engleski

MCMV transcriptome analysis and identification of a new, highly abundant transcript with multiple coding and non-coding functions

Human cytomegalovirus (HCMV) is an important human pathogen, infecting over 70% of human population and responsible for devastating congenital disease and organ transplant failure in immunologically immature or suppressed patients. Unfortunately, it infects only humans and is therefore hard to study. Murine cytomegalovirus (MCMV) is the most widely used model to study HCMV) pathogenesis in vivo since it shares many biological and pathological features and is genetically related to HCMV. Functions of many of its genes, especially immunoevasins, have been characterized thanks to plethora of deletion mutants. Although generation of deletion mutants relies on genomic maps, our currently used map of MCMV genome has many drawbacks. Current annotations were constructed using bioinformatics tools to predict ORFs with verification by genomic tilling arrays. Such analyses are poorly adapted to condensed viral genomes that contain many overlapping genes and can therefore miss ORFs or rare alternative splicing sites. With this in mind we have set to analyze the transcriptome of MCMV using two different approaches: cDNA library and deep sequencing. Based on 448 cDNA clones and deep sequencing data we have constructed a map of MCMV transcriptome which differs significantly with currently used annotations. We have detected multiple instances where transcriptional products differed from predicted ORFs (longer or shorter, transcripts overlapping several predicted ORFs), new splice variants, transcripts in regions annotated as non-coding and coding regions with very low transcription. While we have detected several antisense transcripts, the level of antisense transcription was significantly lower than was observed for HCMV. Surprisingly, one transcript dominated MCMV transcriptome comprising over 1/3 of all transcripts. Despite its high abundance, its function was unknown. 1.7 kb in length, most abundant transcript (MAT) overlaps predicted m168 in antisense and m169 in sense orientation. We have previously observed that this region of MCMV genome is essential for activating Ly49 (P, L and D2) recognition of m04/MHC I complexes. We have shown that MAT transcript encodes poorly translated 17 kDa protein of unknown function and contains binding site for cellular microRNA miR-27 in its 3’-UTR leading to its degradation. Due to MATs abundance, it effectively depletes cellular levels of miR-27 upon infection. To our knowledge MAT is the first transcript described that contains both coding and non-coding functions. Interestingly neither function of miR sponge nor the produced protein are responsible for recognition by activating Ly49 receptors but 5’-UTR. Mechanisms by which 5’UTR mediates activating Ly49 recognition is currently being investigated.

cytomegalovirus transcriptomics MCMV RNASeq expression immunoevasion NK cells

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