Influenza A virus induced cellular and viral microRNAs in viral replication and host defence
Final Report Abstract
Within the scope of this grant we were able to uncover the reasons for severe biases observed in high throughput sequencing of small non-protein coding RNAs (npcRNAs). The use of different adapters and barcodes during ligation as well as complex RNA structures and modifications drastically influence cDNA synthesis efficacies. In addition, variable specific RNA G/C-content is associated with unequal polymerase chain reaction amplification efficiencies. This knowledge leads to enhanced reproducability of RNA deep sequencing approaches and helps to evaluate relative changes in the quantification of the resulting data. Furthermore, for verification of true RNA abundance, RNA-seq data need to be validated by an additional method such as quantitative real-time PCR (qRT-PCR). The selection of suitable internal controls for data normalization is pivotal to reduce errors caused by experimental variations. Therefore, we evaluated and proposed a set of ubiquitously expressed housekeeping npcRNAs as an ‘Expression Ruler’ that allows to cover a wide expression range for analysis of novel or yet poorly characterized transcripts. We envisage that this multiple reference gene concept will be a valuable tool for expression analysis and validation of the human transcriptome. Applying this RNA-seq method on npcRNAs obtained from influenza infected cells, we uncovered a few miRNAs that were differentially regulated upon infection. Interestingly, all let-7 family members detected were downregulated. The inhibition of members let-7b, -7g, or -7i resulted in reduced viral replication and a suppression of viral protein expression. Let-7 family members already have been linked to cytokine expression under certain conditions. Therefore, downregulation of let-7 miRNAs might be part of a defense mechanism of the host, thereby enhancing the expression of pro-inflammatory cytokines. However, in the case of highly pathogenic avian influenza viruses such as H5N1, where the downregulation of let-7 family members was most elevated, suppressed miRNA expression might contribute to overwhelming cytokine responses leading to severe immunopathology. Therefore, modulation of let-7 miRNA expression might be a promising tool to treat influenza infections. In addition, we uncovered the presence of a so far unknown viral RNA derived from the viral genomic PB2 segment of a highly pathogenic H5N1 influenza A virus. This PB2Δ RNA belongs to the group of defective interfering RNAs that are generated due to error-prone polymerase function. This RNA encodes a 10 kDa protein sharing the N-terminal amino acid sequence of the parental PB2 protein followed by frameshift after internal deletion. We provided evidence that the PB2Δ protein induces the expression of interferon (IFN) beta and that of antivirally acting IFN-stimulated genes by direct interaction with the cellular adapter protein MAVS, a key component of the IFN-inducing RIG-I pathway. Thus, IFN-sensitive viruses such as influenza or vesicular stomatitis virus show reduced viral replication in the presence of PB2Δ. This is the first time that such a function was described for a DI RNA-encoded protein, a finding that has several important implications with regard to deciphering viral protein functions and options for immunestimulatory approaches. Furthermore, this is an example of how influenza viruses may acquire novel polypeptides with altered functions from its limited genome.
Publications
- (2011): The rocks and shallows of deep RNA sequencing: Examples in the Vibrio cholerae RNome. RNA 17(7): 1357-66
Raabe CA, Hoe CH, Randau G, Brosius J, Tang TH and TS Rozhdestvensky
- (2013): Einfluss zellulärer und viraler Faktoren auf die Virulenz hochpathogener aviärer Influenza A Viren
Börgeling Y
- (2014) Differential regulation of non-protein coding RNAs from Prader-Willi Syndrome locus. Scientific Reports, volume 4, Article number: 6445 (23 September 2014), 9 S.
Chenna R. Galiveti, Carsten A. Raabe, Zoltán Konthur & Timofey S. Rozhdestvensky
(See online at https://doi.org/10.1038/srep06445) - (2014): Biases in small RNA deep sequencing data. Nucleic Acids Res. 42(3): 1414-26
Raabe CA, Tang TH, Brosius J and TS Rozhdestvensky
(See online at https://doi.org/10.1093/nar/gkt1021) - (2014): Inhibition of p38 mitogen-activated protein kinase impairs influenza virus-induced primary and secondary host gene responses and protects mice from lethal H5N1 infection. J Biol Chem 289: 13-27
Börgeling Y, Schmolke M, Viemann D, Nordhoff C, Roth J and S Ludwig
(See online at https://doi.org/10.1074/jbc.M113.469239) - Evidence for a novel mechanism of influenza virus-induced type I interferon expression by a defective RNA-encoded protein. (PLoS Pathogens 11(5): e1004924)
Yvonne Boergeling, Timofey S. Rozhdestvensky, Mirco Schmolke, Patricia Resa-Infante, Thomas Robeck, Gerrit Randau, Thorsten Wolff, Gülsah Gabriel, Jürgen Brosius, Stephan Ludwig
(See online at https://doi.org/10.1371/journal.ppat.1004924)
