engleski

The transcriptome of herpes simplex virus - focus on non-coding RNAs

Herpes simplex virus 1 (HSV-1) is a human pathogen with a very complex life cycle in two phases. The primary productive phase is characterised by intensive gene expression and replication of the virus at the site of the infection, most often oral mucosa. In the second phase, the virus migrates to sensory neurons and establishes latency, a dormant state in which the majority of HSV-1 genes are suppressed. Non- coding RNAs (ncRNA), including latency associated transcripts (LATs) and microRNAs (miRNAs), are the only abundantly detected gene products in latency. The exact function of ncRNAs for HSV-1 replication is poorly understood. Our long-term aim is to reveal the complete transcriptome of HSV-1, with a specific focus on regulatory ncRNAs and their functions. Firstly, to generate a comprehensive map of the HSV-1 transcriptome, we utilized massive parallel sequencing on Illumina platform (NextSeq 500) of RNA extracted from cells infected with HSV-1 at different time points after infection. Our work in progress shows that we generated a high quality sequence dataset based on the FastQC quality control tools (e.g. per sequence quality, per base sequence content, per base N content, etc.). The obtained sequence-reads we mapped to HSV-1 genome using TopHat spliced junction mapper (CCB - Johns Hopkins University), tool which aligns the obtained sequencing reads to genome. The aligned reads were visualised with the Integrative Genomics Viewer – IGV (Broad Institute), a visualisation tool suitable for large datasets, together with previously annotated HSV-1 open reading frames (ORFs). Currently we are comparing our sequence reads to the reference annotation using Cufflinks (Trapnell Lab), a suite of tools for transcriptome assembly and differential analyses of RNA-sequencing data. Of note, long ncRNAs (lncRNAs) and miRNAs, since newly discovered, were not included in the previous transcriptome analyses. In addition, several new miRNAs have been discovered lately, which we failed to detect in our previous studies. Thus, to generate the complete map of HSV-1 transcriptome, we are currently performing a re-analyses of our datasets for the presence of all currently described HSV-1 miRNAs. We hypothesize that some described miRNAs are not genuine regulatory molecules, but rather degradation products or artefacts. To test this hypothesis we are currently analysing miRNAs bound to the RNA silencing complex (RISC).

Transcriptome, herpes simplex virus

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