Figure 2. Construction of Plscr1 knockout mice. (A) The schematic drawing of the strategy for Plscr1 gene disruption. The target sites of clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 small guide RNAs (sgRNAs) in the Plscr1 gene are indicated in red, and the deleted region in the Plscr1 gene of knockout mice is indicated by dashes. The positions of RT-PCR primers are indicated by arrows. (B) The schematic drawing of the domain structure of PLSCR1 in wildtype and knockout mice. (C)The expression ofPlscr1 mRNA in the tail of P42 mice was determined by RT-PCR. β-actin was included as the internal control. (D) Western blot showed the expression in the liver and lung of P42 mice. (E) PLSCR1 was detected by immunohistology using rabbit anti-mPLSCR1, visualized with 3,3,-diaminobenzidine, and counterstained with hematoxylin. Scale bar: 20 μm. +/+:wild type mice; +/-: heterozygous mice; -/-:Plscr1knockout mice.

We then examined whether Plscr1 knockout affects H1N1 SIV infection. Control and Plscr1 ^-/- mice were infected intranasally with 10³ pfu H1N1 SIV, and the survival rate, body weight and lung virus load were determined over a 28-day period. The results show that the survival rate ofPlscr1 ^-/- mice is much lower than that of control mice after SIV infection (Figure 3A).Plscr1^-/- mice start to die at 3 dpi, and the survival rate drops to 50% at 28 dpi. Meanwhile, similar body weight was observed between survived Plscr1 ^-/- mice and control mice (Figure 3B). Consistent with the decreased survival rate, higher lung viral load was observed inPlscr1 ^-/- mice (Figure 3C). Titers of SIV increase in the lungs of both genotypes and peak at 5 dpi, then start to decrease and not detectable at 14 and 28 dpi. Virus titers are significantly higher in Plscr1 ^-/- mice at all time points examined (Figure 3C). Consistently, western blotting shows that the level of NP in Plscr1^-/- mice was significantly higher than that in wild mice at 3 dpi (Figure 3D and 3E). Taken together, our present data suggest that H1N1 SIV replication is increased in Plscr1 knockout mice that leads to lower survival rate.