteins and a long 3' ORF encoding the replication polyprotein. Members of species within the family have all been detected in ants or via arthropod transcriptomic datasets. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the Polycipiviridae, which is available at www.ictv.global/report/polycipiviridae.

Multiscale modelization in a small virus: Mechanism of proton channeling and its role in triggering capsid disassembly.

In this work, we assess a previously advanced hypothesis that predicts the existence of ion channels in the capsid of small and non-enveloped icosahedral viruses. With this purpose we examine Triatoma Virus (TrV) as a case study. This virus has a stable capsid under highly acidic conditions but disassembles and releases the genome in alkaline environments. Our calculations range from a subtle sub-atomic proton interchange to the dismantling of a large-scale system representing several million of atoms. Our results provide structure-based explanations for the three roles played by the capsid to enable genome release. First, we observe, for the first time, the formation of a hydrophobic gate in the cavity along the five-fold axis of the wild-type virus capsid, which can be disrupted by an ion located in the pore. Second, the channel enables protons to permeate the capsid through a unidirectional Grotthuss-like mechanism, which is the most likely process through which the capsid senses pH. Finally, assuming that the proton leak promotes a charge imbalance in the interior of the capsid, we model an internal pressure that forces shell cracking using coarse-grained simulations. Although qualitatively, this last step could represent the mechanism of capsid opening that allows RNA release. All of our calculations are in agreement with current experimental data obtained using TrV and describe a cascade of events that could explain the destabilization and disassembly of similar icosahedral viruses.

Exploring the role of genome and structural ions in preventing viral capsid collapse during dehydration.

Even though viruses evolve mainly in liquid milieu, their horizontal transmission routes often include episodes of dry environment. Along their life cycle, some insect viruses, such as viruses from the Dicistroviridae family, withstand dehydrated conditions with presently unknown consequences to their structural stability. Here, we use atomic force microscopy to monitor the structural changes of viral particles of Triatoma virus (TrV) after desiccation. Our results demonstrate that TrV capsids preserve their genome inside, conserving their height after exposure to dehydrating conditions, which is in stark contrast with other viruses that expel their genome when desiccated. Moreover, empty capsids (without genome) resulted in collapsed particles after desiccation. We also explored the role of structural ions in the dehydration process of the virions (capsid containing genome) by chelating the accessible cations from the external solvent milieu. We observed that ion suppression helps to keep the virus height upon desiccation. Our results show that under drying conditions, the genome of TrV prevents the capsid from collapsing during dehydration, while the structural ions are responsible for promoting solvent exchange through the virion wall.

X-ray structure of Triatoma virus empty capsid: insights into the mechanism of uncoating and RNA release in dicistroviruses.

In viruses, uncoating and RNA release are two key steps of successfully infecting a target cell. During these steps, the capsid must undergo the necessary conformational changes to allow RNA egress. Despite their importance, these processes are poorly understood in the family Dicistroviridae. Here, we used X-ray crystallography to solve the atomic structure of a Triatoma virus(TrV) empty particle (Protein Data Bank ID 5L7O), which is the resulting capsid after RNA release. It is observed that the overall shape of the capsid and of the three individual proteins is maintained in comparison with the mature virion. Furthermore, no channels indicative of RNA release are formed in the TrV empty particle. However, the most prominent change in the empty particle when compared with the mature virion is the loss of order in the N-terminal domain of the VP2 protein. In mature virions, the VP2 N-terminal domain of one pentamer is swapped with its twofold related copy in an adjacent pentamer, thereby stabilizing the binding between the pentamers. The loss of these interactions allows us to propose that RNA release may take place through transient flipping-out of pentameric subunits. The lower number of stabilizing interactions between the pentamers and the lack of formation of new holes support this model. This model differs from the currently accepted model for rhinoviruses and enteroviruses, in which genome externalization occurs by extrusion of the RNA through capsid channels.

Triatomines intrahaemocoelic inoculation protocol: a useful tool to check infectivity in insects.

Vectors of Chagas disease are currently controlled by employing several chemical insecticides though there is a continuing search for alternative ecological methods against disease causing vectors. An effective method includes the use of specific pathogens as biological control agents. The aim of this work was to describe a complete experimental inoculation protocol in triatomines. The intrahaemocoelic inoculation technique can be applied to inoculate different kinds of microorganisms such as viruses, fungi, bacteria and protozoa; so it could be considered a useful tool in infective bioassays. This article includes results from evaluations of Triatoma virus (TrV, Dicistroviridae: Triatovirus) infectivity in several triatomine species. The protocol, also suitable for any other kind of insects, describes the materials and steps required to safely inoculate the insects, preventing any damage and/or contamination.

Triatoma virus recombinant VP4 protein induces membrane permeability through dynamic pores.

UNLABELLED: In naked viruses, membrane breaching is a key step that must be performed for genome transfer into the target cells. Despite its importance, the mechanisms behind this process remain poorly understood. The small protein VP4, encoded by the genomes of most viruses of the order Picornavirales, has been shown to be involved in membrane alterations. Here we analyzed the permeabilization activity of the natively nonmyristoylated VP4 protein from triatoma virus (TrV), a virus belonging to the Dicistroviridae family within the Picornavirales order. The VP4 protein was produced as a C-terminal maltose binding protein (MBP) fusion to achieve its successful expression. This recombinant VP4 protein is able to produce membrane permeabilization in model membranes in a membrane composition-dependent manner. The induced permeability was also influenced by the pH, being greater at higher pH values. We demonstrate that the permeabilization activity elicited by the protein occurs through discrete pores that are inserted on the membrane. Sizing experiments using fluorescent dextrans, cryo-electron microscopy imaging, and other, additional techniques showed that recombinant VP4 forms heterogeneous proteolipidic pores rather than common proteinaceous channels. These results suggest that the VP4 protein may be involved in the membrane alterations required for genome transfer or cell entry steps during dicistrovirus infection. IMPORTANCE: During viral infection, viruses need to overcome the membrane barrier in order to enter the cell and replicate their genome. In nonenveloped viruses membrane fusion is not possible, and hence, other mechanisms are implemented. Among other proteins, like the capsid-forming proteins and the proteins required for viral replication, several viruses of the order Picornaviridae contain a small protein called VP4 that has been shown to be involved in membrane alterations. Here we show that the triatoma virus VP4 protein is able to produce membrane permeabilization in model membranes by the formation of heterogeneous dynamic pores. These pores formed by VP4 may be involved in the genome transfer or cell entry steps during viral infection.

Seroprevalence of Triatoma virus (Dicistroviridae: Cripaviridae) antibodies in Chagas disease patients.

BACKGROUND: Chagas disease is caused by Trypanosoma cruzi, and humans acquire the parasite by exposure to contaminated feces from hematophagous insect vectors known as triatomines. Triatoma virus (TrV) is the sole viral pathogen of triatomines, and is transmitted among insects through the fecal-oral route and, as it happens with T. cruzi, the infected insects release the virus when defecating during or after blood uptake. METHODS: In this work, we analysed the occurrence of anti-TrV antibodies in human sera from Chagas disease endemic and non-endemic countries, and developed a mathematical model to estimate the transmission probability of TrV from insects to man, which ranged between 0.00053 and 0.0015. RESULTS: Our results confirm that people with Chagas disease living in Bolivia, Argentina and Mexico have been exposed to TrV, and that TrV is unable to replicate in human hosts. CONCLUSIONS: We presented the first experimental evidence of antibodies against TrV structural proteins in human sera.

Triatoma virus structural polyprotein expression, processing and assembly into virus-like particles.

In contrast to the current wealth of structural information concerning dicistrovirus particle structure, very little is known about their morphogenetic pathways. Here, we describe the expression of the two ORFs encoded by the Triatoma virus (TrV) genome. TrV, a member of the Cripavirus genus of the Dicistroviridae family, infects blood-sucking insects belonging to the Triatominae subfamily that act as vectors for the transmission of Trypanosoma cruzi, the aetiological agent of the Chagas disease. We have established a baculovirus-based model for the expression of the NS (non-structural) and P1 (structural) polyproteins. A preliminary characterization of the proteolytic processing of both polyprotein precursors has been performed using this system. We show that the proteolytic processing of the P1 polyprotein is strictly dependent upon the coexpression of the NS polyprotein, and that NS/P1 coexpression leads to the assembly of virus-like particles (VLPs) exhibiting a morphology and a protein composition akin to natural TrV empty capsids. Remarkably, the unprocessed P1 polypeptide assembles into quasi-spherical structures conspicuously larger than VLPs produced in NS/P1-coexpressing cells, likely representing a previously undescribed morphogenetic intermediate. This intermediate has not been found in members of the related Picornaviridae family currently used as a model for dicistrovirus studies, thus suggesting the existence of major differences in the assembly pathways of these two virus groups.

The C-terminal transmembrane domain of human phospholipid scramblase 1 is essential for the protein flip-flop activity and Ca²âº-binding.

Human phospholipid scramblase 1 (SCR) is a 318 amino acid protein that was originally described as catalyzing phospholipid transbilayer (flip-flop) motion in plasma membranes in a Ca²âº-dependent, ATP-independent way. Further studies have suggested an intranuclear role for this protein in addition. A putative transmembrane domain located at the C terminus (aa 291-309) has been related to the flip-flop catalysis. In order to clarify the role of the C-terminal region of SCR, a mutant was produced (SCRΔ) in which the last 28 amino acid residues were lacking, including the α-helix. SCRΔ had lost the scramblase activity and its affinity for Ca²âº was decreased by one order of magnitude. Fluorescence and IR spectroscopic studies revealed that the C-terminal region of SCR was essential for the proper folding of the protein. Moreover, it was found that Ca²âº exerted an overall destabilizing effect on SCR, which might facilitate its binding to membranes.

Structure of the Triatoma virus capsid.

The members of the Dicistroviridae family are non-enveloped positive-sense single-stranded RNA (+ssRNA) viruses pathogenic to beneficial arthropods as well as insect pests of medical importance. Triatoma virus (TrV), a member of this family, infects several species of triatomine insects (popularly named kissing bugs), which are vectors for human trypanosomiasis, more commonly known as Chagas disease. The potential use of dicistroviruses as biological control agents has drawn considerable attention in the past decade, and several viruses of this family have been identified, with their targets covering honey bees, aphids and field crickets, among others. Here, the crystal structure of the TrV capsid at 2.5 Å resolution is reported, showing that as expected it is very similar to that of Cricket paralysis virus (CrPV). Nevertheless, a number of distinguishing structural features support the introduction of a new genus (Triatovirus; type species TrV) under the Dicistroviridae family. The most striking differences are the absence of icosahedrally ordered VP4 within the infectious particle and the presence of prominent projections that surround the fivefold axis. Furthermore, the structure identifies a second putative autoproteolytic DDF motif in protein VP3, in addition to the conserved one in VP1 which is believed to be responsible for VP0 cleavage during capsid maturation. The potential meaning of these new findings is discussed.

Molecular techniques for dicistrovirus detection without RNA extraction or purification.

Dicistroviridae is a new family of small, nonenveloped, and +ssRNA viruses pathogenic to both beneficial arthropods and insect pests as well. Triatoma virus (TrV), a dicistrovirus, is a pathogen of Triatoma infestans (Hemiptera: Reduviidae), one of the main vectors of Chagas disease. In this work, we report a single-step method to identify TrV, a dicistrovirus, isolated from fecal samples of triatomines. The identification method proved to be quite sensitive, even without the extraction and purification of RNA virus.

Inoculation of Triatoma virus (Dicistroviridae: Cripavirus) elicits a non-infective immune response in mice.

BACKGROUND: Dicistroviridae is a new family of small, non-enveloped, +ssRNA viruses pathogenic to both beneficial arthropods and insect pests. Little is known about the dicistrovirus replication mechanism or gene function, and any knowledge on these subjects comes mainly from comparisons with mammalian viruses from the Picornaviridae family. Due to its peculiar genome organization and characteristics of the per os viral transmission route, dicistroviruses make good candidates for use as biopesticides. Triatoma virus (TrV) is a pathogen of Triatoma infestans (Hemiptera: Reduviidae), one of the main vectors of the human trypanosomiasis disease called Chagas disease. TrV was postulated as a potential control agent against Chagas' vectors. Although there is no evidence that TrV nor other dicistroviruses replicate in species outside the Insecta class, the innocuousness of these viruses in humans and animals needs to be ascertained. METHODS: In this study, RT-PCR and ELISA were used to detect the infectivity of this virus in Mus musculus BALB/c mice. RESULTS: In this study we have observed that there is no significant difference in the ratio IgG2a/IgG1 in sera from animals inoculated with TrV when compared with non-inoculated animals or mice inoculated only with non-infective TrV protein capsids. CONCLUSIONS: We conclude that, under our experimental conditions, TrV is unable to replicate in mice. This study constitutes the first test to evaluate the infectivity of a dicistrovirus in a vertebrate animal model.

Pores of the toxin FraC assemble into 2D hexagonal clusters in both crystal structures and model membranes.

The recent high-resolution structure of the toxin FraC derived from the sea anemone Actinia fragacea has provided new insight into the mechanism of pore formation by actinoporins. In this work, we report two new crystal forms of FraC in its oligomeric prepore conformation. Together with the previously reported structure, these two new structures reveal that ring-like nonamers of the toxin assemble into compact two-dimensional hexagonal arrays. This supramolecular organization is maintained in different relative orientations adopted by the oligomers within the crystal layers. Analyses of the aggregation of FraC pores in both planar and curved (vesicles) model membranes show similar 2D hexagonal arrangements. Our observations support a model in which hexagonal pore-packing is a clustering mechanism that maximizes toxin-driven membrane damage in the target cell.