RESUMEN

Plant viruses such as brome mosaic virus and cowpea chlorotic mottle virus are effectively purified through PEG precipitation and sucrose cushion ultracentrifugation. Increasing ionic strength and an alkaline pH cause the viruses to swell and disassemble into coat protein subunits. The coat proteins can be reassembled into stable virus-like particles (VLPs) that carry anionic molecules at low ionic strength and through two-step dialysis from neutral pH to acidic buffer. VLPs have been extensively studied due to their ability to protect and deliver cargo, particularly RNA, while avoiding degradation under physiological conditions. Furthermore, chemical functionalization of the surface of VLPs allows for the targeted drug delivery. VLPs derived from plants have demonstrated great potential in nanomedicine by offering a versatile platform for drug delivery, imaging, and therapeutic applications.

Asunto(s)

Virus de Plantas , Virus de Plantas/genética , Proteínas de la Cápside/química , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Virión/química , Virión/genética , Bromovirus/química , Bromovirus/genética , ARN/química , Concentración de Iones de Hidrógeno , ARN Viral/genética

RESUMEN

There is an urgent need for ultrarapid testing regimens to detect the severe acute respiratory syndrome coronavirus 2 [SARS-CoV-2] infections in real-time within seconds to stop its spread. Current testing approaches for this RNA virus focus primarily on diagnosis by RT-qPCR, which is time-consuming, costly, often inaccurate, and impractical for general population rollout due to the need for laboratory processing. The latency until the test result arrives with the patient has led to further virus spread. Furthermore, latest antigen rapid tests still require 15-30 min processing time and are challenging to handle. Despite increased polymerase chain reaction (PCR)-test and antigen-test efforts, the pandemic continues to evolve worldwide. Herein, we developed a superfast, reagent-free, and nondestructive approach of attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy with subsequent chemometric analysis toward the prescreening of virus-infected samples. Contrived saliva samples spiked with inactivated γ-irradiated COVID-19 virus particles at levels down to 1582 copies/mL generated infrared (IR) spectra with a good signal-to-noise ratio. Predominant virus spectral peaks are tentatively associated with nucleic acid bands, including RNA. At low copy numbers, the presence of a virus particle was found to be capable of modifying the IR spectral signature of saliva, again with discriminating wavenumbers primarily associated with RNA. Discrimination was also achievable following ATR-FTIR spectral analysis of swabs immersed in saliva variously spiked with virus. Next, we nested our test system in a clinical setting wherein participants were recruited to provide demographic details, symptoms, parallel RT-qPCR testing, and the acquisition of pharyngeal swabs for ATR-FTIR spectral analysis. Initial categorization of swab samples into negative versus positive COVID-19 infection was based on symptoms and PCR results (n = 111 negatives and 70 positives). Following training and validation (using n = 61 negatives and 20 positives) of a genetic algorithm-linear discriminant analysis (GA-LDA) algorithm, a blind sensitivity of 95% and specificity of 89% was achieved. This prompt approach generates results within 2 min and is applicable in areas with increased people traffic that require sudden test results such as airports, events, or gate controls.

Asunto(s)

Algoritmos , COVID-19/diagnóstico , SARS-CoV-2/fisiología , Espectroscopía Infrarroja por Transformada de Fourier/métodos , Virión/química , COVID-19/virología , Análisis Discriminante , Rayos gamma , Humanos , Pruebas en el Punto de Atención , Análisis de Componente Principal , SARS-CoV-2/aislamiento & purificación , Saliva/virología , Sensibilidad y Especificidad , Relación Señal-Ruido , Virión/efectos de la radiación , Inactivación de Virus

RESUMEN

BACKGROUND: After the isolation of Acanthamoeba polyphaga mimivirus (APMV), the study and search for new giant viruses has been intensified. Most giant viruses are associated with free-living amoebae of the genus Acanthamoeba; however other giant viruses have been isolated in Vermamoeba vermiformis, such as Faustovirus, Kaumoebavirus and Orpheovirus. These studies have considerably expanded our knowledge about the diversity, structure, genomics, and evolution of giant viruses. Until now, there has been only one Orpheovirus isolate, and many aspects of its life cycle remain to be elucidated. METHODS: In this study, we performed an in-depth characterization of the replication cycle and particles of Orpheovirus by transmission and scanning electron microscopy, optical microscopy and IF assays. RESULTS: We observed, through optical and IF microscopy, morphological changes in V. vermiformis cells during Orpheovirus infection, as well as increased motility at 12 h post infection (h.p.i.). The viral factory formation and viral particle morphogenesis were analysed by transmission electron microscopy, revealing mitochondria and membrane recruitment into and around the electron-lucent viral factories. Membrane traffic inhibitor (Brefeldin A) negatively impacted particle morphogenesis. The first structure observed during particle morphogenesis was crescent-shaped bodies, which extend and are filled by the internal content until the formation of multi-layered mature particles. We also observed the formation of defective particles with different shapes and sizes. Virological assays revealed that viruses are released from the host by exocytosis at 12 h.p.i., which is associated with an increase of particle counts in the supernatant. CONCLUSIONS: The results presented here contribute to a better understanding of the biology, structures and important steps in the replication cycle of Orpheovirus.

Asunto(s)

Virus ADN/crecimiento & desarrollo , Virus Gigantes/crecimiento & desarrollo , Replicación Viral , Antígenos Virales/análisis , Virus ADN/ultraestructura , Virus Gigantes/ultraestructura , Lobosea/virología , Microscopía , Microscopía Electrónica de Rastreo , Microscopía Electrónica de Transmisión , Microscopía Fluorescente , Virión/química , Virión/ultraestructura

RESUMEN

In the last few decades, the isolation of amoebae-infecting giant viruses has challenged established principles related to the definition of virus, their evolution, and their particle structures represented by a variety of shapes and sizes. Tupanviruses are one of the most recently described amoebae-infecting viruses and exhibit a peculiar morphology with a cylindrical tail attached to the capsid. Proteomic analysis of purified viral particles revealed that virions are composed of over one hundred proteins with different functions. The putative origin of these proteins had not yet been investigated. Here, we provide evidences for multiple origins of the proteins present in tupanvirus particles, wherein 20% originate from members of the archaea, bacteria and eukarya.

Asunto(s)

Virus Gigantes/química , Virus Gigantes/genética , Proteoma , Proteínas Virales/genética , Virión/química , Amoeba/virología , Archaea/genética , Bacterias/genética , Eucariontes/genética , Proteómica , Proteínas Virales/química , Virión/genética

RESUMEN

The geminiviruses are a family of small, non-enveloped viruses with single-stranded, circular DNA genomes of 2500-5200 bases. Geminiviruses are transmitted by various types of insect (whiteflies, leafhoppers, treehoppers and aphids). Members of the genus Begomovirus are transmitted by whiteflies, those in the genera Becurtovirus, Curtovirus, Grablovirus, Mastrevirus and Turncurtovirus are transmitted by specific leafhoppers, the single member of the genus Topocuvirus is transmitted by a treehopper and one member of the genus Capulavirus is transmitted by an aphid. Geminiviruses are plant pathogens causing economically important diseases in most tropical and subtropical regions of the world. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the Geminiviridae which is available at www.ictv.global/report/geminiviridae.

Asunto(s)

Geminiviridae/clasificación , Enfermedades de las Plantas/virología , Animales , Microscopía por Crioelectrón , ADN de Cadena Simple/química , ADN de Cadena Simple/genética , ADN Viral/química , ADN Viral/genética , Geminiviridae/genética , Geminiviridae/fisiología , Geminiviridae/ultraestructura , Orden Génico , Genoma Viral , Insectos/virología , Virión/química , Virión/genética , Virión/ultraestructura , Replicación Viral , Zea mays/virología

RESUMEN

Virus-like particles (VLPs) have demonstrated to be valuable scaffolds for the display of heterologous peptides for vaccine development and other specific interactions. VLPs of primate erythroparvovirus 1, generally referred as parvovirus B19 (B19V), have already been produced in-vivo and in-vitro from the recombinant VP2 protein of this virus. In this study, chimeric forms of B19V VP2 were constructed, and their ability to assemble into VLPs was evaluated. Chimeras were composed of the VP2 protein fused, at its N-terminus, with two peptides derived from the fusion glycoprotein (F) of the respiratory syncytial virus (RSV). The chimeric proteins self-assembled into VLPs morphologically similar to B19V virions. Stability of these VLPs was analyzed under denaturation conditions with guanidinium chloride (GdnHCl). Our results indicate that the presence of the heterologous fragments increased the stability of VLPs assembled by any of the VP2 chimeras. Specific proteolysis assays shown that a fraction of the N-termini of the chimeric proteins is located on the outer surface of the VLPs. Immunogenicity of VLPs against RSV was evaluated and the results indicate that the particles can elicit a humoral immune response, although these antibodies did not cross-react with RSV in ELISA tests. These results provide novel insights into the localization of the N-termini of B19V VP2 protein after in vitro assembly into VLPs, and point them to be attractive sites to display peptides or proteins without compromise the assembly or stability of VLPs.

Asunto(s)

Parvovirus B19 Humano/química , Parvovirus B19 Humano/inmunología , Biblioteca de Péptidos , Vacunas de Partículas Similares a Virus/química , Vacunas de Partículas Similares a Virus/inmunología , Vacunas Virales/química , Vacunas Virales/inmunología , Animales , Anticuerpos Antivirales/análisis , Proteínas de la Cápside/química , Proteínas de la Cápside/genética , Proteínas de la Cápside/inmunología , Guanidina/química , Inmunidad Humoral , Inmunogenicidad Vacunal/inmunología , Ratones , Ratones Endogámicos BALB C , Desnaturalización Proteica , Estabilidad Proteica , Proteolisis , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Virus Sincitial Respiratorio Humano/química , Virus Sincitial Respiratorio Humano/inmunología , Virión/química , Virión/genética , Virión/inmunología

RESUMEN

Virus-like particles (VLPs) are the product of the self-assembly, either in vivo or in vitro, of structural components of viral capsids. These particles are excellent scaffolds for surface display of biomolecules that can be used in vaccine development and tissue-specific drug delivery. Surface engineering of VLPs requires structural stability and chemical reactivity. Herein, we report the enhanced assembly, colloidal stabilization and fluorescent labeling of primate erythroparvovirus 1 (PE1V), generally referred to as parvovirus B19. In vitro assembly of the VP2 protein of PE1V produces VLPs, which are prone to flocculate and hence undergo limited chemical modification by thiol-specific reagents like the fluorogenic monobromobimane (mBBr). We determined that the addition of 0.2M l-arginine during the assembly process produced an increased yield of soluble VLPs with good dispersion stability. Fluorescent labeling of VLPs suspended in phosphate buffered saline (PBS) added with 0.2M l-Arg was achieved in significantly shorter times than the flocculated VLPs assembled in only PBS buffer. Finally, to demonstrate the potential application of this approach, mBBr-labeled VLPs were successfully used to tag human hepatoma HepG2 cells. This new method for assembly and labeling PE1V VLPs eases its applications and provides insights on the manipulation of this biomaterial for further developments. STATEMENT OF SIGNIFICANCE: Application of virus-derived biomaterials sometimes requires surface modification for diverse purposes, including enhanced cell-specific interaction, the inclusion of luminescent probes for bioimaging, or the incorporation of catalytic properties for the production of enzyme nanocarriers. In this research, we reported for the first time the colloidal stabilization of the primate erythroparvovirus 1 (PE1V) virus-like particles (VLPs). Also, we report the chemical modification of the natural Cys residues located on the surface of these VLPs with a fluorescent probe, as well as its application for tagging hepatoma cells in vitro. Keeping in mind that PE1V is a human pathogen, virus-host interactions already exist in human cells, and they can be exploited for therapeutic and research aims. This study will impact on the speed in which the scientific community will be able to manipulate PE1V VLPs for diverse purposes. Additionally, this study may provide insights on the colloidal properties of these VLPs as well as in the effect of different protein additives used for protein stabilization.

Asunto(s)

Coloides/química , Parvoviridae/química , Ingeniería de Proteínas/métodos , Virión/química , Animales , Arginina/farmacología , Compuestos Bicíclicos con Puentes/metabolismo , Centrifugación , Cristalografía por Rayos X , Endocitosis , Filtración , Fluorescencia , Glicerol/farmacología , Células Hep G2 , Humanos , Concentración de Iones de Hidrógeno , Modelos Moleculares , Primates , Compuestos de Sulfhidrilo/metabolismo , Propiedades de Superficie , Proteínas Virales/química , Proteínas Virales/metabolismo , Virión/efectos de los fármacos , Virión/ultraestructura

RESUMEN

Virus-like particles (VLPs) have vast potential for applications in nanoscience and nanomedicine. These biological nanoparticles may be used for medical imaging, vaccination, or tissue-specific delivery of drugs or other bioactive molecules. VLPs of Human parvovirus B19 (B19 V) can be assembled in vitro from the recombinant VP2 protein. In this research, we describe a simple method for the encapsulation of heterologous linear dsDNA fragments of different sizes into B19 V-VP2 VLPs, in which the DNA and denatured VP2 protein are co-incubated and the assembly process is conducted by one dialysis step. Characterization of the particles by qPCR demonstrated the encapsulation of dsDNA, and indicates that the length of the dsDNA is critical for the encapsulation process. The strategy presented here opens the possibility to use this VLPs as a delivery system with future therapeutically applications.

Asunto(s)

ADN/química , Nanopartículas/química , Parvovirus B19 Humano/química , Virión/química , ADN/genética , ADN/metabolismo , Modelos Moleculares , Nanopartículas/metabolismo , Nanopartículas/ultraestructura , Tamaño de la Partícula , Parvovirus B19 Humano/metabolismo , Electricidad Estática , Virión/aislamiento & purificación , Virión/metabolismo , Virión/ultraestructura

RESUMEN

The feline immunodeficiency virus (FIV) envelope glycoprotein (Env) possesses a short cytoplasmic domain of 53 amino acids containing four highly conserved cysteines at Env positions 804, 811, 815 and 848. Since palmitoylation of transmembrane proteins occurs at or near the membrane anchor, we investigated whether cysteines 804, 811 and 815 are acylated and analyzed the relevance of these residues for Env functions. Replacement of cysteines 804, 811 and 815 individually or in combination by serine residues resulted in Env glycoproteins that were efficiently expressed and processed. However, mutations C804S and C811S reduced Env fusogenicity by 93% and 84%, respectively, compared with wild-type Env. By contrast, mutant C815S exhibited a fusogenic capacity representing 50% of the wild-type value. Remarkably, the double mutation C804S/C811S abrogated both Env fusion activity and Env incorporation into virions. Finally, by means of Click chemistry assays we demonstrated that the four FIV Env cytoplasmic cysteines are palmitoylated.

Asunto(s)

Síndrome de Inmunodeficiencia Adquirida del Felino/virología , Virus de la Inmunodeficiencia Felina/metabolismo , Proteínas del Envoltorio Viral/metabolismo , Virión/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Gatos , Fusión Celular , Línea Celular , Humanos , Virus de la Inmunodeficiencia Felina/química , Virus de la Inmunodeficiencia Felina/genética , Lipoilación , Datos de Secuencia Molecular , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/genética , Virión/química , Virión/genética

RESUMEN

Virus-like particles (VLPs) are biological nanoparticles identical to the natural virions, but without genetic material. VLPs are suitable for the analysis of viral infection mechanisms, vaccine production, tissue-specific drug delivery, and as biological nanomaterials. Human parvovirus B19 (B19) infects humans; therefore VLPs derived from this virus have enormous potential in medicine and diagnostics. Current production of self-assembled VLPs derived from B19 is typically carried out in eukaryotic expression systems. However many applications of VLPs require access to its internal core. Consequently, the processes of disassembly and further reassembly of VLPs are critical both for purification of viral proteins, and for encapsulation purposes. Herein we report the in vitro self-assembly of B19 VLPs derived from the recombinant VP2 protein expressed in Escherichia coli and the effects of pH and ionic strength on the assembly process. Our results demonstrate that VP2 is able to form VLPs completely in vitro. At neutral pH, homogeneous VLPs assemble, while at acidic and basic pHs, with low ionic strength, the major assemblies are small intermediates. The in vitro self-assembled VLPs are highly stable at 37°C, and a significant fraction of particles remain assembled after 30min at 80°C.

Asunto(s)

Nanopartículas/química , Parvovirus B19 Humano/química , Virión/química , Escherichia coli/genética , Escherichia coli/metabolismo , Concentración de Iones de Hidrógeno , Nanotecnología , Concentración Osmolar

RESUMEN

The interaction between dengue virus particles (DENV), sedimentation hemagglutinin particles (SHA), dengue virus envelope protein (Eprot), and solid surfaces was investigated by means of ellipsometry and atomic force microscopy (AFM). The surfaces chosen are bare Si/SiO2 wafers and Si/SiO2 wafers covered with concanavalin A (ConA), jacalin (Jac), polystyrene (PS), or poly(styrene sulfonate) (PSS) films. Adsorption experiments at pH 7.2 and pH 3 onto all surfaces revealed that (i) adsorption of DENV particles took place only onto ConA under pH 7.2, because of specific recognition between glycans on DENV surface and ConA binding site; (ii) DENV particles did not attach to any of the surfaces at pH 3, suggesting the presence of positive charges on DENV surface at this pH, which repel the positively charged lectin surfaces; (iii) SHA particles are positively charged at pH 7.2 and pH 3 because they adhered to negatively charged surfaces at pH 7.2 and repelled positively charged layers at pH 3; and (iv) SHA particles carry polar groups on the surface because they attached to silanol surfaces at pH 3 and avoided hydrophobic PS films at pH 3 and pH 7.2. The adsorption behavior of Eprot at pH 7.2 revealed affinity for ConA>Jac>PSS>PS≈bare Si/SiO2 layers. These findings indicate that selectivity of the Eprot adsorption is higher when it is part of virus structure than when it is free in solution. The correlation between surface energy values determined by means of contact angle measurements and DENV, SHA, or Eprot adsorption behavior was used to understand the intermolecular forces at the interfaces. A direct correlation was not found because the contributions from surface energy were probably surpassed by specific contributions.

Asunto(s)

Materiales Biocompatibles/química , Virus del Dengue/química , Proteínas Virales/química , Virión/química , Adsorción , Ensayo de Materiales , Unión Proteica , Propiedades de Superficie

RESUMEN

Formation of stable thin films of mixed xyloglucan (XG) and alginate (ALG) onto Si/SiO(2) wafers was achieved under pH 11.6, 50mM CaCl(2), and at 70 degrees C. XG-ALG films presented mean thickness of (16+/-2)nm and globules rich surface, as evidenced by means of ellipsometry and atomic force microscopy (AFM), respectively. The adsorption of two glucose/mannose-binding seed (Canavalia ensiformis and Dioclea altissima) lectins, coded here as ConA and DAlt, onto XG-ALG surfaces took place under pH 5. Under this condition both lectins present positive net charge. ConA and DAlt adsorbed irreversibly onto XG-ALG forming homogenous monolayers approximately (4+/-1)nm thick. Lectins adsorption was mainly driven by electrostatic interaction between lectins positively charged residues and carboxylated (negatively charged) ALG groups. Adhesion of four serotypes of dengue virus, DENV (1-4), particles to XG-ALG surfaces were observed by ellipsometry and AFM. The attachment of dengue particles onto XG-ALG films might be mediated by (i) H bonding between E protein (located at virus particle surface) polar residues and hydroxyl groups present on XG-ALG surfaces and (ii) electrostatic interaction between E protein positively charged residues and ALG carboxylic groups. DENV-4 serotype presented the weakest adsorption onto XG-ALG surfaces, indicating that E protein on DENV-4 surface presents net charge (amino acid sequence) different from E proteins of other serotypes. All four DENV particles serotypes adsorbed similarly onto lectin films adsorbed. Nevertheless, the addition of 0.005mol/L of mannose prevented dengue particles from adsorbing onto lectin films. XG-ALG and lectin layers serve as potential materials for the development of diagnostic methods for dengue.

Asunto(s)

Alginatos/química , Virus del Dengue/metabolismo , Glucanos/química , Lectinas/química , Virión/metabolismo , Xilanos/química , Alginatos/metabolismo , Concanavalina A/química , Concanavalina A/metabolismo , Dengue/metabolismo , Virus del Dengue/química , Virus del Dengue/ultraestructura , Glucanos/metabolismo , Ácido Glucurónico/química , Ácido Glucurónico/metabolismo , Ácidos Hexurónicos/química , Ácidos Hexurónicos/metabolismo , Concentración de Iones de Hidrógeno , Lectinas/metabolismo , Microscopía de Fuerza Atómica , Dióxido de Silicio/química , Dióxido de Silicio/metabolismo , Espectroscopía Infrarroja por Transformada de Fourier , Virión/química , Xilanos/metabolismo

RESUMEN

In this article we studied, by nuclear magnetic resonance relaxation measurements, the disassembly of a virus particle-the MS2 bacteriophage. MS2 is one of the single-stranded RNA bacteriophages that infect Escherichia coli. At pH 4.5, the phage turns to a metastable state, as is indicated by an increase in the observed nuclear magnetic resonance signal intensity upon decreasing the pH from 7.0 to 4.5. Steady-state fluorescence and circular dichroism spectra at pH 4.5 show that the difference in conformation and secondary structure is not pronounced if compared with the phage at pH 7.0. At pH 4.5, two-dimensional (15)N-(1)H heteronuclear multiple quantum coherence (HMQC) spectrum shows approximately 40 crosspeaks, corresponding to the most mobile residues of MS2 coat protein at pH 4.5. The (15)N linewidth is approximately 30 Hz, which is consistent with an intermediate with a rotational relaxation time of 100 ns. The average spin lattice relaxation time (T(1)) of the mobile residues was measured at different temperatures, clearly distinguishing between the dimer and the equilibrium intermediate. The results show, for the first time, the presence of intermediates in the process of dissociation of the MS2 bacteriophage.

Asunto(s)

Cristalografía/métodos , Escherichia coli/virología , Levivirus/química , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular/métodos , Desnaturalización Proteica , Proteínas Virales/química , Virión/química , Simulación por Computador , Dimerización , Sustancias Macromoleculares , Movimiento (Física) , Unión Proteica , Conformación Proteica , Pliegue de Proteína , Ensamble de Virus

RESUMEN

Research about the kinetoplast of trypanosomatids has yielded valuable information about the organization of extranuclear structure. However, the ultrastructural localization of nucleic acids within these protozoa remains uncertain. We have applied cytochemical and immunocytochemical approaches to precisely identify DNA and RNA in lower endosymbiont-bearing trypanosomatids. Using the Terminal deoxynucleotidyl Transferase (TdT) immunogold technique, we showed that nuclear DNA is seen associated with the nuclear envelope during the trypanosomatid cell cycle. By combining the TdT technique with the acetylation method, which improves the contrast between structures containing fibrils and granules, we have demonstrated that the nucleolus of endosymbiont-bearing trypanosomatids is composed of two constituents: a granular component and a DNA-positive fibrillar zone. Moreover, we revealed that DNA of endosymbiotic bacteria consisted of electron-dense filaments which are usually in close contact with the prokaryote envelope. Using a Lowicryl post-embedding immunogold labeling procedure with anti-RNA antibodies, we showed the presence of RNA not only over the cytoplasm, the interchromatin spaces and the nucleolus, but also over the kinetoplast and virus-like particles present in Crithidia desouzai.

Asunto(s)

Crithidia/citología , ADN Protozoario/análisis , ARN Protozoario/análisis , Trypanosomatina/citología , Animales , Bacterias/química , Ciclo Celular , Crithidia/química , Crithidia/microbiología , Crithidia/virología , ADN Nucleotidilexotransferasa , ADN Bacteriano/análisis , ADN de Cinetoplasto/análisis , Inmunohistoquímica , Simbiosis , Trypanosomatina/química , Trypanosomatina/microbiología , Virión/química

RESUMEN

In the present work, we demonstrate virus-like particles (VLPs) with various morphological variations in Trichomonas vaginalis. The VLPs were distinct based on size, shape and electron density, with VLPs being either electron-dense or electron-lucent. We used electron microscopy thin sections of several T. vaginalis strains virus-infected, and also negative staining of fractions obtained after purification by CsCl buoyant density gradient centrifugation. The particles observed in fractions are identical to those previously described, but by thin sections, we found new forms. The shapes found were icosahedral, spherical and oblong, and the sizes varied from 33 to 120nm in diameter with the most common VLP being spherical and having a size range from 83 to 104nm. The VLPs were found in the cytoplasm closely associated with the Golgi complex, with some VLPs budding from the Golgi, and other VLPs were detected adjacent to the plasma membrane. Unidentified cytoplasmic inclusions were observed in the region close to the VLPs and Golgi. Clusters of the already described icosahedral virus were also observed in the cytoplasm, although less frequently. These results indicate that T. vaginalis organisms may be infected with different dsRNA viruses simultaneously.

Asunto(s)

Trichomonas vaginalis/virología , Virión/ultraestructura , Animales , Proteínas de la Cápside/análisis , Microscopía Electrónica , Microscopía Inmunoelectrónica , ARN Bicatenario/análisis , Trichomonas vaginalis/crecimiento & desarrollo , Trichomonas vaginalis/ultraestructura , Virión/química , Virión/aislamiento & purificación

RESUMEN

Little is known about the mechanism of hepatitis C virion assembly. So the capacity of the entire Hepatitis C virus core protein (HCcAg) produced in Pichia pastoris to form particles either in its native soluble state or after detergent treatment of HCcAg associated to cell debris were studied. Size exclusion chromatography suggested that HCcAg assembled into high molecular weight structures. HCcAg was also specifically recognized by a serum from a chronic HCV carrier patient. This antigen migrated with buoyant density values similar to those obtained for native nucleocapsid particles from infected patients when analyzed using sucrose density gradient centrifugation. The analysis by electron microscopy of purified HCcAg showed aggregates resembling virus-like particles (VLPs) with an average diameter of 30 nm. These results indicated that the HCcAg obtained from P. pastoris assembled into VLPs resembling HCV nucleocapsid particles in a mature stage. Such HCcAg aggregates characterized here could be a valuable tool to elucidate the mechanisms of HCV nucleocapsid assembly.

Asunto(s)

Hepacivirus/química , Pichia/virología , Proteínas del Núcleo Viral/química , Virión/química , Immunoblotting , Peso Molecular , Renaturación de Proteína , Proteínas del Núcleo Viral/metabolismo

RESUMEN

We have re-evaluated the relationships among the polypeptides of eastern equine encephalitis (EEE) viruses using SDS-PAGE and peptide mapping of individual virion proteins. Four to five distinct polypeptide bands were detected upon SDS-PAGE analysis of viruses: the E1, E2 and C proteins normally associated with alphavirus virions, as well as an additional more rapidly-migrating E2-associated protein and a high M(r) (HMW) protein. In contrast with previous findings by others, the electrophoretic profiles of the virion proteins of EEE viruses displayed a marked correlation with serotype. The protein profiles of the 33 North American (NA)-serotype viruses examined were remarkably homogeneous, with variation detected only in the E1 protein of two isolates. In contrast, considerable heterogeneity was observed in the migration profiles of both the E1 and E2 glycoproteins of the 13 South American (SA)-type viruses examined. Peptide mapping of individual virion proteins using limited proteolysis with Staphylococcus aureus V8 protease confirmed that, in addition to the homogeneity evident among NA-type viruses and relative heterogeneity among SA-type viruses, the E1 and E2 proteins of NA- and SA-serotype viruses exhibited serotype-specific structural variation. The C protein was highly conserved among isolates of both virus serotypes. Endoglycosidase analyses of intact virions did not reveal substantial glycosylation differences between the glycoproteins of NA- and SA-serotype viruses. Both the HMW protein and the E2 protein (doublet) of EEE virus appeared to contain, at least in part, high-mannose type N-linked oligosaccharides. No evidence of O-linked glycans was found on either the E1 or the E2 glycoprotein. Despite the observed structural differences between proteins of NA- and SA-type viruses, Western blot analyses utilizing polyclonal antibodies indicated that immunoreactive epitopes appeared to be conserved.

Asunto(s)

Virus de la Encefalitis Equina del Este/clasificación , Animales , Aves/virología , Culicidae/virología , Perros/virología , Electroforesis en Gel de Poliacrilamida , Virus de la Encefalitis Equina del Este/aislamiento & purificación , Glicoproteínas/análisis , Glicoproteínas/aislamiento & purificación , Caballos/virología , Humanos , Mapeo Peptídico , Serotipificación , América del Sur , Estados Unidos , Proteínas Virales/análisis , Proteínas Virales/aislamiento & purificación , Virión/química