Discussion
Influenza A virus has caused several outbreaks of influenza in human history, including the 2009 H1N1 Influenza pandemics. As a single-strand negative-strand, segmented RNA virus, its high mutation rate and gene rearrangement properties can continuously produce new virus subtypes, imposing a long-term threat to human and animal health. Swine have receptors for both human influenza (SAα-2,6-Gal) and avian influenza (SAα-2,3-Gal), hence are referred to as ”mixers” of influenza viruses (Castrucci, 1993). They play an important role in the recombination of influenza viruses. In 2009, the H1N1pdm09 IV pandemic broke out in the world, and researchers found that many of its gene fragments are derived from SIV(Vincent ,2014). In addition, the H1N1 subtype SIV has always maintained a high prevalence rate in the pig herd (Sadler, 2011), and has been involved in the recombination of a variety of new viruses during its evolution (Zimmermann, 2011). The experimental strain used in the present work was collected from a sick pig farm, and shows homology of more than 90% with the 2009 human epidemic strains. Therefore, our work is of great significance for exploring the pathogenic mechanism of H1N1.
Influenza A virus ribonucleoprotein (vRNP) complex is the structural basis of viral RNA transcription and replication, and plays an important role in the process of virus infection (Chutiwitoonchai , 2016). During the early stage of viral infection, the vRNP complex is released into the cytoplasm of the host cell via endocytosis and demembrane, and then enters the nucleus with the help of nuclear localization signals and transport proteins (Wang, 1997). To date, several host proteins have been reported to be potential binding partners of the vRNP complex, and regulate viral infection through various mechanisms (Davis ,2017). For example, PLSCR1 negatively regulates virus replication by interacting with NP in the cytoplasm and preventing its nuclear import in vitro (Luo, 2018). In our present work, we established Plscr1knockout mice and found that it is indeed more susceptible to SIV. By examining the survival rate, body weight and lung virus load of the mice, we presented in vivo evidence for the first time that PLSCR1 is important for inhibiting influenza virus replication.
Moreover, we identified ILDR1 as a novel PLSCR1-binding partner. ILDR1 has been shown to mediate fat-stimulated cholecystokinin (CCK) secretion (Chandra ,2013) and play an important role in regulating the integration of tTJs(Higashi ,2015). Ildr1 knockout mice show profound hearing loss accompanied with tTJs destruction in the inner ear (Mehrjoo ,2015; Morozko ,2015) and polyuria due to renal concentrating defects in kidneys (Hempstock ,2020). ILDR1 is a putative type I transmembrane protein containing an immunoglobulin (Ig)-like domain (Hauge ,2004). ILDR1 belongs to angulin protein family, which also includes immunoglobulin-like domain-containing receptor 2 (ILDR2) and lipolysis-stimulated lipoprotein receptor (LSR). The immunomodulatory effect of its homologous protein ILDR2 has been extensively studied in recent years. It has been confirmed that ILDR2 is a new B7 family protein, expressed in immune cells and inflammatory tissues, and has the activity of suppressing T cells (Hecht ,2018). ILDR2-Fc regulates the function of immunity in the treatment of autoimmune diseases by regulating the stability of the immune internal environment and rebuilding the balance of immune tolerance (Podojil ,2018).In our previous study, we detected high expression of ILDR1 in lungs of mice (Liu, 2017). However, its function in the lungs and whether it participates in disease infection has not been reported so far. Therefore, we observed the role of ILDR1 in influenza virus infection. We first determined the level of ILDR1 after H1N1 SIV infection by western blot and immunohistochemistry. The results showed that ILDR1 levels significantly increased by more than 15-fold on the first day after infection and it decreased as the viral load decreased. The same results were obtained using Plscr1 knockout mice. Moreover, we examined the effect of ILDR1 overexpression on virus infection, and confirmed that virus replication was significantly promoted and the viability of virus-infected cells is decreased, suggesting that ILDR1 might play a role in Influenza virus replication.
ILDR1 could interact with PLSCR1, which has been reported to impair nuclear import of influenza A virus NP. To explore whether ILDR1 affects virus infection through regulating NP importation, we first detected the interaction between ILDR1 and NP using co-IP. The results show that PLSCR1 could interact with NP protein as previously reported, but ILDR1 could not directly interact with NP. Then, the effect of ILDR1 and NP on PLSCR1 was determined, and we found that they could bind NP protein competitively. At present it remains unclear why ILDR1 affects the replication of influence virus, and what role does it play in other virus? Further investigation is needed to clarify the mechanism by which ILDR1 regulates viral replication.