Targeting the chromatin environment of the influenza virus transcriptase to interfere with the virus cycle – FluChromatin

The objective of the present proposal is to determine how the transcriptase machinery of Influenza A virus (IAV) is targeted to chromatin, to explore the global impact of the virus on host chromatin structure, and to gain preliminary insight on the effect of a selection of epi-drugs on the viral cycle.
The 2009 A/H1N1 pandemic and the recent species barrier crossings of avian influenza A infection in human have highlighted the unpredictability and potential threat for human health posed by influenza viruses. Despite numerous studies, many aspects associated with viral genome transcription and replication remain unexplored. Fundamental knowledge on these issues is needed for improving vaccine efficiency, anti-viral drug discovery and diagnostics tools to protect from a new severe pandemic provoked by a pathogenic influenza.
IAV is a negative-sense single-stranded RNA virus. Its genome consists of eight genomic segments individually encapsidated into ribonucleoprotein (RNP) complexes. Each RNP is comprised of a single heterotrimeric RNA-dependent RNA-polymerase (RdRp) bound to the complementary viral RNA termini and multiple copies of the viral nucleoprotein (NP) thus forming an intricate RNA-protein complex. In contrast to many RNA viruses, the influenza virus genome is transcribed and replicates in the nucleus of the infected cell. Viral transcription involves a cap-snatching function to steal short 5’-capped RNA primers (10-15 nt) from host RNAs through association of a RdRp subunit with the cellular RNA polymerase II. Intriguingly, noncoding host RNAs seem the preferred cap-snatching source over mRNAs or pre-mRNAs, suggesting a certain degree of specificity in the binding of the RdRp either to the snatched RNAs or to the site of snatching. The snatching of non-coding RNAs also suggests a possible impact of the snatching on the chromatin structure.
Thus, in this collaborative FluChromatin project, we will use state of the art genome-wide and proteomic approaches to characterize the chromatin environment required for (primary) viral transcription, determining which functional domains (promoters, enhancers, etc) are preferred by RdRp when binding to chromatin, identifying histone modifications possibly involved in the targeting, and screening for novel interactions between viral proteins and chromatin components. Furthermore, we will use genome-wide approaches to explore the impact of RdRp recruitment on chromatin compaction/DNA accessibility and the subsequent impact on the expression of host defense genes. Finally, we will evaluate the potency of selected small molecules active on chromatin (epi-drugs) in interfering with the life cycle of the virus.
The collected data is expected to put the RdRp-chromatin interactions in a completely new perspective, with possible insight in unsuspected viral strategies to highjack host defense mechanisms. We also anticipate that our studies will provide new approaches in the field of anti-influenza drug discovery and attenuated live vaccines.