Conclusion and Future Perspectives

The present work includes new insights into the transcription and translation of the influenza A virus and points the way to an quantitative and temporal characterization of new as well as previously described virus-host interaction pathways and hypotheses behind the mystery how viruses control cellular processes.

Apart from a previous proteomic study using SILAC to analyse the global changes in host protein abundance upon influenza A virus infection [244] 12 h p.i. this work revealed a strong regulation of distinct proteins rather than a complete decrease in host cell protein levels. In particular, the dynamics in protein expression during the first 6 to 8 h of the viral infection (see figure 40) indicate strong viral host interaction mechanisms where the virus hijacks cellular processes and resources to efficiently replicate. The question how the in this work identified strong regulation of antiviral response proteins like SERPIN B6, CAD, SF1 and /or ZER 1 is controlled and imbedded into this interaction network remains to be further investigated.

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The innovative FIT-PNA technique enables detailed temporal, spatial and quantitative investigation on different viral mRNA molecules in living cells simultaneously. This sequence specific method is applicable to a wide range of viruses, in particular with mRNA intermediate step, and nucleic acid variants. The relevance of these probes for nucleic acid imaging is reflected by the article in ChemBioChem written by Jens Tilsner and Cristina Flors 2011 [245] highlighting our publication on influenza A virus mRNA imaging in living infected cells using FIT-PNAs [183]. They assume that studies in cellular complex environments benefit from the high specificity and increased sensitivity of FIT-PNAs and conclude that these probes might be suitable for single-molecule approaches.

For the first time a time-resolved analysis of the cellular and viral proteomic dynamics in the early stages of influenza A virus infection in MDCK cells was performed. The recently by Schwanhäußer et al. 2009 [246] described pulsed SILAC technique might improve the flexibility and temporal resolution. This method enables the direct and quantitative determination of protein translation and turnover rates on a proteome-wide scale. In concert with data obtained from single cell studies using FIT-PNAs this global approach might complete an overall description of the host and influenza A proteome dynamics in time and space.

Further studies could focus on the detailed analysis of functional protein phosphorylation, nucleic and cytosolic protein fractionation, other cell types and virus strains. In particular, pandemic variants compared to seasonal flu causing strains might identify unique features of highly pathogenic viruses and help to improve antiviral therapies.

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Whether the presented results obtained in an immortalized cell line can be applied to the in vivo situation remains to be clarified. Pan et al. [194] ruled out that the lack of tissue architecture and a heterogeneous population of different cell types in cell culture generally impedes cell-cell interaction and other functions based on tissue context. Therefore cells in culture might acquire a molecular phenotype varied from cells in vivo that requires a careful choice of the most appropriate experimental system.  

An improvement of the quantitative analysis to determine the fluorescence intensity of FIT-PNAs in cells might be achieved by automation of the measurement. This requires the identification of cells by the software and the recognition of cell borders to distinguish between the cytosol and the background.


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