Abstract
The family Poxviridae consists of large double-stranded DNA containing viruses that replicate exclusively in the cytoplasm of infected cells. Members of the orthopox genus include variola, the causative agent of human small pox, monkeypox, and vaccinia (VAC), the prototypic member of the virus family. Within the relatively large (~ 200 kb) vaccinia genome, three classes of genes are encoded: early, intermediate, and late. While all three classes are transcribed by virally-encoded RNA polymerases, each class serves a different function in the life cycle of the virus. Poxviruses utilize multiple strategies for modulation of the host cellular environment during infection. In order to understand regulation of both host and virus gene expression, we have utilized genome-wide approaches to analyze transcript abundance from both virus and host cells. Here, we demonstrate time course infections of HeLa cells with Vaccinia virus and sampling RNA at several time points post-infection. Both host and viral total RNA is isolated and amplified for hybridization to microarrays for analysis of gene expression.
Highlights
The family Poxviridae consists of large double-stranded DNA containing viruses that replicate exclusively in the cytoplasm of infected cells
The infection can be performed with either a crude stock of vaccinia virus with a known titer, or sucrose purified virus with a known titer if you're concerned about host gene expression
A mixture of infected and uninfected cells will lead to multiple rounds of infection, heterogeneous mixtures of time points, and asynchronous viral and host transcriptional responses
Summary
2. Add the desired amount of sucrose purified virus or trypsinized crude virus to 37°C viral growth media and mix well. 3. Remove media from the cells and rinse with room temperature PBS. 4. Add the virus/viral growth media to each flask. Tilt and swirl plates every 15 minutes to spread virus uniformly and keep cells moist. For a 0hr/Mock time point, add the viral growth media only, with no virus added. 5. After the 1 hour incubation, remove the media containing the virus, and rinse three times with room temperature PBS. 30 mL of media for a T-175 flask. Start counting this as your 0 hr time point
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