Protocol for Vaccinia infection of HeLa cells and analysis of host and viral gene expression. Part 1 of 3.
Part 1: Setting up the infection
Part 2: Infecting cells
Part 3: Harvesting cells
Part 4: RNA extraction of samples in TRIzol
Critical Steps
Part 1 & 2
There are several critical steps to setting up a synchronous vaccinia infection, the first being careful sonication (or trypzinizing) of the virus, in order to disaggregate virus particles. Vaccinia is highly prone to aggregating, and disruption of virus particles is important for ensuring even infection of cells. In order to achieve a synchronous infection, a high MOI (greater than 2) should be used to ensure each cell is infected. 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. The infection should be carried out in minimal amounts of media to allow maximum adsorption of virus onto the cells. In addition, regular shaking of the flasks or culture dishes (every 10 minutes) enables distribution of virus across the flask and ensures that the cells do not dry out.
Part 4
1-Bromo-3-chloropropane (BCP) is used in place of phenol to reduce genomic DNA contamination. A subsequent optional DNAse treatment (Qiagen RNAase Free-DNAse) may also be performed to eliminate any remaining genomic DNA. A second chloroform extraction is used to remove any traces of organic solvent from the extraction, as even trace amounts can inhibit subsequent amplification steps. Traces of phenol/BCP can be detected as a spike at 270nm (beyond the standard peak at 260nm) when measuring absorbance of total RNA after extraction. If such contamination appears, re-extract the RNA (using a filter or column-based RNA extraction method) before proceeding to amplification. Minimum amount of RNA needed to perform an amplification reaction is 100ng; however, 500-1000ng is preferred.
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 array1, 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 approaches2,3,4.
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 stimuli5,6,7,8. 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.
Whitehead Institute Fellows Funds
Material Name | Tipo | Company | Catalogue Number | Comment |
---|---|---|---|---|
TRIzol Reagent | Reagent | Invitrogen | 15596-026 | Similar reagents, such as TriPure from Roche, will also work. |
BCP Phase Separation Reagent | Reagent | Molecular Research Center | BP151 | |
RNase-Free DNase Set | Reagent | Qiagen | 79254 | DNase treatment is an optional step. |