Cryo-electron microscopy reconstructions of triatoma virus particles: a clue to unravel genome delivery and capsid disassembly

The pollination services provided by the western honeybee (Apis mellifera) are critical for agricultural production and the diversity of wild flowering plants. However, honeybees suffer from environmental pollution, habitat loss, and pathogens, including viruses that can cause fatal diseases. Israeli acute bee paralysis virus (IAPV), from the family Dicistroviridae, has been shown to cause colony collapse disorder in the United States. Here, we present the IAPV virion structure determined to a resolution of 4.0 Å and the structure of a pentamer of capsid protein protomers at a resolution of 2.7 Å. IAPV has major capsid proteins VP1 and VP3 with noncanonical jellyroll β-barrel folds composed of only seven instead of eight β-strands, as is the rule for proteins of other viruses with the same fold. The maturation of dicistroviruses is connected to the cleavage of precursor capsid protein VP0 into subunits VP3 and VP4. We show that a putative catalytic site formed by the residues Asp-Asp-Phe of VP1 is optimally positioned to perform the cleavage. Furthermore, unlike many picornaviruses, IAPV does not contain a hydrophobic pocket in capsid protein VP1 that could be targeted by capsid-binding antiviral compounds.IMPORTANCE Honeybee pollination is required for agricultural production and to sustain the biodiversity of wild flora. However, honeybee populations in Europe and North America are under pressure from pathogens, including viruses that cause colony losses. Viruses from the family Dicistroviridae can cause honeybee infections that are lethal, not only to individual honeybees, but to whole colonies. Here, we present the virion structure of an Aparavirus, Israeli acute bee paralysis virus (IAPV), a member of a complex of closely related viruses that are distributed worldwide. IAPV exhibits unique structural features not observed in other picorna-like viruses. Capsid protein VP1 of IAPV does not contain a hydrophobic pocket, implying that capsid-binding antiviral compounds that can prevent the replication of vertebrate picornaviruses may be ineffective against honeybee virus infections.

Section: Resultssupporting

confidence: 66%

“…The genome release of many picornaviruses results in the formation of empty capsids, the so-called B particles (54). However, the empty capsids of some picornaviruses and dicistroviruses disassemble into pentamers of capsid protein protomers (55, 56). Furthermore, pentamers of capsid protomers were also shown to be capsid precursors (57, 58).…”

Section: Resultsmentioning

confidence: 99%

Virion Structure of Israeli Acute Bee Paralysis Virus

Mullapudi

Přidal

Pálková

et al. 2016

“…These picornavirus entry steps can be mimicked in the laboratory setting by heating infectious particles to produce A-particle and 80S. Heating TrV virus particles has also been shown to release the genome, resulting in a stable empty capsid (24).…”

mentioning

confidence: 99%

Honey Bee Deformed Wing Virus Structures Reveal that Conformational Changes Accompany Genome Release

Organtini

Shingler

Ashley

et al. 2017

The picornavirus-like deformed wing virus (DWV) has been directly linked to colony collapse; however, little is known about the mechanisms of host attachment or entry for DWV or its molecular and structural details. Here we report the three-dimensional (3-D) structures of DWV capsids isolated from infected honey bees, including the immature procapsid, the genome-filled virion, the putative entry intermediate (A-particle), and the empty capsid that remains after genome release. The capsids are decorated by large spikes around the 5-fold vertices. The 5-fold spikes had an open flower-like conformation for the procapsid and genome-filled capsids, whereas the putative A-particle and empty capsids that had released the genome had a closed tube-like spike conformation. Between the two conformations, the spikes undergo a significant hinge-like movement that we predicted using a Robetta model of the structure comprising the spike. We conclude that the spike structures likely serve a function during host entry, changing conformation to release the genome, and that the genome may escape from a 5-fold vertex to initiate infection. Finally, the structures illustrate that, similarly to picornaviruses, DWV forms alternate particle conformations implicated in assembly, host attachment, and RNA release. IMPORTANCE Honey bees are critical for global agriculture, but dramatic losses of entire hives have been reported in numerous countries since 2006. Deformed wing virus (DWV) and infestation with the ectoparasitic mite Varroa destructor have been linked to colony collapse disorder. DWV was purified from infected adult worker bees to pursue biochemical and structural studies that allowed the first glimpse into the conformational changes that may be required during transmission and genome release for DWV.

“…The TrV capsid structure was solved at high resolution (10), and it was shown to be composed of 60 copies of the three major structural proteins (VP1, VP2, and VP3), all of which are folded in a jelly roll-like core. Surprisingly the small protein VP4 (5.5 kDa) was not observed either in the X-ray crystallographic structure or in cryo-electron microscopy (cryo-EM) three-dimensional reconstructions (10,11), even though it is known to be present in infectious TrV particles (8). These data showed that the TrV VP4 protein is disordered within the capsid, contrary to picornavirus VP4, which is largely ordered inside the particle in a manner that matches the icosahedral capsid symmetry (12,13).…”

mentioning

confidence: 99%

“…Moreover, empty capsids (after genome delivery) do not display any conformational changes similar to those in picornaviruses (11). Therefore, a different mechanism of uncoating was proposed by characterizing the capsid disassembly and genome release using atomic force microscopy (AFM) and native mass spectrometry techniques (22).…”

mentioning

confidence: 99%

Triatoma Virus Recombinant VP4 Protein Induces Membrane Permeability through Dynamic Pores

Sánchez-Eugenia

Goikolea

Gil

et al. 2015

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 orderPicornaviridae 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.T he positive single-stranded RNA (ssRNA) virus triatoma virus (TrV) belongs to the Dicistroviridae family in the Picornavirales order. It is a lethal pathogen of the bloodsucking insect Triatoma infestans and of other insect species belonging to the Triatominae subfamily (1, 2). These insects are the main vectors for the transmission of the protozoon Trypanosoma cruzi, the causative agent of Chagas disease (American trypanosomiasis), a neglected tropical disease endemic in Latin America (3). Thus, TrV has been proposed to be a biological control agent that could be used to prevent the spread of this pathogen (4-6).Dicistroviruses were initially referred to as picorna-like viruses due to their similarities with members of the Picornaviridae family: the nonenveloped icosahedral capsid structure, the positive ssRNA genome, and the capsid protein composition. However, dic...