This work was supported by the Academy of Finland (130750 and 218080 to J.T.H.), the Wellcome Trust (090532/Z/09/Z; 089026/Z/09/Z to T.A.B.), and by the MRC (MR/J007897/1 to J.T.H and T.A.B; MR/L009528/1 to T.A.B.).

<ol>
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The authors have nothing to disclose.

<html> El conocimiento de la estructura de espiga glicoproteína viral en la membrana del virión es esencial para la comprensión de la replicación del virus y el desarrollo de terapias para tratar y prevenir la infección. Electron crio-microscopía combinado con solo un promedio de partículas se ha convertido en el método más utilizado para resolver las estructuras de partículas de virus envueltos, incluyendo los picos de glicoproteína. Sin embargo, este método se limita a icosahedrally virus simétricas. Aquí, a través de la aplicación de la crio-tomografía de electrones y subtomogram promedio en Jsubtomo, hemos descrito un protocolo general para la determinación de los picos de glicoproteína en pleomórfico virus envueltos que no son susceptibles a otros métodos de biología estructurales actuales. Nuestros resultados representativos demuestran que la resolución de este método es suficiente para revelar conocimientos sobre arquitectura de dominio, oligomerización, y de mayor orden organización de espigas de glicoproteína en viriones intactos. jove_content &quot;&gt; El paso más crítico dentro de este protocolo está construyendo dos modelos de partida fiables que son estadísticamente independientes una de la otra. ejecución exitosa de este paso se supone que los picos de glicoproteína son suficientemente grandes y no está llena uno contra el otro con demasiada fuerza, de modo que los picos individuales puede ser reconocidos visualmente y manualmente recogidos en el tomografías, y dos modelos independientes en promedio. Si esto no es posible, dos modificaciones en el protocolo se puede intentar. Primera, dos modelos aleatorias independientes se pueden construir mediante la definición de primera dos subconjuntos aleatorios de subtomograms y luego promediando los subtomograms dentro de estos subconjuntos 30. segundo lugar, si una estructura de la espiga aislado ha sido derivado por otros medios, por ejemplo mediante cristalografía de rayos X, que puede ser utilizado como un modelo de partida. Sin embargo, se debe tener cuidado para bajos filtro de paso este modelo utilizando un punto de corte de baja resolución (50-70 Å), ya que los dos modelos resultantes en la próxima ronda de refinamiento voluntadestadísticamente independiente sólo más allá de esta resolución. Debido a esta advertencia, se recomienda el enfoque anterior. La resolución que se puede obtener a partir de este protocolo depende de cuatro factores principales: i. estrategia de recopilación de datos y la calidad de los datos de entrada, ii. número de los subtomograms, iii. exactitud de la alineación de los subtomograms, y iv. la heterogeneidad de las estructuras. Mientras que la primera y segunda limitación se puede superar en gran medida mediante el uso de detectores de alta de señal a ruido directa de electrones en combinación con tomografía CTF corregido y recolección de datos automatizado, la alineación de precisión se ve afectada más por el tamaño y forma de la estructura de la propia interés. Al aplicar este protocolo en pequeñas espigas que carecen de características destacadas, puede ser ventajoso para unir los fragmentos Fab a la punta para mejorar la precisión de la alineación y por lo tanto la resolución 31. Por último, si las estructuras para obtener el promedio de exposición múltiples conformaciones, sub-tomogrmétodos de clasificación am se pueden utilizar para promediar diferentes conformaciones separado. Con ese fin, Jsubtomo integra con el paquete Dynamo, ofreciendo clasificación poderosa subtomogram 9. El protocolo anterior es complementaria a la cristalografía de rayos X de las glicoproteínas virales aisladas. Estructuras cristalográficas pueden ensamblarse en promedios sub-tomografía para obtener la orientación precisa de la glicoproteína con respecto a la membrana del virión. La aplicación de esta metodología, sin duda, seguirá arrojar luz sobre la estructura del virus envuelto y biopatología.

Electron crio-tomografía es una técnica de imagen electrónica de crio-microscopía que permite el cálculo de una en tres dimensiones (3D) la reconstrucción de muestras biológicas complejas. Muestras adecuadas varían de complejos purificados macromoleculares 1, filamentos 2, vesículas recubiertas 3, y virus de membrana pleomórficas 4 a 5 células procariotas enteros e incluso zonas delgadas de células enteras eucariotas 6. Tras la recogida de datos de una serie de posiciones, volúmenes tomográficos 3D, o tomografías, puede calcularse utilizando varios paquetes de software establecidas, incluida Bsoft 7 y 8 IMOD. Dos aspectos inherentes al estudio de muestras biológicas por el límite de la crio-tomografía de electrones de la interpretación biológica de los volúmenes correspondientes tomográficas. En primer lugar, debido a la dosis de electrones limitada que se puede aplicar a materiales biológicos antes de introducir el daño por radiación significativa, signa-l-a ruido en los datos tomográficos son típicamente muy baja. En segundo lugar, como resultado de la geometría de inclinación limitado de la muestra durante la recolección de datos, algunos puntos de vista del objeto permanecen ausente, dando lugar a un denominado artefacto &#39;falta de cuña&#39; en el volumen tomográfica. Sin embargo, ambas de estas limitaciones se pueden superar si el volumen tomográfica contiene la repetición de estructuras idénticas, tales como complejos macromoleculares, que pueden ser promediados con éxito 9-12. Antes de un promedio de estructuras de reconstrucciones tomograma, objetos de interés deben ser encontrados y alineados con la misma orientación. Localización de tales estructuras se puede lograr mediante la correlación cruzada de una estructura de plantilla en el volumen tomográfica utilizando un enfoque a menudo referido como plantilla de juego 13. La plantilla utilizada en este proceso de correspondencia se puede derivar de la crio-microscopía de electrones de electrones o crio-tomografía combinado con reconstrucción 3D, o puede ser un mapa de densidad simulado desdeuna estructura atómica. Varios paquetes computacionales se han desarrollado para llevar a cabo estas tareas 11. Promediando de espigas de glicoproteína de membrana de virus, como el VIH-1, ha sido un enfoque particularmente exitoso para el estudio de su estructura 14-16. La comprensión de la estructura es integral tanto para revelar las bases moleculares de las interacciones virus-huésped y guiar el desarrollo del diseño y la vacuna antiviral. Mientras que la cristalografía macromolecular es la técnica de elección para alta resolución (generalmente mejor que 4 Å) análisis estructural de las glicoproteínas virales individuales y sus complejos, las estructuras de rayos X resultantes de este método son de proteínas aisladas del ambiente membranosa natural sobre el virión . Por lo tanto, los detalles importantes, tales como la arquitectura más alto orden de glicoproteínas virales, en el contexto del virión, siguen siendo deficiente. Por otro lado, electrón crio-microscopía y la reconstrucción de una sola partículade la totalidad de los virus envueltos se limita a viriones con simetría icosaédrica 17,18. Por lo tanto cryotomography Electron combinado con la alineación sub-volumen ha surgido como una técnica complementaria que permite el estudio de los picos de glicoproteína de forma irregular, virus pleomórficos in situ. Hemos desarrollado un software llamado Jsubtomo (www.opic.ox.ac.uk/jsubtomo) para la detección, la alineación, y un promedio de sub-volúmenes tomográficos. Jsubtomo se ha utilizado en la determinación de la estructura de un número de estructuras celulares y virales 19-26. Aquí, presentamos un protocolo detallado, que permite la determinación de la superficie viral estructuras pico. Para evitar un exceso de refinamiento de estructuras promediadas mediante la correlación de ruido, el esquema de refinamiento &#39;estándar de oro&#39; se adoptó 10,27. Por último, se discuten las estrategias para la visualización e interpretación de los resultados típicos.

<table border="0" cellpadding="0" cellspacing="0" width="686">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:184px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Jsubtomo (ver 1.3.1)</td>
			<td style="width:184px;">University of Oxford</td>
			<td style="width:119px;">n/a</td>
			<td>www.opic.ac.uk/jsubtomo</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Bsoft (ver 1.8.7)</td>
			<td style="width:184px;">NIAMS, NIH</td>
			<td style="width:119px;">n/a</td>
			<td>bsoft.ws</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">UCSF Chimera</td>
			<td style="width:184px;">UCSF</td>
			<td style="width:119px;">n/a</td>
			<td>www.cgl.ucsf.edu/chimera</td>
		</tr>
	</tbody>
</table>

averaging of viral envelope glycoprotein spikes from electron cryotomography reconstructions using jsubtomo

Los virus envueltos utilizan glicoproteínas de la membrana en su superficie para mediar en la entrada en las células huésped. Análisis estructural tridimensional de estas glicoproteína &#39;picos&#39; es a menudo un reto técnico, pero importante para la comprensión de la patogénesis viral y en el diseño de fármacos. Aquí, un protocolo se presenta para la determinación estructural repunte viral a través de un promedio de cálculo de los datos de la crio-tomografía electrónica. Electron crio-tomografía es una técnica en la microscopía electrónica utilizada para obtener reconstrucciones tridimensionales de tomografía de volumen, o tomografías, de muestras biológicas pleomórficas, como los virus de la membrana en un estado casi nativo, congelado-hidratado. Estas tomografías revelan estructuras de interés en tres dimensiones, aunque a baja resolución. Promediado Computacional de sub-volúmenes, o sub-tomografías, es necesario obtener una resolución más alta detalle de repetir motivos estructurales, tales como puntas de glicoproteína viral. Un enfoque computacional detallada para aliGning y un promedio de sub-tomografías utilizando el paquete de software Jsubtomo se perfila. Este enfoque permite la visualización de la estructura de espigas glicoproteína viral a una resolución en el intervalo de 20-40 Å y el estudio del estudio de las interacciones de orden superior-pico-a pico sobre la membrana del virión. Los resultados típicos se presentan para el virus de Bunyamwera, un virus con envuelta de la familia Bunyaviridae. Esta familia es un grupo estructuralmente diverso de patógenos que suponen una amenaza para la salud humana y animal.

Se demuestra la aplicación de la promediación de flujo de trabajo sub-tomografía descrito anteriormente para el complejo de glicoproteína de la envoltura del virus de Bunyamwera (Orthobunyavirus, Bunyaviridae) usando un conjunto de datos previamente publicados 24. Parámetros de recolección y refinamiento de datos se enumeran en la Tabla 1. Una tomografía representativo se muestra en la Figura 2. <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> Parámetro (unidad) </td><td> Valor </td></tr><tr><td> La recolección de datos </td><td></td></tr><tr><td> Voltaje (kV) </td><td> 300 </td></tr><tr><td> Ampliación calibrada (X) </td><td> 111000 </td></tr><tr><td> Tamaño de píxel (Å) </td><td> 5.4 </td></tr><tr><td> Dosis estimada (e - / Å 2) </td><td> 100 </td></tr><tr><td> Underfocus (micras) </td><td> 4,0-4,5 </td&gt; </tr><tr><td> Primera cero CTF (A) un </td><td> 26-30 </td></tr><tr><td> Rango de inclinación (°) </td><td> -60-60 </td></tr><tr><td> Muestreo Inclinación (°) </td><td> 3 </td></tr><tr><td> De datos y el refinamiento </td><td></td></tr><tr><td> Las tomografías </td><td> 11 </td></tr><tr><td> Los viriones </td><td> 29 </td></tr><tr><td> Semillas por virión </td><td> 106 </td></tr><tr><td> Número total de semillas </td><td> 3074 </td></tr><tr><td> Picos detectados </td><td> 1346 </td></tr><tr><td> Pinchos después de la eliminación de superposiciones </td><td> 1401 </td></tr><tr><td> Pinchos después de la selección basada correlación cruzada </td><td> 1022 </td></tr><tr><td> Espigas incluidos en el promedio final </td><td> 1022 </td></tr><tr><td> Simetría </td><td> C3 </td></tr><tr><td> Muestreo angular (°) </td><td> 8 </td></tr><tr><td> Resoluciónrango utilizado en el refinamiento (Å) </td><td> 42-334 </td></tr><tr><td> Estimación final de resolución (A) b </td><td> 35 </td></tr></tbody></table> Tabla 1: la recopilación de datos y estadísticas Bunyamwera refinamiento. un CTF, función de transferencia de contraste. b Calculado usando Fourier correlación shell entre dos estructuras de forma independiente refinados en un umbral de 0.143. <img alt="Figura 2" fo:content-width="6in" src="/files/ftp_upload/51714/51714fig2highres.jpg" width="600" /> Figura 2:. Rebanada a través de una tomografía de los viriones Bunyamwera Varias vistas laterales pico evidente en la periferia de cada virión se indican con flechas. La tomografía ha sido filtrado de paso bajo a 60 Å. Barra de escala 100 nm. En primer lugar, estamos perfeccionando un modelo inicial con 205 picos recogidos manualmente ( <strong&gt; Figura 3). La simetría de tres veces del centro-pico más era evidente sin aplicar ninguna simetría (Figura 3B) y se impuso en las rondas posteriores de refinamiento (Figura 3C). Para la detección de todos los picos de forma automática en las superficies de virión, se generaron 106 semillas para cada virión en el radio de 43 nm y el espaciamiento de 20 grados (Figura 4A) y iterativamente refinado sus posiciones con respecto a la membrana (Figura 4B). <img alt="Figura 3" fo:content-width="6in" src="/files/ftp_upload/51714/51714fig3highres.jpg" width="600" /> Figura 3: El perfeccionamiento de la estructura inicial de la plantilla. (A) cilíndrica promedio (C100) plantilla construida a partir de las posiciones definidas manualmente de picos. (B) densidad promediada después de cinco rondas de refinamiento sin ninguna simetría (C1) impuso muestra una espiga con triplecaracterísticas simétricas. La resolución del modelo es de 48 Å. (C) Promedio de la espiga se resolvió en 41 Å después de cinco rondas de refinamiento con simetría triple. <img alt="Figura 4" fo:content-width="6in" src="/files/ftp_upload/51714/51714fig4highres.jpg" width="600" /> Figura 4:. Refinamiento de las semillas AB) Un subconjunto de las semillas antes (A) y después (B) el refinamiento se muestran en una densidad virión en la Figura 2 Semillas en (B) han sido codificados por color con base en el respectivo cruzada. coeficientes de correlación (Azules, baja correlación; rojo, alta correlación). Los mejores parches correlacionar glicoproteína pico (superior al 75% después de la eliminación de superposiciones; ~ 1,000 picos) se utilizaron para calcular el promedio final. El promedio se resolvió a 35 Å de resolución (Figura 5). Se reveló una estructura trimérica pico en el medio, enAdemás de alguna contribución de seis picos vecinos. Modelos de compuestos de los viriones, calculados mediante la colocación de la estructura en las posiciones conocidas, revelaron colocación de los picos en la superficie del virión (Figura 6A). Ocasionalmente, los parches ordenados localmente de picos eran evidentes (Figura 6B). <img alt="Figura 5" fo:content-width="6in" src="/files/ftp_upload/51714/51714fig5highres.jpg" width="600" /> Figura 5: Estructura de refinado de un parche de la capa de glicoproteína pico después de la adaptación de la plantilla. (AB) Dos mapas &#39;incluso&#39; y &#39;extraño&#39;, reconstruido a partir de dos mitades de los datos se muestran. Los dos mapas muestran un notable grado de similitud, la verificación de la validness del enfoque. La orientación alrededor del eje largo pico es diferente ya que los dos mapas se han reconstruido totalmente independientemente uno de otro. (C) Media de los dos mapas se muestra enla resolución final de 35 Å. <img alt="Figura 6" fo:content-width="6in" src="/files/ftp_upload/51714/51714fig6highres.jpg" width="600" /> Figura 6: Colocación de los picos en el virión Bunyamwera. (A) Modelo Compuesto de un virión. Ver vectores (palos) indican las orientaciones de los picos. Los colores indican la correlación cruzada entre cada pico y la estructura de la plantilla (azul, baja correlación; rojo, alta correlación). (B) Un primer plano de un parche ordenada de los picos.

Watch this Scientific Journal Video about Averaging of Viral Envelope Glycoprotein Spikes from Electron Cryotomography Reconstructions using Jsubtomo at JoVE.com

Averaging of Viral Envelope Glycoprotein Spikes from Electron Cryotomography Reconstructions using Jsubtomo

An approach is presented for determining structures of viral membrane glycoprotein complexes using a combination of electron cryo-tomography and sub-tomogram averaging with the computational package Jsubtomo.

Un promedio de glicoproteína de la envoltura viral Espigas de Electron cryotomography reconstrucciones utilizando Jsubtomo

Enveloped viruses utilize membrane glycoproteins on their surface to mediate entry into host cells. Three-dimensional structural analysis of these ...

averaging-viral-envelope-glycoprotein-spikes-from-electron

Research

JoVE Journal

Immunology and Infection

12.0K vistas.  University of Oxford. An approach is presented for determining structures of viral membrane glycoprotein complexes using a combination of electron cryo-tomography and sub-tomogram averaging with the computational package Jsubtomo.

Video: Averaging of Viral Envelope Glycoprotein Spikes from Electron Cryotomography Reconstructions using Jsubtomo

Using a Pan-Viral Microarray Assay (Virochip) to Screen Clinical Samples for Viral Pathogens

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence of novel viral pathogens, such as SARS coronavirus and 2009 pandemic H1N1 influenza virus, that are not detectable by traditional methods. To address these challenges, we have previously developed and validated a pan-viral microarray platform called the Virochip with the capacity to detect all known viruses as well as novel variants on the basis of conserved sequence homology1. Using the Virochip, we have identified the full spectrum of viruses associated with respiratory infections, including cases of unexplained critical illness in hospitalized patients, with a sensitivity equivalent to or superior to conventional clinical testing2-5. The Virochip has also been used to identify novel viruses, including the SARS coronavirus6,7, a novel rhinovirus clade5, XMRV (a retrovirus linked to prostate cancer)8, avian bornavirus (the cause of a wasting disease in parrots)9, and a novel cardiovirus in children with respiratory and diarrheal illness10. The current version of the Virochip has been ported to an Agilent microarray platform and consists of ~36,000 probes derived from over ~1,500 viruses in GenBank as of December of 2009. Here we demonstrate the steps involved in processing a Virochip assay from start to finish (~24 hour turnaround time), including sample nucleic acid extraction, PCR amplification using random primers, fluorescent dye incorporation, and microarray hybridization, scanning, and analysis.

Using a Pan-Viral Microarray Assay Virochip to Screen Clinical Samples for Viral Pathogens

The Virochip is a pan-viral microarray designed to simultaneously detect all known viruses as well as novel viruses on the basis of conserved sequence homology. Here we demonstrate how to run a Virochip assay to analyze clinical samples for the presence of both known and unknown viruses.

El uso de un ensayo de microarrays Pan-viral (Virochip) a la pantalla de muestras clínicas de los patógenos virales

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence ...

Investigación

Inmunología e Infección

Determination of Molecular Structures of HIV Envelope Glycoproteins using Cryo-Electron Tomography and Automated Sub-tomogram Averaging

Since its discovery nearly 30 years ago, more than 60 million people have been infected with the human immunodeficiency virus (HIV) <a href="http://www.usaid.gov">(www.usaid.gov)</a>. The virus infects and destroys CD4+ T-cells thereby crippling the immune system, and causing an acquired immunodeficiency syndrome (AIDS) 2. Infection begins when the HIV Envelope glycoprotein "spike" makes contact with the CD4 receptor on the surface of the CD4+ T-cell. This interaction induces a conformational change in the spike, which promotes interaction with a second cell surface co-receptor 5,9. The significance of these protein interactions in the HIV infection pathway makes them of profound importance in fundamental HIV research, and in the pursuit of an HIV vaccine.
The need to better understand the molecular-scale interactions of HIV cell contact and neutralization motivated the development of a technique to determine the structures of the HIV spike interacting with cell surface receptor proteins and molecules that block infection. Using cryo-electron tomography and 3D image processing, we recently demonstrated the ability to determine such structures on the surface of native virus, at &tilde;20 &Aring; resolution 9,14. This approach is not limited to resolving HIV Envelope structures, and can be extended to other viral membrane proteins and proteins reconstituted on a liposome. In this protocol, we describe how to obtain structures of HIV envelope glycoproteins starting from purified HIV virions and proceeding stepwise through preparing vitrified samples, collecting, cryo-electron microscopy data, reconstituting and processing 3D data volumes, averaging and classifying 3D protein subvolumes, and interpreting results to produce a protein model. The computational aspects of our approach were adapted into modules that can be accessed and executed remotely using the Biowulf GNU/Linux parallel processing cluster at the NIH <a href="http://biowulf.nih.gov">(http://biowulf.nih.gov)</a>. This remote access, combined with low-cost computer hardware and high-speed network access, has made possible the involvement of researchers and students working from school or home.

The protocol describes a high-throughput approach to determining structures of membrane proteins using cryo-electron tomography and 3D image processing. It covers the details of specimen preparation, data collection, data processing and interpretation, and concludes with the production of a representative target for the approach, the HIV-1 Envelope glycoprotein. These computational procedures are designed in a way that enables researchers and students to work remotely and contribute to data processing and structural analysis.

Determinación de estructuras moleculares de glicoproteínas de la envoltura del VIH con Cryo-tomografía electrónica y automatizada promedio Sub-tomografía

Since its discovery nearly 30 years ago, more than 60 million people have been infected with the human immunodeficiency virus (HIV) (www.usaid.gov). The ...

Cost-effective Method for Microbial Source Tracking Using Specific Human and Animal Viruses

Microbial contamination of the environment represents a significant health risk. Classical bacterial fecal indicators have shown to have significant limitations, viruses are more resistant to many inactivation processes and standard fecal indicators do not inform on the source of contamination. The development of cost-effective methods for the concentration of viruses from water and molecular assays facilitates the applicability of viruses as indicators of fecal contamination and as microbial source tracking (MST) tools. Adenoviruses and polyomaviruses are DNA viruses infecting specific vertebrate species including humans and are persistently excreted in feces and/or urine in all geographical areas studied. In previous studies, we suggested the quantification of human adenoviruses (HAdV) and JC polyomaviruses (JCPyV) by quantitative PCR (qPCR) as an index of human fecal contamination. Recently, we have developed qPCR assays for the specific quantification of porcine adenoviruses (PAdV) and bovine polyomaviruses (BPyV) as animal fecal markers of contamination with sensitivities of 1-10 genome copies per test tube. In this study, we present the procedure to be followed to identify the source of contamination in water samples using these tools. As example of representative results, analysis of viruses in ground water presenting high levels of nitrates is shown.
Detection of viruses in low or moderately polluted waters requires the concentration of the viruses from at least several liters of water into a much smaller volume, a procedure that usually includes two concentration steps in series. This somewhat cumbersome procedure and the variability observed in viral recoveries significantly hamper the simultaneous processing of a large number of water samples.
In order to eliminate the bottleneck caused by the two-step procedures we have applied a one-step protocol developed in previous studies and applicable to a diversity of water matrices. The procedure includes: acidification of ten-liter water samples, flocculation by skimmed milk, gravity sedimentation of the flocculated materials, collection of the precipitate and centrifugation, resuspension of the precipitate in 10 ml phosphate buffer. The viral concentrate is used for the extraction of viral nucleic acids and the specific adenoviruses and polyomaviruses of interest are quantified by qPCR. High number of samples may be simultaneously analyzed using this low-cost concentration method.
The procedure has been applied to the analysis of bathing waters, seawater and river water and in this study, we present results analyzing groundwater samples. This high-throughput quantitative method is reliable, straightforward, and cost-effective.

The study describes a cost-effective method for the identification of the source of fecal/urine contamination or contamination by nitrates in water using qPCR for the specific quantification of human/porcine/bovine DNA viruses, adenoviruses and polyomaviruses, proposed as MST tools.

Método rentable para el Rastreo de uso microbiana específica virus humanos y animales

Microbial contamination of the environment represents a significant health risk. Classical bacterial fecal indicators have shown to have significant ...

Isolation of Fidelity Variants of RNA Viruses and Characterization of Virus Mutation Frequency

RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the generation of extreme population diversity that facilitates virus adaptation and evolution. Increasing evidence shows that the intrinsic error rates, and the resulting mutation frequencies, of RNA viruses can be modulated by subtle amino acid changes to the viral polymerase. Although biochemical assays exist for some viral RNA polymerases that permit quantitative measure of incorporation fidelity, here we describe a simple method of measuring mutation frequencies of RNA viruses that has proven to be as accurate as biochemical approaches in identifying fidelity altering mutations. The approach uses conventional virological and sequencing techniques that can be performed in most biology laboratories. Based on our experience with a number of different viruses, we have identified the key steps that must be optimized to increase the likelihood of isolating fidelity variants and generating data of statistical significance. The isolation and characterization of fidelity altering mutations can provide new insights into polymerase structure and function1-3. Furthermore, these fidelity variants can be useful tools in characterizing mechanisms of virus adaptation and evolution4-7.

The present article describes the steps required to isolate and characterize RNA polymerase fidelity variants of RNA viruses and how to use mutation frequency data to confirm fidelity changes in tissue culture.

El aislamiento de las variantes de los virus de ARN de fidelidad y caracterización de la frecuencia de mutación de virus

RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the ...

Separation of Single-stranded DNA, Double-stranded DNA and RNA from an Environmental Viral Community Using Hydroxyapatite Chromatography

Viruses, particularly bacteriophages (phages), are the most numerous biological entities on Earth1,2. Viruses modulate host cell abundance and diversity, contribute to the cycling of nutrients, alter host cell phenotype, and influence the evolution of both host cell and viral communities through the lateral transfer of genes 3. Numerous studies have highlighted the staggering genetic diversity of viruses and their functional potential in a variety of natural environments. 
Metagenomic techniques have been used to study the taxonomic diversity and functional potential of complex viral assemblages whose members contain single-stranded DNA (ssDNA), double-stranded DNA (dsDNA) and RNA genotypes 4-9. Current library construction protocols used to study environmental DNA-containing or RNA-containing viruses require an initial nuclease treatment in order to remove nontargeted templates 10. However, a comprehensive understanding of the collective gene complement of the virus community and virus diversity requires knowledge of all members regardless of genome composition. Fractionation of purified nucleic acid subtypes provides an effective mechanism by which to study viral assemblages without sacrificing a subset of the community&#x2019;s genetic signature. 
Hydroxyapatite, a crystalline form of calcium phosphate, has been employed in the separation of nucleic acids, as well as proteins and microbes, since the 1960s11. By exploiting the charge interaction between the positively-charged Ca2+ ions of the hydroxyapatite and the negatively charged phosphate backbone of the nucleic acid subtypes, it is possible to preferentially elute each nucleic acid subtype independent of the others. We recently employed this strategy to independently fractionate the genomes of ssDNA, dsDNA and RNA-containing viruses in preparation of DNA sequencing 12. Here, we present a method for the fractionation and recovery of ssDNA, dsDNA and RNA viral nucleic acids from mixed viral assemblages using hydroxyapatite chromotography.

We describe an efficient method to separate single-stranded DNA, double-stranded DNA and RNA molecules from environmental viral communities. Nucleic acids are fractionated using hydroxyapatite chromatography with increasing concentrations of phosphate-containing buffers. This method permits the isolation of all viral nucleic acid types from environmental samples.

La separación de una sola hebra de ADN, el ADN de doble cadena y el ARN de una comunidad ambiental viral mediante cromatografía de hidroxiapatita

Viruses, particularly bacteriophages (phages), are the most numerous biological entities on Earth1,2.  Viruses modulate host cell abundance and diversity, ...

Preparation of Viral DNA from Nucleocapsids

Viruses are obligate cellular parasites, and thus the study of their DNA requires isolating viral material away from host cell contaminants and DNA. Several downstream applications require large quantities of pure viral DNA, which is provided by this protocol. These applications include viral genome sequencing, where the removal of host DNA is crucial to optimize data output for viral sequences, and the production of new viral recombinant strains, where co-transfection of purified plasmid and linear viral DNA facilitates recombination.1,2,3 
This procedure utilizes a combination of extractions and density-based centrifugation to isolate purified linear herpesvirus nucleocapsid DNA from infected cells.4,5 The initial purification steps aim to isolate purified viral capsids, which contain and protect the viral DNA during the extractions and centrifugation steps that remove cellular proteins and DNA. Lysis of nucleocapsids then releases viral DNA, and two final phenol-chloroform steps remove remaining proteins. The final DNA captured from solution is highly concentrated and pure, with an average OD260/280 of 1.90. Depending on the quantity of infected cells used, yields of viral DNA range from 150-800 &mu;g or more. The purity of this DNA makes it stable during long-term storage at 4C. This DNA is thus ideally suited for high-throughput sequencing, high fidelity PCR reactions, and transfections. 
Prior to beginning the protocol, it is important to know the average number of cells per dish (e.g. an average of 8 x 106 PK-15 cells in a confluent 15 cm dish), and the titer of the viral stock to be used (e.g. 1 x 108 plaque-forming units per ml). These are necessary to calculate the appropriate multiplicity of infection (MOI) for the protocol.6 For instance, to infect one 15 cm dish of PK-15 cells with the above viral stock, at an MOI of 5, you would use 400 &mu;l of viral stock and dilute it with 3.6 ml of medium (total inoculation volume of 4 ml for one 15 cm plate).
Multiple viral DNA preparations can be prepared at the same time. The number of simultaneous preparations is limited only by the number of tubes held by the ultracentrifuge rotor (one per virus; see step 3.9 below). Here we describe the procedure as though being done for one virus.

We describe the process of isolating high purity herpesvirus nucleocapsid DNA from infected cells. The final DNA captured from solution is of high concentration and purity, making it ideally suited for high-throughput sequencing, high fidelity PCR reactions, and transfections to produce new viral recombinants.

Preparación de ADN viral de nucleocápside

Viruses are obligate cellular parasites, and thus the study of their DNA requires isolating viral material away from host cell contaminants and DNA. ...

Imaging of HIV-1 Envelope-induced Virological Synapse and Signaling on Synthetic Lipid Bilayers

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between CD4+ T cells involves the formation of a virological synapse (VS), which is an F-actin-dependent cell-cell junction formed upon the engagement of HIV-1 envelope gp120 on the infected cell with CD4 and the chemokine receptor (CKR) CCR5 or CXCR4 on the target cell 8. In addition to gp120 and its receptors, other membrane proteins, particularly the adhesion molecule LFA-1 and its ligands, the ICAM family, play a major role in VS formation and virus transmission as they are present on the surface of virus-infected donor cells and target cells, as well as on the envelope of HIV-1 virions1,4,5,6,7,13. VS formation is also accompanied by intracellular signaling events that are transduced as a result of gp120-engagement of its receptors. Indeed, we have recently showed that CD4+ T cell interaction with gp120 induces recruitment and phosphorylation of signaling molecules associated with the TCR signalosome including Lck, CD3&zeta;, ZAP70, LAT, SLP-76, Itk, and PLC&gamma;15.
In this article, we present a method to visualize supramolecular arrangement and membrane-proximal signaling events taking place during VS formation. We take advantage of the glass-supported planar bi-layer system as a reductionist model to represent the surface of HIV-infected cells bearing the viral envelope gp120 and the cellular adhesion molecule ICAM-1. The protocol describes general procedures for monitoring HIV-1 gp120-induced VS assembly and signal activation events that include i) bi-layer preparation and assembly in a flow cell, ii) injection of cells and immunofluorescence staining to detect intracellular signaling molecules on cells interacting with HIV-1 gp120 and ICAM-1 on bi-layers, iii) image acquisition by TIRF microscopy, and iv) data analysis. This system generates high-resolution images of VS interface beyond that achieved with the conventional cell-cell system as it allows detection of distinct clusters of individual molecular components of VS along with specific signaling molecules recruited to these sub-domains.

This article describes a method to visualize formation of an HIV-1 envelope-induced virological synapse on glass supported planar bilayers by total internal reflection fluorescence (TIRF) microscopy. The method can also be combined with immunofluorescence staining to detect activation and redistribution of signaling molecules that occur during HIV-1 envelope-induced virological synapse formation.

Imagen del VIH-1 sobre inducida sinapsis virológica y señalización en la bicapa lipídica sintéticas

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between ...

Conformational Evaluation of HIV-1 Trimeric Envelope Glycoproteins Using a Cell-based ELISA Assay

HIV-1 envelope glycoproteins (Env) mediate viral entry into target cells and are essential to the infectious cycle. Understanding how those glycoproteins are able to fuel the fusion process through their conformational changes could lead to the design of better, more effective immunogens for vaccine strategies. Here we describe a cell-based ELISA assay that allows studying the recognition of trimeric HIV-1 Env by monoclonal antibodies. Following expression of HIV-1 trimeric Env at the surface of transfected cells, conformation specific anti-Env antibodies are incubated with the cells. A horseradish peroxidase-conjugated secondary antibody and a simple chemiluminescence reaction are then used to detect bound antibodies. This system is highly flexible and can detect Env conformational changes induced by soluble CD4 or cellular proteins. It requires minimal amount of material and no highly-specialized equipment or know-how. Thus, this technique can be established for medium to high throughput screening of antigens and antibodies, such as newly-isolated antibodies.

Understanding viral surface antigens conformations is required to evaluate antibody neutralization and guide the design of effective vaccine immunogens. Here we describe a cell-based ELISA assay that allows the study of the recognition of trimeric HIV-1 Env expressed at the surface of transfected cells by specific anti-Env antibodies.

Evaluación conformacional del VIH-1 glicoproteínas de la envoltura trimérica El uso de un ELISA ensayo basado en células

HIV-1 envelope glycoproteins (Env) mediate viral entry into target cells and are essential to the infectious cycle. Understanding how those glycoproteins ...

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

Coxiella burnetii, the agent causing Q fever, is an obligate intracellular bacterium. PCR based diagnostic assays have been developed for detecting C. burnetii DNA in cell cultures and clinical samples. PCR requires specialized equipment and extensive end user training, and therefore, it is not suitable for routine work especially in a resource-constrained area. We have developed a loop-mediated isothermal amplification (LAMP) assay to detect the presence of C. burnetii in patient samples. This method is performed at a single temperature around 60 &#176;C in a water bath or heating block. The sensitivity of this LAMP assay is very similar to PCR with a detection limit of about 25 copies per reaction. This report describes the preparation of the reaction using lyophilized reagents and visualization of results using hydroxynaphthol blue (HNB) or a UV lamp with fluorescent intercalating dye in the reaction. The LAMP reagents were lyophilized and stored at room temperature (RT) for one month without loss of detection sensitivity. This LAMP assay is particularly robust because the reaction mixture preparation does not involve complex steps. This method is ideal for use in resource-limited settings where Q fever is endemic.

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

We described here a simple loop-mediated isothermal amplification (LAMP) method using lyophilized reagents for the detection of C. burnetii in patient samples.

El desarrollo de la mediación de la Loop liofilizada amplificación isotérmica reactivos para la detección de<em&gt; Coxiella burnetii</em

Coxiella burnetii, the agent causing Q fever, is an obligate intracellular bacterium. PCR based diagnostic assays have been developed for detecting C. ...

Measuring Influenza Neuraminidase Inhibition Antibody Titers by Enzyme-linked Lectin Assay

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional methods to measure NA inhibiting (NI) antibody titers are not practical for routine serology. This protocol describes the enzyme-linked lectin assay (ELLA), a practical alternative method to measure NI titers that is performed in 96 well plates coated with a large glycoprotein substrate, fetuin. NA cleaves terminal sialic acids from fetuin, exposing the penultimate sugar, galactose. Peanut agglutinin (PNA) is a lectin with specificity for galactose and therefore the extent of desialylation can be quantified using a PNA-horseradish peroxidase conjugate, followed by addition of a chromogenic peroxidase substrate. The optical density that is measured is proportional to NA activity. To measure NI antibody titers, serial dilutions of sera are incubated at 37 &#176;C O/N on fetuin-coated plates with a fixed amount of NA. The reciprocal of the highest serum dilution that results in &#8805;50% inhibition of NA activity is designated as the NI antibody titer. The ELLA provides a practical format for routine evaluation of human antibody responses following influenza infection or vaccination.

We describe the enzyme-linked lectin assay (ELLA) for measuring influenza neuraminidase (NA)-inhibition antibody titers in sera. The assay uses peanut agglutinin to quantify galactose residues that become accessible when NA removes sialic acid from fetuin-coated, 96-well plates.

La medición de la influenza neuraminidasa inhibición títulos de anticuerpos mediante un ensayo de lectina ligado a enzimas

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional ...