Diego M.A. Guérin

Probing the biophysical interplay between a viral genome and its capsid

The interaction between a viral capsid and its genome governs crucial steps in the life cycle of a virus, such as assembly and genome uncoating. Tuning cargo-capsid interactions is also essential for successful design and cargo delivery in engineered viral systems. Here we investigate the interplay between cargo and capsid for the picorna-like Triatoma virus using a combined native mass spectrometry and atomic force microscopy approach. We propose a topology and assembly model in which heterotrimeric pentons that consist of five copies of structural proteins VP1, VP2 and VP3 are the free principal units of assembly. The interpenton contacts are established primarily by VP2. The dual role of the genome is first to stabilize the densely packed virion and, second, on an increase in pH to trigger uncoating by relaxing the stabilizing interactions with the capsid. Uncoating occurs through a labile intermediate state of the virion that reversibly disassembles into pentons with the concomitant release of protein VP4.

Capsid protein identification and analysis of mature Triatoma virus (TrV) virions and naturally occurring empty particles

Triatoma virus (TrV) is a non-enveloped +ssRNA virus belonging to the insect virus family Dicistroviridae. Mass spectrometry (MS) and gel electrophoresis were used to detect the previously elusive capsid protein VP4. Its cleavage sites were established by sequencing the N-terminus of the protein precursor and MS, and its stoichiometry with respect to the other major capsid proteins (VP1-3) was found to be 1:1. We also characterized the polypeptides comprising the naturally occurring non-infectious empty capsids, i.e., RNA-free TrV particles. The empty particles were composed of VP0-VP3 plus at least seven additional polypeptides, which were identified as products of the capsid precursor polyprotein. We conclude that VP4 protein appears as a product of RNA encapsidation, and that defective processing of capsid proteins precludes genome encapsidation.

Structure of the Triatoma virus capsid

The crystallographic structure of TrV shows specific morphological and functional features that clearly distinguish it from the type species of the Cripavirus genus, CrPV.

AC-ELISA and RT-PCR assays for the diagnosis of triatoma virus (TrV) in triatomines (Hemiptera: Reduviidae) species

Triatoma virus (TrV) is the only entomopathogenic virus found in triatomines. TrV replicates in cells of the midgut epithelium of triatomines, causing a high mortality rate and delayed development of the infected insect. In this work, we report an antigen-capture enzyme-linked immunosorbent assay (AC-ELISA) and a reverse transcription-polymerase chain reaction (RT-PCR) assay for detection of TrV infection. For antiserum production, rabbits and hens where inoculated with purified TrV. Antiserum reactivity was checked by immunodiffusion, and its specificity was confirmed by western blot and AC-ELISA. Totally 90 fecal samples from T. infestans were analysed. AC-ELISA and RT-PCR results correlated well with transmission electron microscopy (EM) observations, which are considered the gold standard, with Kappa values of 0.73 for AC-ELISA and 0.93 for RT-PCR when compared with EM. Applications and complementary uses of the two techniques reported in this work are discussed.

Interdomain Ca2+ effects in Escherichia coli α-haemolysin: Ca2+ binding to the C-terminal domain stabilizes both C- and N-terminal domains

alpha-Haemolysin (HlyA) is a toxin secreted by pathogenic Escherichia coli, whose lytic activity requires submillimolar Ca(2+) concentrations. Previous studies have shown that Ca(2+) binds within the Asp and Gly rich C-terminal nonapeptide repeat domain (NRD) in HlyA. The presence of the NRD puts HlyA in the RTX (Repeats in Toxin) family of proteins. We tested the stability of the whole protein, the amphipathic helix domain and the NRD, in both the presence and absence of Ca(2+) using native HlyA, a truncated form of HlyADeltaN601 representing the C-terminal domain, and a novel mutant HlyA W914A whose intrinsic fluorescence indicates changes in the N-terminal domain. Fluorescence and infrared spectroscopy, tryptic digestion, and urea denaturation techniques concur in showing that calcium binding to the repeat domain of alpha-haemolysin stabilizes and compacts both the NRD and the N-terminal domains of HlyA. The stabilization of the N-terminus through Ca(2+) binding to the C-terminus reveals long-range inter-domain structural effects. Considering that RTX proteins consist, in general, of a Ca(2+)-binding NRD and separate function-specific domains, the long-range stabilizing effects of Ca(2+) in HlyA may well be common to other members of this family.

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

BackgroundDicistroviridae 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.MethodsIn this study, RT-PCR and ELISA were used to detect the infectivity of this virus in Mus musculus BALB/c mice.ResultsIn 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.ConclusionsWe 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.

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

Triatoma virus (TrV) is a member of the insect virus family Dicistroviridae and consists of a small, non-enveloped capsid that encloses its positive-sense ssRNA genome. Using cryo-transmission electron microscopy and three-dimensional reconstruction techniques combined with fitting of the available crystallographic models, this study analysed the capsids corresponding to mature and several RNA-empty TrV particles. After genome release, the resulting reconstruction of the empty capsids displayed no prominent conformational changes with respect to the full virion capsid. The results showed that RNA delivery led to empty capsids with an apparent overall intact protein shell and suggested that, in a subsequent step, empty capsids disassemble into small symmetrical particles. Contrary to what is observed upon genome release in mammalian picornaviruses, the empty TrV capsid maintained a protein shell thickness and size identical to that in full virions.

Phasing of the Triatoma virus diffraction data using a cryo-electron microscopy reconstruction

The blood-sucking reduviid bug Triatoma infestans, one of the most important vector of American human trypanosomiasis (Chagas disease) is infected by the Triatoma virus (TrV). TrV has been classified as a member of the Cripavirus genus (type cricket paralysis virus) in the Dicistroviridae family. This work presents the three-dimensional cryo-electron microscopy (cryo-EM) reconstruction of the TrV capsid at about 25 A resolution and its use as a template for phasing the available crystallographic data by the molecular replacement method. The main structural differences between the cryo-EM reconstruction of TrV and other two viruses, one from the same family, the cricket paralysis virus (CrPV) and the human rhinovirus 16 from the Picornaviridae family are presented and discussed.

The C-terminal transmembrane domain of human phospholipid scramblase 1 is essential for the protein flip-flop activity and Ca2+-binding

Abstract 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 Ca2+-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 Ca2+ 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 Ca2+ exerted an overall destabilizing effect on SCR, which might facilitate its binding to membranes.

Exploration for Triatoma virus (TrV) infection in laboratory-reared triatomines of Latin America: a collaborative study*

Triatoma virus (TrV) is a small, non-enveloped virus that has a +ssRNA genome and is currently classified under the Cripavirus genus of the Dicistroviridae family. TrV infects haematophagous triatomine insects (Hemiptera: Reduviidae), which are vectors of American trypanosomosis (Chagas disease). TrV can be transmitted through the horizontal faecal-oral route, and causes either deleterious sublethal effects or even the death of laboratory insect colonies. Various species of triatomines from different regions of Latin America are currently being reared in research laboratories, with little or no awareness of the presence of TrV; therefore, any biological conclusion drawn from experiments on insects infected with this virus is inherently affected by the side effects of its infection. In this study, we developed a mathematical model to estimate the sample size required for detecting a TrV infection. We applied this model to screen the infection in the faeces of triatomines belonging to insectaries from 13 Latin American countries, carrying out the identification of TrV by using RT-PCR. TrV was detected in samples coming from Argentina, which is where the virus was first isolated from Triatoma infestans (Hemiptera: Reduviidae) several years ago. Interestingly, several colonies from Brazil were also found infected with the virus. This positive result widens the TrV’s host range to a total of 14 triatomine species. Our findings suggest that many triatomine species distributed over a large region of South America may be naturally infected with TrV.