Background: Virus:host interactions occur at many levels, including viral entry, viral transcription and replication, and host immune response. Whereas, molecular assays (RT-PCR, northern blot) have been typically used to investigate viral and host transcription. Here we present new next-generation sequencing (NGS) approaches to studying virus:host interactions at the transcriptional level for Influenza and Ebola virus.
Aims: Using NGS coupled with custom bioinformatics pipelines, we set out to examine the interplay between host and viral transcription during infection. Focusing on Influenza A virus (IAV), we investigated the substrate preferences for IAV cap-snatching - the process by which the viral RNA-dependent RNA polymerase (RdRP) complex cleaves the first 10-15 nucleotides to prime viral messenger RNA transcription - and its effect on host gene expression and viral protein diversity. In addition, through the application of Gene Co-Expression Network Analysis to a unique sample set of peripheral blood mononuclear cells (PBMC) obtained from Ebola virus-infected patients from Sierra Leone, we explored mechanisms of Ebola pathogenesis and differences in host responses between patients that survived or succumbed to infection.
Results: Utilizing a 5’ end “cap-snatch” sequencing technique, CS-Seq, we characterized RNA kinetics throughout infection across cellular compartments. We not only comprehensively profiled viral transcripts but, unlike previous studies, we also quantified the host response at the same time. Analysis of viral mRNAs produced by cap-snatching revealed RdRP’s preference for host transcripts starting with a purine, and the reciprocal avoidance of pyrimidine-rich 5’ TOP genes that play an important role in translation. These nucleotide preferences were conserved amongst other cap-snatching viruses, despite differences in viral polymerases and their subcellular localization. Furthermore, we show that cap-snatching primarily targets short nascent mRNAs (<200nt), as longer transcripts are protected by the cellular Cap Binding Complex. This same mechanism also ensures that viral mRNAs are protected from self-cleavage. We then go on to show that the addition of a short host-oligo increases the probability of introducing a start codon prior to the native start codon, allowing the production of extended viral proteins or out-of-frame products from upstream ORFs (uORFs). The detection of the products of one of these uORFs in mass spectrometry data indicates that the cap-snatching mechanism has the potential to increase viral protein diversity. Because these proteins have not been characterized, they open up new avenues of investigation regarding their role in infectivity. Lastly, we were able to acquire PBMC RNA samples from patients in Sierra Leone’s 2014 Ebola outbreak to study host transcriptional response to infection. By employing gene co-expression network analysis, we were able to observe differential host-immune responses to Ebola infection, correlate them with patient outcome and support them with other OMICs data from the same patients.
Conclusions Genomic approaches in the form of sequencing methods and analysis pipelines have provided valuable insight into virus-host interactions that may ultimately result in new targets for drug discovery
|Advisor:||van Bakel, Harm|
|Commitee:||Brown, Brian, Garcia-Sastre, Adolfo, Medina, Rafael, Pinto, Dalila, Sharp, Andrew|
|School:||Icahn School of Medicine at Mount Sinai|
|Department:||Genetics and Genomic Sciences|
|School Location:||United States -- New York|
|Source:||DAI-B 79/02(E), Dissertation Abstracts International|
|Keywords:||Cap-snatching, Influenza, Rna-seq, Transcription|
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