Reduced Nucleoprotein availability impairs negative-sense RNA virus replication and promotes host recognition

Negative-sense RNA viruses (NSVs) rely on prepackaged viral RNA-dependent RNA polymerases (RdRp) to replicate and transcribe their viral genomes. Their replication machinery consists of an RdRp bound to viral RNA which is wound around a nucleoprotein (NP) scaffold, forming a viral ribonucleoprotein complex. NSV NP is known to regulate transcription and replication of genomic RNA, however its role in maintaining and protecting the viral genetic material is unknown. Here, we exploited host microRNA expression to target NP of influenza A virus and Sendai virus to ascertain how this would impact genomic levels and the host response to infection. We find that in addition to inducing a drastic decrease in genome replication, the antiviral host response in the absence of NP is dramatically enhanced. Additionally, our data shows that insufficient levels of NP prevent the replication machinery of these NSVs to process full-length genomes, resulting in aberrant replication products which form pathogen-associated molecular patterns in the process. These dynamics facilitate immune recognition by cellular pattern recognition receptors leading to a strong host antiviral response. Moreover, we observe that the consequences of limiting NP levels are universal amongst NSVs including Ebola virus, Lassa virus and Measles virus. Overall, these results provide new insights into viral genome replication of negative-sense RNA viruses and highlight novel avenues towards developing effective antiviral strategies, adjuvants, and/or live-attenuated vaccines. Independent biological duplicates of Lung adenocarcinoma-derived cell lines (A549) were mock treated or infected with wild-type IAV (A/Puerto Rico/8/1934 (H1N1)), SeV (Fushimi), or recombinant IAV and SeV containing a microRNA cassette targeting their correspondant NP/N mRNAs. Additionally, Independent biological triplicates of Lung adenocarcinoma-derived cell lines (A549) transduced with a vector expressing human ACE2, were transfected with siRNAs targeting the SARS-CoV-2 N mRNAs (or a scramble control) and infected with SARS-CoV-2 (USA-WA1/2020).