Sizun, C. | bioRxiv Science

bioRxiv2021

Respiratory Syncytial virus NS1 protein targets the transactivator binding domain of MED25

Respiratory syncytial virus has evolved a unique strategy to evade host immune response by coding for two non-structural proteins NS1 and NS2. Recently it was shown that in infected cells, nuclear NS1 could be involved in transcription regulation of host genes linked to innate immune response, via an interaction with chromatin and the Mediator complex. Here we identified the MED25 Mediator subunit as an NS1 interactor in a yeast two-hybrid screen. We demonstrate that NS1 directly interacts with MED25 in vitro and in cellula, and that this interaction involves the C-terminal 3 helix of NS1 and the MED25 ACID domain. More specifically we showed by NMR that the NS1 3 sequence primarily binds to the MED25 ACID H2 face, which is a transactivation domain (TAD) binding site for transcription regulators such as ATF6, a master regulator of ER stress response activated upon viral infection. Moreover, we found out that the NS1 3 helix could compete with ATF6 TAD binding to MED25. This finding points to a mechanism of NS1 interfering with innate immune response by impairing recruitment by cellular TADs of the Mediator via MED25 and hence transcription of specific genes by RNA polymerase II. ImportanceHuman RSV is the leading cause of infantile bronchiolitis in the world and one of the major causes of childhood deaths in resource-poor settings. It is a major unmet target for vaccines and anti-viral drugs. RSV non-structural protein NS1 is known to antagonize the cellular immune response and was recently shown to be involved in transcription regulation of infected cells. However, the exact mechanism of this regulation is not well defined. Here we show that nuclear NS1 interacts directly with the Mediator subunit MED25 and is able to compete with a cellular transcription activator, which is activated during viral infection. We hypothesize that this interaction may underlie regulation of the expression of genes involved in the innate immune response.

microbiology↗

bioRxiv2020

Tetramerization of Phosphoprotein is essential for Respiratory Syncytial virus budding while its N terminal region mediates direct interactions with the Matrix protein.

It was shown previously that the Matrix (M), Phosphoprotein (P), and the Fusion (F) proteins of Respiratory syncytial virus (RSV) are sufficient to produce virus-like particles (VLPs) that resemble the RSV infection-induced virions. However, the exact mechanism and interactions among the three proteins are not known. This work examines the interaction between P and M during RSV assembly and budding. We show that M interacts with P in the absence of other viral proteins in cells using a Split Nano Luciferase assay. By using recombinant proteins, we demonstrate a direct interaction between M and P. By using Nuclear Magnetic Resonance (NMR) we identify three novel M interaction sites on P, namely site I in the N2 region, site II in the 115-125 region, and the oligomerization domain (OD). We show that the OD, and likely the tetrameric structural organization of P, is required for virus-like filament formation and VLP release. Although sites I and II are not required for VLP formation, they appear to modulate P levels in RSV VLPs. ImportanceHuman RSV is the commonest cause of infantile bronchiolitis in the developed world and of childhood deaths in resource-poor settings. It is a major unmet target for vaccines and anti-viral drugs. The lack of knowledge of RSV budding mechanism presents a continuing challenge for VLP production for vaccine purpose. We show that direct interaction between P and M modulates RSV VLP budding. This further emphasizes P as a central regulator of RSV life cycle, as an essential actor for transcription and replication early during infection and as a mediator for assembly and budding in the later stages for virus production.

microbiology↗