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. 2017 Dec 14;92(1):e01690-17.
doi: 10.1128/JVI.01690-17. Print 2018 Jan 1.

Innate Sensing of Influenza A Virus Hemagglutinin Glycoproteins by the Host Endoplasmic Reticulum (ER) Stress Pathway Triggers a Potent Antiviral Response via ER-Associated Protein Degradation

Dylan A Frabutt #  1 , Bin Wang #  2 , Sana Riaz  1 , Richard C Schwartz  1 , Yong-Hui Zheng  3   1
Affiliations

Innate Sensing of Influenza A Virus Hemagglutinin Glycoproteins by the Host Endoplasmic Reticulum (ER) Stress Pathway Triggers a Potent Antiviral Response via ER-Associated Protein Degradation

Dylan A Frabutt et al. J Virol. .

Abstract

Innate immunity provides an immediate defense against infection after host cells sense danger signals from microbes. Endoplasmic reticulum (ER) stress arises from accumulation of misfolded/unfolded proteins when protein load overwhelms the ER folding capacity, which activates the unfolded protein response (UPR) to restore ER homeostasis. Here, we show that a mechanism for antiviral innate immunity is triggered after the ER stress pathway senses viral glycoproteins. When hemagglutinin (HA) glycoproteins from influenza A virus (IAV) are expressed in cells, ER stress is induced, resulting in rapid HA degradation via proteasomes. The ER-associated protein degradation (ERAD) pathway, an important UPR function for destruction of aberrant proteins, mediates HA degradation. Three class I α-mannosidases were identified to play a critical role in the degradation process, including EDEM1, EDEM2, and ERManI. HA degradation requires either ERManI enzymatic activity or EDEM1/EDEM2 enzymatic activity when ERManI is not expressed, indicating that demannosylation is a critical step for HA degradation. Silencing of EDEM1, EDEM2, and ERManI strongly increases HA expression and promotes IAV replication. Thus, the ER stress pathway senses influenza HA as "nonself" or misfolded protein and sorts HA to ERAD for degradation, resulting in inhibition of IAV replication.IMPORTANCE Viral nucleic acids are recognized as important inducers of innate antiviral immune responses that are sensed by multiple classes of sensors, but other inducers and sensors of viral innate immunity need to be identified and characterized. Here, we used IAV to investigate how host innate immunity is activated. We found that IAV HA glycoproteins induce ER stress, resulting in HA degradation via ERAD and consequent inhibition of IAV replication. In addition, we have identified three class I α-mannosidases, EDEM1, EDEM2, and ERManI, which play a critical role in initiating HA degradation. Knockdown of these proteins substantially increases HA expression and IAV replication. The enzymatic activities and joint actions of these mannosidases are required for this antiviral activity. Our results suggest that viral glycoproteins induce a strong innate antiviral response through activating the ER stress pathway during viral infection.

Keywords: EDEM1; EDEM2; EDEM3; ER stress; ERAD; ERManI; HA; NA; PAMP; PRR; UPR; hemagglutinin; influenza; innate immunity.

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Figures

FIG 1
FIG 1
Induction of ER stress by IAV infection. (A) BiP expression was analyzed in H1N1 A/WSN/33 virus-infected A549 cells by real-time qPCR at the indicated time points postinfection. (B) XBP1 splicing was analyzed in the same H1N1 A/WSN/33 virus-infected A549 cells by PCR. The spliced (S) XBP1, unspliced (U) XBP1, and actin bands are indicated. Error bars represent standard deviations (SD) from three independent experiments. y axes represent normalized fold changes, where mock infection is considered 1-fold.
FIG 2
FIG 2
Activation of the IRE1 pathway by HA. (A) Wild-type (WT), ΔCNX, ΔCRT, and ΔΔ 293T cell lines were transfected with an IAV HA (H5), NA (N1), or NP expression vector, and protein expression was analyzed via Western blotting using the indicated antibodies. (B) HA cell surface expression in WT, ΔCNX, ΔCRT, and ΔΔ 293T cells was determined by flow cytometry. (C) HA expression in ΔΔ cells was rescued by ectopic expression of CNX or CRT in a dose-dependent manner. (D) The XBP1 splicing was determined after transient transfection of the pXBP1u-FLuc reporter and viral glycoprotein expression vectors for HIV-1 and IAV into WT, ΔCNX, ΔCRT, and ΔΔ 293T cells. HA proteins from IAV subtypes H1 and H5 and Env proteins from HIV-1 NL43, JRFL, and SF162 were used. NHK, terminally misfolded human alpha1-antitrypsin variant null (Hong Kong). Control 1 (Ctrl 1) was from untransfected cells, and Ctrl 2 was from cells only transfected with the pXBP1u-FLuc vector. Results are displayed as the means ± standard errors of the means (SEM) (n = 3). *, P < 0.05 by unpaired two-tailed t test. The y axis represents normalized fold changes, where Ctrl 1 is considered 1-fold.
FIG 3
FIG 3
HA degradation via ERAD. (A) WT and ΔΔ 293T cells were transfected with an H5 expression vector, and the HA steady state was chased at the indicated time points by Western blotting after treatment with cycloheximide (CHX) at 50 μg/ml. The relative HA expression was measured by quantification of the intensity of each protein band on the blot using ImageJ. Error bars represent SD from three independent experiments. y axes represent percent changes, where the value at time zero is considered 100%. (B) After transfection of WT or ΔΔ 293T cells with an H5 expression vector, cells were incubated with MG132 (25 μM), kifunensine (5 μM), eeyarestatin (50 μM), or bafilomycin A1 (100 nM) for 4 h and analyzed by Western blotting.
FIG 4
FIG 4
Inhibition of HA expression by EDEM1, EDEM2, and ERManI. (A) 293T cells were transfected with an H5 expression vector and a vector expressing EDEM1, EDEM2, or ERManI with a C-terminal FLAG tag. After 48 h, cells were lysed and analyzed via Western blotting. APOBEC3A (A3A) was used as a control. (B) HA expression on Western blots shown in panel A was quantified with ImageJ and is presented as relative values. Results are displayed as the means ± SD (n = 3). *, P < 0.05 by unpaired two-tailed t test. (C) A similar experiment was conducted by replacing H5 with H1 and human EDEM3 with murine EDEM3 (mEDEM3). An unspecific band, which overlaps the EDEM2 band, is labeled with an asterisk. (D) 293T cells were transfected with a fixed amount of H5 and increasing amounts of EDEM1, EDEM2, or ERManI expression vector (in micrograms), and HA expression was analyzed via Western blotting. (E and F) The effect of EDEM and ERManI on IAV N1 expression (E) and that of EDEM and ERManI on HIV-1 Env expression (F) were similarly determined. (G) Effect of EDEM and ERManI on single-cycle viral replication. HIV-1 with authentic Env or HIV-1 pseudoviruses with IAV H5 and N1 were produced by transfection of an EDEM or ERManI expression vector plus pNL4-3 or pNL-GFP proviral vector, respectively. The infectivity of natural HIV-1 was determined after infection of HIV-1 luciferase reporter cell line TZM-b1 cells and measurement of the intracellular luciferase activity; the infectivity of HA-pseudotyped HIV-1 was determined after infection of MDCK cells and measurement of the intracellular green fluorescent protein (GFP) expression. Results are displayed as the means ± SEM (n = 3). *, P < 0.05 by unpaired two-tailed t test. y axes in panels B and G represent percent changes, where the value from the vector control is considered 100%.
FIG 5
FIG 5
Requirement of mannosidase activity for HA inhibition. (A) 293T cells were transfected with an H5 vector and the indicated EDEM and ERManI expression vectors. After 24 h, cells were treated with kifunensine (5 μM), lactacystin (25 μM), or MG132 (25 μM) for 6 h, and protein expression was analyzed by Western blotting. (B) 293T cells were transfected with an H5 vector and the indicated EDEM and ERManI WT or catalytic mutant expression vector, and protein expression was analyzed by Western blotting. (C) ERManI-KO 293T cells were transfected with an H5 vector and the indicated EDEM WT or mutant expression vector, and protein expression was analyzed by Western blotting. (D) The relative expression of ERManI, EDEM1, EDEM2, and EDEM3 in the human lung epithelial cell line A549 was determined by real-time qPCR. Results are displayed as means ± SEM (n = 3). *, P < 0.05 by unpaired two-tailed t test. The y axis represents relative mRNA levels (%) after normalization to the levels of GAPDH.
FIG 6
FIG 6
Inhibition of HA expression by endogenous EDEM1, EDEM2, and ERManI. (A) WT 293T cells were transfected with EDEM1, EDEM2, and ERManI expression vectors plus a lentiviral vector expressing the specific shRNAs or a scrambled shRNA as a control (ctrl). Protein expression was analyzed by Western blotting. (B) ΔΔ 293T cells were transfected with an H5 or HIV-1 proviral vector plus a lentiviral vector expressing the indicated shRNAs. Protein expression was analyzed by Western blotting. (C) WT A549 and 293T cells were stably transduced with a lentiviral vector expressing the indicated shRNAs, and HA expression in these cells was analyzed by Western blotting.
FIG 7
FIG 7
Inhibition of IAV replication by EDEM1, EDEM2, and ERManI. (A) Stable A549 cell lines expressing the indicated shRNAs were infected with H1N1 A/WSN/33 viruses at an MOI of 0.5. Viral supernatants were sampled at the specified time points. Viral titers were determined by a hemagglutination assay using turkey red blood cells and plaque-forming cell assay after infecting MDCK cells. Results are displayed as means ± SD (n = 2). *, P < 0.05 by unpaired two-tailed t test. (B) Infected cells at 24 h and 48 h from the same infection experiments were collected and analyzed by Western blotting using the indicated antibodies. The relative intensity of HA0 was measured using ImageJ software.
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