Scientia Agricultura Sinica ›› 2022, Vol. 55 ›› Issue (10): 2067-2076.doi: 10.3864/j.issn.0578-1752.2022.10.016

• ANIMAL SCIENCE·VETERINARY SCIENCE·RESOURCE INSECT • Previous Articles    

Mechanism of NMRAL1 Regulating Influenza Virus Replication

YAN Ya(),WANG GuangWen,KONG FanDi,WANG XuYuan,WANG YiHan,LI JunPing,ZHAO YuHui,LI ChengJun,CHEN HuaLan,JIANG Li()   

  1. State Key Laboratory Veterinary Biotechology/Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150069
  • Received:2021-03-09 Accepted:2021-05-14 Online:2022-05-16 Published:2022-06-02
  • Contact: Li JIANG E-mail:yanhs2019@163.com;jiangli@caas.cn

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Abstract:

【Objective】Influenza virus is a zoonotic pathogen that often causes a pandemic and poses a great threat to human health, and the influenza viruses are prone to variants and can constantly escape the host cell immune response and develop resistance to existing anti-influenza drugs, so the search for new ways to fight influenza is imminent. This study aimed to explore the effect of NMRAL1 (NmrA-like family domain-containing protein 1) on influenza virus replication, and to reveal the molecular mechanism by which it functioned, so as to provide a potential target for anti-influenza drugs development. 【Method】In this study, siRNA interference technology was used to down regulate the expression of NMRAL1 in A549 cells, and the expression levels of NMRAL1 were detected by Western Blot. Virus titers in cell supernatants at 24 h and 48 h after infection with two different subtypes influenza viruses, including a/Anhui/ 2/2005(AH05)(H5N1) and a/WSN/33(H1N1), were detected using the plaque assay. To determine the specific stage at which NMRAL1 affected influenza virus replication, NMRAL1 was overexpressed by transiently transfecting NMRAL1-Myc-pCAGGS plasmid in HEK293T cells, and the effect of overexpressing NMRAL1 on influenza virus polymerase activity was examined by luciferase reporter system. The influenza virus NP protein was stained by using immunofluorescence, and the down-regulated expression of NMRAL1 on the localization of NP protein at 3, 4, 5, 6 and 8 h post infection was assessed respectively by confocal assay to determine whether down-regulated expression of NMRAL1 affected the process of influenza virus vRNP import and export. Western Blot was used to detect the effect of NMRAL1 knockdown on the expression of viral proteins and on the expression of IFN stimulated genes (ISGs) downstream of type I interferon pathway activated by influenza virus. Indirect immunofluorescence assay was utilized to further verify the effect of NMRAL1 on influenza virus replication. 【Result】Western Blot assay showed that NMRAL1 siRNA could significantly down regulate NMRAL1 expression in A549 cells. With the down-regulated expression of NMRAL1, A549 cells were infected with H5N1 and H1N1 viruses, respectively. Then the virus titers in the cell supernatant were measured by plaque assay, which showed that the virus titers in the supernatant of cells at 24 and 48 h after infection with H5N1 or H1N1 were significantly decreased, meaning that NMRAL1 could promote the replication of different subtypes influenza viruses. To further explore the specific mechanism by which NMRAL1 regulated influenza virus replication, a luciferase reporter system was used to detect influenza virus polymerase activity, and it was found that the overexpression of NMRAL1 had no effect on influenza virus polymerase activity. The results of confocal assay showed that the down-regulated expression of NMRAL1 did not affect the process of NP nuclear import and export, meanwhile Western Blot assay indicated that down-regulated expression of NMRAL1 did not affect the expression of each viral protein. However, the results of the fluorescence quantitative PCR assay showed that down-regulated expression of NMRAL1 was able to promote the up-regulation of IFN-β mRNA levels induced by influenza virus infection, and Western Blot assay found that down expression of NMRAL1 promoted the expression of MxA and IFITM3 antiviral proteins downstream of type I interferon pathway. Meanwhile, the indirect immunofluorescence assay showed that the down expression of NMRAL1 could significantly inhibit influenza virus replication. 【Conclusion】 Those results demonstrated that, during influenza virus infection, NMRAL1 did not affect the process of influenza virus invasion as well as transcription translation, but rather inhibited the expression of antiviral factors, such as MxA and IFITM3, by inhibiting type I interferon pathway activation, which ultimately promoted influenza virus replication. This study confirmed that the host factor NMRAL1 positively regulated influenza virus replication and enriched the network of host factors involved in influenza virus replication.

Key words: NMRAL1, influenza virus, virus replication, interferon β, antiviral gene

Table 1

Primers and siRNA sequences"

引物Primers/siRNA 序列Sequence (5'-3')
NMRAL1-Myc F CCGGAATTCATGGTGGACAAGAAACTGGTGGTGG
NMRAL1-Myc R CTAGCTAGCCAGCAGGTTGAAGTCCCCTTTGTG
NMRAL1 RT F ACGCCACCTTCATCGTGAC
NMRAL1 RT R AACGCCAATGTCCCGGAAAT
IFN-β RT F TCTGGCACAACAGGTAGTAGGC
IFN-β RT R GAGAAGCACAACAGGAGAGCAA
GAPDH RT F GGAGCGAGATCCCTCCAAAAT
GAPDH RT R GGCTGTTGTCATACTTCTCATGG
NMRAL1 siRNA CCUUCAUCGUGACCAAUUAtt
Scrambled siRNA UUCUUCGAACGUGUCACGTtt

Fig. 1

Effect of NMRAL1 knockdown on influenza virus replication titer in A549 cells A: siRNA knockdown effect of NMRAL1 in A549 cells detected by Western Blot; B: Effect of NMRAL1 knockdown on H5N1 influenza virus replication titer in A549 cells; C: Effect of NMRAL1 knockdown on H1N1 influenza virus replication titer in A549 cells"

Fig. 2

Effect of up-regulated NMRAL1 expression on influenza virus polymerase activity in HEK293T cells"

Fig. 3

Effect of NMRAL1 knockdown on the NP nuclear import and export in A549 cells（Bars: 50 μm）"

Fig. 4

Effect of NMRAL1 knockdown on the expression of viral proteins in A549 cells"

Fig. 5

Effect of NMRAL1 knockdown on the IFN-β expression during influenza virus infection in A549 cells"

Fig. 6

Effect of NMRAL1 knockdown on influenza virus replication in A549 cells A: Effect of NMRAL1 knockdown on anti-viral protein expression during influenza virus infection in A549 cells B: Effect of NMRAL1 knockdown on influenza virus infection ratio in A549 cells (Bars: 400 μm)"

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