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.