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EXAMINING THE ROLE OF ADENOVIRUS E4 11K PROTEIN ON IFN-BETA EXPRESSION

Abstract

Reverse Transcription quantitative PCR (RT-qPCR) is an effective method for studying changes in gene expression through quantification of specific mRNAs. As viruses alter gene expression during an infection, we used RT-qPCR to study the effects of adenovirus, a DNA virus that causes the common cold. RIG-I is a viral RNA sensor that can inhibit viral replication by triggering a signaling cascade to stimulate a type I interferon (IFN) response. Type I IFN transcription is potentially enhanced by the binding of RIG-I to DEAD-box helicase (Ddx6). An adenoviral protein, E4 11k, has been shown to colocalize with Ddx6. We hypothesize that this interaction may modulate RIG-I, decreasing IFN-beta expression. To examine this, we used RT-qPCR to observe the levels of RIG-I and IFN-beta mRNA. Human lung carcinoma cells (A549) were infected with 3 virus variants (an Ad5 wildtype virus (wt), a virus that only expresses E4 11k, and an E4 11k-deleted virus) for 12, 24, 30, 36, and 48 hours. At each time point, cells were lysed, RNA was isolated, and cDNA was synthesized. IFN-beta and housekeeping primers were added to each sample with Maxima SYBR green master mix in triplicate. Initially, GAPDH primers were used but were concerned about cellular mRNA reductions at late time points. Some studies have shown that PPIA is more consistent so we tested its use as an alternate housekeeping gene. Samples were subjected to 40 cycles of qPCR with an annealing temperature of 62˚C. By measuring the threshold cycle (CT) of each IFN-beta sample and normalizing it to GAPDH, we found that E4 11k-deleted virus revealed a significant increase in IFN-beta expression at 48 hpi compared to wt (p=0.00113, via ANOVA and Tukey Test). Initial findings suggest that RT-qPCR is an effective method in determining the role of E4 11K 48hpi. We anticipate that PPIA will yield more reliable results supporting GAPDH findings.

Acknowledgements

GC Journeys Mini Grant, ​GCSU Academic Affairs Small Grant, GCSU MURACE Summer Research Grant, GC Journeys Mini Grant, GCSU Academic Affairs Small Grant ​, GCSU MURACE Summer Research Grant, and Tri Beta Research Grant.

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