Name: isolation of viral replication compartment-enriched sub-nuclear fractions from adenovirus-infected normal human cells
Uploaded: 2015-11-12T14:00:00
Description: Watch this Scientific Journal Video about Isolation of Viral Replication Compartment-enriched Sub-nuclear Fractions from Adenovirus-infected Normal Human Cells at JoVE.com

This work was supported by grants from CONACyT-SEP (SEP-2008-84582; CB-2011-01-168497) and Promep-SEP for R.A.G.; P.H. received a scholarship from CONACyT (447442).

1. HFF Cultivo Celular y Ad-infección <ol><li> Propagar virus Ad5 WT en monocapas de células HEK-293 y título como unidades formadoras de fluorescentes (FFU) sobre las células HFF tal como se describe anteriormente 8. </li><li> Crecer fibroblastos de prepucio humano (HFF) en 10 ml de DMEM / 10% suero bovino fetal (FBS) en placas estériles de cultivo de 100 mm a 37 ºC y 5% de CO 2 en un incubador humidificado. Determinar el número de células usando una cámara de Neubauer contando las cél...

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In order to elucidate the molecular mechanisms that govern regulation of cellular activities by viral infection understanding the composition and activities associated with RC would be instrumental. Therefore, to make a detailed analysis of RC, we established a cell-free system that takes advantage of the size and biochemical composition of these virus-induced structures, to isolate subnuclear fractions enriched with RC using a simple procedure that relies on velocity gradients with sucrose cushions. Critical steps of th...

Los adenovirus contienen un genoma de doble cadena de ADN que se replica en el núcleo de la célula infectada. Cuando el ADN viral entra en el núcleo, se localiza adyacente a cuerpos nucleares de PML 1. Después de la expresión génica viral temprana, la arquitectura nuclear se reorganiza de manera espectacular, inducir la formación de microambientes virales, denominado compartimentos de replicación viral (RC) 2. Desde adenovirus (Ad) RC son sitios donde la replicación del genoma viral y la expresión de genes tardíos virales tienen lugar, que proporcionan un entorno para el reclutamiento de todos los factores virales y celulares necesarias que participan en estos procesos. Curiosamente, una variedad de proteínas celulares responsables de la respuesta antiviral celular, tales como la respuesta al daño del ADN, la respuesta inmune innata y la supresión de tumores son cooptados a estos sitios virales 2. Hubs reguladoras Por lo tanto, Ad RC se puede considerar que promueven la replicación viral eficaz, mientras que de forma concomitante la regulación de larespuesta antiviral celular, lo que indica que estas estructuras son la clave para la comprensión de las interacciones de células virus-huésped. Sin embargo, los mecanismos moleculares de la formación de RC, su composición y actividades asociadas, son poco conocidos. Adenoviral RC, así como RC de otros virus de ADN que se replican en el núcleo no están asociados a las membranas, en contraste con RC citoplasmática 3. Por otra parte, estas estructuras inducidas por virus son susceptibles de ser compuesto en su totalidad de proteínas y ácidos nucleicos. RC formado en las células infectadas con los virus de ARN (generalmente denominado fábricas víricas) han sido aislados, tomando ventaja de su localización citoplasmática y el estado unido a la membrana, que ha facilitado su morfológica detallada, caracterización funcional y bioquímico 4. Para nuestro conocimiento, nuclear viral RC no se han aislado, tal vez debido a la complejidad de la arquitectura nuclear y la ausencia de m intranuclearembranes que facilitarían su aislamiento. Su estudio se ha basado más bien en microscopía de inmunofluorescencia, FISH y microscopía electrónica de transmisión. Sin embargo, a pesar de las complicaciones inherentes a aislar estructuras subnuclear, otros ámbitos nucleares como nucleolos y Cuerpos de Cajal se han aislado antes de 5,6. Desde nucleolos y RC están ambas compuestas de proteínas y ácidos nucleicos, y tienen un diámetro entre 0.5 - 5 micras, la hipótesis de que RC también debe ser susceptible de aislamiento. Por lo tanto, con el fin de caracterizar con más precisión la composición molecular y funciones asociadas a RC, establecimos un nuevo método para aislar fracciones enriquecidas con subnuclear RC. Con este fin, hemos preparado fracciones sub-nuclear utilizando gradientes de velocidad y cojines de sacarosa similares a los procedimientos utilizados para aislar nucleolos 7 u otros dominios nucleares 6 y establecimos un sistema libre de células que permite el estudio de la composición molecular y actividades asociadas deRC. Por tanto, esta técnica debe avanzar en la comprensión de las interacciones celulares virus-huésped y representa una poderosa herramienta que también debe facilitar el análisis detallado de RC de otros virus que se replican en el núcleo e inducir la formación de compartimentos de replicación de dimensiones similares a las formadas en adenovirus las células infectadas, como, herpesvirus, papilomavirus o poliomavirus.

<table border="0">
	<tbody>
		<tr>
			<td>DMEM</td>
			<td>Gibco</td>
			<td>12100-046</td>
			<td>Warm in 37 &#186;C water bath before use</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Gibco</td>
			<td>12484-028</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose, Ultra Pure</td>
			<td>Research Organics</td>
			<td>0928S</td>
			<td>Prepare a 2.55 M stock solution and store at 4 &#186;C</td>
		</tr>
		<tr>
			<td>Dounce homogenizer</td>
			<td>Kontess Glass Company</td>
			<td>884900-0000</td>
			<td></td>
		</tr>
		<tr>
			<td>Branson 1800 Ultrasonic Bath</td>
			<td>Branson</td>
			<td>Z769533&#160;SIGMA</td>
			<td>Turn on 15 min before use.</td>
		</tr>
		<tr>
			<td>Peroxidase AffiniPure F(ab&#39;)&#8322; Fragment Goat Anti-Mouse IgG (H+L)</td>
			<td>Jackson ImmunoResearch</td>
			<td>115-036-003</td>
			<td>Use at a 1:10,000 dilution in PBS/0.03% non-fat milk</td>
		</tr>
		<tr>
			<td>Goat anti-Mouse IgG1 Secondary Antibody, Alexa Fluor 488 conjugate</td>
			<td>Life Technologies</td>
			<td>A-21121</td>
			<td>Use at a 1:2,000 dilution in PBS</td>
		</tr>
		<tr>
			<td>Silane-Prep Slides</td>
			<td>Sigma</td>
			<td>S4651-72EA</td>
			<td>Open in a laminar flow cabinet</td>
		</tr>
		<tr>
			<td>SuperSignal West Pico Chemiluminescent Substrate</td>
			<td>Pierce ThermoScientific</td>
			<td>34080</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of viral replication compartment-enriched sub-nuclear fractions from adenovirus-infected normal human cells

During infection of human cells by adenovirus (Ad), the host cell nucleus is dramatically reorganized, leading to formation of nuclear microenvironments through the recruitment of viral and cellular proteins to sites occupied by the viral genome. These sites, called replication compartments (RC), can be considered viral-induced nuclear domains where the viral genome is localized and viral and cellular proteins that participate in replication, transcription and post-transcriptional processing are recruited. Moreover, cellular proteins involved in the antiviral response, such as tumor suppressor proteins, DNA damage response (DDR) components and innate immune response factors are also co-opted to RC. Although RC seem to play a crucial role to promote an efficient and productive replication cycle, a detailed analysis of their composition and associated activities has not been made. To facilitate the study of adenoviral RC and potentially those from other DNA viruses that replicate in the cell nucleus, we adapted a simple procedure based on velocity gradients to isolate Ad RC and established a cell-free system amenable to conduct morphological, functional and compositional studies of these virus-induced subnuclear structures, as well as to study their impact on host-cell interactions.

Puesto que los compartimentos de replicación viral (RC) son estructuras inducidas por virus subnuclear compuestas de proteínas y ácidos nucleicos, similar a otros dominios nucleares, que resultó ser susceptible de aislamiento mediante gradientes de velocidad en base a características bioquímicas. Los pasos críticos en el protocolo de fraccionamiento se ilustran en la figura 1. En cada paso las muestras deben ser supervisados ​​por microscopía de campo claro para garantizar la integridad de l...

Watch this Scientific Journal Video about Isolation of Viral Replication Compartment-enriched Sub-nuclear Fractions from Adenovirus-infected Normal Human Cells at JoVE.com

Isolation of Viral Replication Compartment-enriched Sub-nuclear Fractions from Adenovirus-infected Normal Human Cells

We provide a novel strategy to isolate viral replication compartments (RC) from adenovirus (Ad)-infected human cells. This approach represents a cell-free system that can help to elucidate the molecular mechanisms regulating viral genome replication and expression as well as regulation of viral-host interactions established at the RC.

Aislamiento de fracciones Sub-nuclear enriquecido compartimentos viral de replicación de infectados por adenovirus células normales Humanos

During infection of human cells by adenovirus (Ad), the host cell nucleus is dramatically reorganized, leading to formation of nuclear microenvironments ...

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