To replicate, all viruses must co-opt the host translational machinery to express viral proteins and peptides. Infection activates innate cellular defense and stress responses that also substantially alter cellular transcription, translation, and the resulting proteome. Studies of host gene expression responses to virus infection are incomplete. Virus infection alters the cellular transcriptome, but this often correlates poorly wit changes in protein levels. Similarly, tandem mass spectrometry lacks the sensitivity to fully characterize the proteome. Regulation of protein abundance is predominantly at the level of translation, although mRNA degradation-, protein degradation-, and transcription rates are important factors. A complete description is lacking of which cellular transcripts are preferentialy translated during virus infection and what open reading frames (ORFs) are used. Recent studies of the cellular translatome have indicated a greater than expected complexity, suggesting that there are viral ORFs that remain undiscovered. The overall goal of this project is to determine and quantify which cellular and viral transcripts are being translated and which open reading frames (ORFs) are being decoded at different times post-infection examining three different human viruses -- influenza A virus (IAV), herpes simplex virus 1 (HSV-1), and mammalian orthoreovirus (REOV). Recently developed approaches based on next generation sequencing of total RNA and ribosome-protected fragments of RNA will be used to test two hypotheses: (1) that virus infection induces a cohort of common changes in global cellular translation patterns overlaid by unique changes specific for each virus type and (2) that viral genomes contain more ORFs than have been identified thus far.
Two specific aims will address these hypotheses.
Aim 1 will determine the host cell translational changes in A549 cells at three different times after infection with IAV, HSV-1, or REOV. To identify changes in translation, the cellular translatome after infection with each virus will be compared to the translatome from uninfected cells. The cellular translatomes of each virus will then be compared to identify common changes and translational patterns.
Aim 2 will test the hypothesis that viral genomes contain more ORFs than are currently known. Translation initiation sites will be identified by deep sequencing libraries prepared from ribosome-protected fragments of RNA recovered from cells treated with drugs that fix ribosomes on start codons. The studies will also provide new information about changes in cellular translation during the three different virus infections and will identify common translational responses to infection. In addition, novel virus-encoded ORFs will likely be discovered. Such ORFs may initiate from alternative near- or non-canonical start sites. This information would provide new insights into translational regulation by viruses, and expand our knowledge of basic virology.
Human viral pathogens cause a significant burden of disease and new therapeutic and vaccine strategies are needed. Knowledge about the cellular or viral proteins that are required for successful infection is a prerequisite for developing new therapeutics or vaccines. By using a highly sensitive and accurate new approach, this project will identify common changes in the protein composition of cells in response to three human viruses (Herpes simplex virus 1, influenza virus, and reovirus) and in addition more completely identify the protein products that are encoded by their genomes.
| Desmet, Emily A; Anguish, Lynne J; Parker, John S L (2014) Virus-mediated compartmentalization of the host translational machinery. MBio 5:e01463-14 |
