Ubiquitin E3 ligase MARCH10 targets influenza hemagglutinin for ubiquitination

Abstract

Influenza virus infection damages the airways and can cause acute lung injury. Influenza virus infection remains difficult to combat since treatment is limited to supportive care or antiviral drugs to prevent influenza early in the condition. Influenza hemagglutinin (HA) is the surface glycoprotein that facilitates viral entry by binding to sialic acid-containing receptors on the host's lung cells. Therefore, it is a promising target for the development of anti-influenza therapeutic drugs. We demonstrate that the understudied E3 ligase MARCH10 destabilizes influenza HA protein in a dose-response manner and decreases the half-life of influenza HA over time. However, it does not affect the mRNA expression of influenza HA. Further, MARCH10 specifically polyubiquitinates influenza HA targeting it for degradation. When BEAS-2B cells ectopically expressed MARCH10 and were infected with PR8 virus, 1378 genes were differentially expressed. In addition, our analysis reveals that MARCH10 upregulates multiple pathways that involved interferon signaling during influenza virus infection. These findings suggest that MARCH10 plays a protective role during influenza virus infection and may enhance airway host defense and innate immunity.

Keywords: E3 ubiquitin ligase; Hemagglutinin; Influenza; Interferon signaling; MARCH10; Ubiquitination.

Figure 1:. Overexpression of MARCH10 destabilizes influenza virus HA protein.

A) HEK293T cells co-transfected with CA09 HA and increasing amounts of nLuc control or MARCH10 plasmids. HEK293T cells co-transfected with Matrix 1 plasmid, as specificity control, along with increasing concentrations of nLuc control or MARCH10 plasmids. B) A puromycin chase was performed on HEK293 cells transfected with either nLuc control or MARCH10 plasmid and infected with PR8 virus. A puromycin chase on HEK293T cells transfected with Matrix 1 plasmid and infected with PR8 was also performed as a specificity control. C) PR8 HA mRNA expression was measured in HEK293T cells transfected with nLuc control or MARCH10 plasmids and infected with PR8 virus. Results are expressed as mean ± SD. *P<0.05 vs. control (nLuc) by multiple unpaired t-tests. Data represents n=3 separate experiments.

Figure 2:. MARCH10 mediates influenza HA ubiquitination and degradation.

A) Shown is polyubiquitination of ectopically expressed FLAG-tagged CA09 virus HA protein in the presence or absence of MARCH10-V5 or HA-tagged ubiquitin plasmid co-transfection. Cells were transfected with plasmids and processed for immunoprecipitation (IP) and immunoblotting. B) IP of ectopically expressed FLAG-tagged CA09 virus HA co-transfected with WT or K →R HA-ubiquitin constructs to identify primary ubiquitin linkages or an HA-ubiquitin construct with all mutated lysines (KO). Data represents n=2 separate experiments.

Figure 3:. Bulk RNA-seq of MARCH10 expressing BEAS-2B infected with influenza virus strain PR8

A) mRNA expression of MARCH10 in BEAS-2B cells following overexpression of MARCH10 and nLuc control expression. Results are expressed as mean ± SD of n-fold MARCH10 mRNA expression vs. nLuc. Replicates of n=4 samples. ****P<0.0001. B) Ingenuity pathway analysis of bulk RNA-seq data in BEAS-2B transfected with MARCH10 vs control plasmids and infected with the influenza virus strain PR8 (fold change > |1.5|, adjusted P value <0.05 (n=4 per group). RNA lysates were collected 24h post infection. The diagram lists the 30 most regulated function terms and shows the number of DEGs in the RNA-seq data set within each category. DEG terms were labeled according to their relevance for development (red), cell movement and molecular transport (orange), biosynthesis and metabolism (green), cell cycle and DNA repair (blue), and other (gray). C) Ingenuity pathway analysis of the most regulated signaling pathways with adjusted P<0.05 and absolute z score cut-off > |2.5| for MARCH10 expression vs. nLuc control in infected BEAS-2B. D) Clustered heatmap shows upregulation of genes involving interferon signaling promoted by MARCH10 overexpression during infection with influenza virus PR8. Heatmap data is normalized log₂-fold expression.