Dissecting the Alternative Splicing mechanism of influenza virus: a new host-targeted antiviral strategy

Influenza A Virus (IAV) is one of the most important causes of respiratory infections and has caused tens of millions of deaths in several pandemic outbreaks throughout human history. In recent years, the recurrence of influenza epidemics in poultry and swine flocks has posed a serious threat to public health safety, thus, research on the prevention, control, and treatment of influenza viruses is of great significance.

 

Alternative splicing of genes is an important mechanism for eukaryotic cells to regulate gene expression and produce protein diversity. When an influenza virus infects a host cell, the virus hijacks the host cell's "splicing machinery" to complete variable splicing of the viral genome, resulting in the production of a variety of proteins essential for viral growth and reproduction, such as M2, M42, and NS2. According to research, the splicing efficiency of these proteins not only determines viral replication and pathogenicity, but also has a significant impact on the host range of influenza viruses infected.

 

Interfering with viral gene splicing is thus the treatment for viral infections. Currently available anti-influenza viral drugs target viral proteins, but viral gene mutation and drug resistance occur occasionally. Parsing the replication process of influenza viruses in the host and screening for drugs that target essential host factors for IAV infection, as a result, holds promise for new viral infection treatment strategies.

 

Prof. Jinhua Liu's team from China Agricultural University recently published a paper in the journal Advanced Science titled "SRSF5-mediated alternative splicing of M gene is essential for influenza A virus replication: a host-directed target against influenza virus". The study elucidates the mechanism by which the host splicing factor SRSF5 protein regulates the variable splicing of influenza A virus's M2 gene, as well as screens and validates small molecule anti-influenza virus drugs targeting SRSF5.

 

The researchers used RNA pull-down combined with mass spectrometry to screen key host RNA-binding proteins in the IAV life cycle and discovered that several proteins in the SRSFs family of host splicing factors can bind influenza virus RNA. Following that, the researchers used conditional knockout mice and knockout cell lines to confirm that SRSF5 is a key host factor regulating IAV infection and replication, and discovered that SRSF5 protein regulates variable splicing and expression of the viral M2 gene. Knocking out the srsf5 gene significantly reduced the variable splicing of the viral M2 gene, as well as the virus's replication ability in cells and pathogenicity in mice.

 

Further investigators found by RNA Pulldown, MST, and RNA FISH that the SRSF5 protein can bind directly to viral M pre-mRNA and complete variable splicing of the viral M gene by recruiting the core subunit U1-A of the host splicing complex U1 snRNP to the influenza virus M pre-mRNA, resulting in the production of the important ion channel protein M2. When the viral M gene is mutated in the RNA motif that binds to the SRSF5 protein, the binding ability of viral M mRNA to the host splicing complex is significantly reduced, and the splicing and expression of the M2 gene is severely impaired, thus affecting viral infection and replication. On this basis, the investigators virtually screened the FDA drug library for small molecule drug inhibitors targeting SRSF5 by molecular docking, and evaluated and validated the antiviral effects of small molecule drugs against IAV in vitro and in vivo.

 

This study identified the key host splicing factors that regulate IAV variable splicing, screened small molecule drug inhibitors targeting the viral splicing process, and validated their antiviral effects in vivo and in vitro, resulting in a new solution for influenza prevention, control, and treatment.