Vaccinia Virus Infection & Temporal Analysis of Virus Gene Expression: Part 2

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.


Critical Steps
A second round of amplification is not advised as biases in array data have been observed.Mixing carefully at each enzymatic step (1 st and 2 nd strand cDNA synthesis, IVT) is critical to obtaining good amplification yields, as is incubation of each enzymatic step at the appropriate temperature.A PCR cycler with adjustable heated lid is preferred -even deviations as small as 2-3 degrees during IVT in an air hybridization oven or water bath hybridization can significantly affect yield of amplified product.

Application/Significance
The labeled RNA resulting from this protocol can be hybridized to human, viral, or custom microarrays to assess gene expression responses to infected cells in culture.Microarray platforms vary, so follow manufacturer instructions for preparation of hybridization mixture from labeled probe.
Using a custom designed poxvirus array 1 , we were able to classify genes into the general categories of "early" or "late" based on timing of hybridization signal and whether or not viral DNA replication was required for transcript detection.We observed the expected functional categories of genes in each temporal class (i.e., expected early, intermediate and late genes) variation as to the exact timing of transcription.
The methods utilized in this work are able to predict virus genes transcribed early or late in the replication cycle, but have more difficulty distinguishing early-only versus genes with an early and late promoter since transcripts with a dual early/late promoter may persist and be detected at late times.In addition, run-through transcription of late viral genes may affect signal at a given probe/spot on the array, as the RNA hybridizing to the array may have come from the designated ORF or an upstream ORF.Tiling arrays have attempted to resolve this issue, however challenges remain in detecting run through transcription using hybridization based approaches 2,3,4 .
Host transcriptional patterns can also be assessed using these methods.However, vaccinia encodes a variety of mechanisms to inhibit host responses, and host transcriptional responses may be diminished compared to other stimuli 5,6,7,8 .Since the expression of many genes involved in host defense is altered after infection, the contribution of viral genes that counteract host immune responses should therefore be taken into consideration.
Utilizing these methods, a map of the transcriptional timing of all viral genes can be identified and used to interrogate functions of unknown viral genes.In addition, these methods can be utilized to dissect the intricate dialogue between virus and host.These methods are broadly applicable to other host-pathogen infection systems.If the pathogen of interest does not have polyadenylated mRNAs, alternative methods can be used to directly label the total RNA, without linear amplification.By analyzing both host and virus gene expression during synchronous infection, these methods allow us to gain insight into virus interaction with the host cellular environment as well as host counter-defenses against virus infection.

Table 1 : cDNA 1 st Strand Synthesis Master Mix Table 2: cDNA 2 nd Strand Synthesis Master MixTable 3 : IVT Master MixTable 4 : aRNA Binding Mix
Mix the RNA binding beads with the bead resuspension solution first.** Add the isopropanol and mix well before adding the aRNA binding buffer concentrate. *