Genome-Wide Screen Identifies RTF2 as a Host Restriction Factor That Restricts Influenza A Virus Replication

Abstract

Viral infection triggers the secretion of type I interferons, which in turn induce expression of hundreds of genes. However, the roles and molecular mechanisms of these induced genes in the context of antiviral immunity remain largely unknown. This has limited our ability to develop host-based antiviral therapeutics against pathogenic viruses such as influenza virus, which causes annual epidemics and recurring pandemics. While recent work has identified antiviral factors that are sufficient to restrict viruses when these factors are overexpressed, the question of whether these factors are indeed inhibiting viruses in a physiological context remains unanswered. Here, we performed a loss-of-function genetic CRISPR screen in cells pre-stimulated with type I interferon to identify antiviral genes that restrict influenza A virus replication. In addition to the key components of the interferon signaling pathway, we found a new factor, Replication Termination Factor 2 (RTF2). Our data reveal that RTF2 restricts influenza, at least, at the nuclear stage of the viral life cycle based on several lines of evidence. First, a deficiency in RTF2 leads to higher levels of viral primary transcription, in the presence of cycloheximide to block genome replication and secondary transcription. Second, cells that lack RTF2 have enhanced activity of a viral reporter that depends solely on four viral proteins that carry out replication and transcription in the nucleus. Third, when RTF2 protein is mislocalized outside the nucleus, it is not able to restrict replication. In addition to restricting transcription, the absence of RTF2 reduces expression of antiviral factors in response to interferon. RTF2 thus inhibits influenza primary transcription, likely acts in the nucleus, and contributes to upregulation of antiviral effectors in response to type I interferons. Influenza A virus remains a global health threat, with an estimated 3-5 million cases of severe illness, and 0.29-0.65 million deaths worldwide annually. This work contributes to the field of antiviral immunology by discovering and characterizing a novel restriction factor of influenza, and may ultimately be useful for understanding how to control a virus that causes significant morbidity and mortality worldwide.