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. 2023 Oct 16:14:1259237.
doi: 10.3389/fimmu.2023.1259237. eCollection 2023.

Direct interaction of the molecular chaperone GRP78/BiP with the Newcastle disease virus hemagglutinin-neuraminidase protein plays a vital role in viral attachment to and infection of culture cells

Chenxin Han  1   2 , Ziwei Xie  1   2 , Yadi Lv  1   2 , Dingxiang Liu  1   2   3 , Ruiai Chen  1   2
Affiliations

Direct interaction of the molecular chaperone GRP78/BiP with the Newcastle disease virus hemagglutinin-neuraminidase protein plays a vital role in viral attachment to and infection of culture cells

Chenxin Han et al. Front Immunol. .

Abstract

Introduction: Glucose Regulated Proteins/Binding protein (GRP78/Bip), a representative molecular chaperone, effectively influences and actively participates in the replication processes of many viruses. Little is known, however, about the functional involvement of GRP78 in the replication of Newcastle disease virus (NDV) and the underlying mechanisms.

Methods: The method of this study are to establish protein interactomes between host cell proteins and the NDV Hemagglutinin-neuraminidase (HN) protein, and to systematically investigate the regulatory role of the GRP78-HN protein interaction during the NDV replication cycle.

Results: Our study revealed that GRP78 is upregulated during NDV infection, and its direct interaction with HN is mediated by the N-terminal 326 amino acid region. Knockdown of GRP78 by small interfering RNAs (siRNAs) significantly suppressed NDV infection and replication. Conversely, overexpression of GRP78 resulted in a significant increase in NDV replication, demonstrating its role as a positive regulator in the NDV replication cycle. We further showed that the direct interaction between GRP78 and HN protein enhanced the attachment of NDV to cells, and masking of GRP78 expressed on the cell surface with specific polyclonal antibodies (pAbs) inhibited NDV attachment and replication.

Discussion: These findings highlight the essential role of GRP78 in the adsorption stage during the NDV infection cycle, and, importantly, identify the critical domain required for GRP78-HN interaction, providing novel insights into the molecular mechanisms involved in NDV replication and infection.

Keywords: GRP78/BiP; Newcastle disease virus; attachment; hemagglutinin-neuraminidase protein; replication.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Identification and analysis of host cell interacting proteins with NDV HN proteins. (A) Schematic diagram showing the IP-MS procedure for mapping HN-host protein interactomes. (B) Separation and silver staining of HN-interacting proteins from NDV-infected DF-1 cells. (C) GO annotation of HN-interacting proteins. GO annotation was used to classify HN-interacting proteins in DF-1 cells. The horizontal coordinate represents the number of proteins and the vertical coordinate represents the GO annotation. (D) COG annotation of HN-interacting proteins. COG function classification was used to classify HN-interacting proteins in DF-1 cells. The horizontal coordinate represents the functional classification of the annotation and the vertical coordinate represents the number of proteins annotated to the corresponding function.
Figure 2
Figure 2
Analysis of interacting proteins with NDV HN proteins. (A) KEGG functional annotation. The horizontal axis represents the number of proteins and the vertical axis represents the KEGG pathway annotation. HN-interacting proteins in DF-1 cells were classified based on the KEGG pathway annotation. (B) Analysis of the interacting proteins with NDV HN proteins using IPR functional annotation. The horizontal axis represents the number of proteins and the vertical axis represents the IPR annotation. HN-interacting proteins in DF-1 cells were classified based on the IPR annotation. (C) Subcellular localization analysis. HN-interacting proteins in DF-1 cells were classified based on their subcellular localization. (D) Venn diagram illustrating the collectively enriched proteins. Each circle in the graph represents the annotation results for a database. The overlapping and the non-overlapping sections represent proteins annotated to multiple databases together and individual databases, respectively.
Figure 3
Figure 3
The interactome map of HN-host protein interactions was generated using Cytoscape software. The map displays the interactions between NDV HN-associated host proteins. Red and yellow nodes represent host proteins with high interaction abundance and intermediate abundance, respectively. The incorporation of host factor interactions was based on interaction data obtained from STRING.
Figure 4
Figure 4
Identification and characterization of GRP78-HN interaction. (A) Schematic diagram illustrating the domain structures of HN and GRP78 as well as truncated GRP78 constructs. The functional domains in HN and GRP78 are shown with different colored boxes, and the positions of the HA and Flag tags added to the two proteins are indicated. (B) Interaction between HN and GRP78 in HEK-293T cells overexpressing the two proteins. Cells transfected with pXJ40-HA-C-HN and pXJ40-Flag-C-GRP78 were immunoprecipitated using anti-HA agarose beads, and the precipitated proteins were subjected to Western blotting with anti-HA and anti-Flag mAbs. β-actin was used as a control. (C) Interaction between HA-HN and the endogenous GRP78. HEK-293T cells transfected with pXJ40-HA-C-HN and pXJ40 vector were immunoprecipitated using anti-HA agarose beads, and the precipitated proteins were subjected to Western blotting with anti-GRP78 pAbs and anti-HA mAbs. β-actin was used as a control. (D) Co-localization of GRP78 and HN proteins in HEK-293T cells. Cells overexpressing Flag-GRP78 (green) and HA-HN (red) were immunostained with mouse anti-Flag monoclonal antibody and rabbit anti-HN pAb, respectively, and examined by confocal microscopy. Nuclei were stained with DAPI (blue). (E) Analysis of the direct binding of recombinant HN protein to GRP78 protein by GST-pulldown assay. The recombinant proteins were purified using GST beads and detected by Western blotting. The purified recombinant proteins were coupled to GST beads, with GST alone as a control. (F) Effects of the N-linked glycosylation of HN protein on its interaction with GRP78. Total cell lysates expressing HN were treated with or without PNGase F, immunoprecipitated using anti-Flag agarose beads, and the precipitated proteins were analyzed by Western blotting with anti-Flag mAb and anti-GRP78 pAb. β-actin expression was used as an internal loading control. (G) Interaction of the GRP78(1-326aa) domain with HN protein. HEK-293T cells co-transfected with HA-HN and Flag-GRP78, Flag-GRP78(1-326aa), or Flag-GRP78(327-652aa) were immunoprecipitated and analyzed by Western blotting using anti-Flag mAb and anti-HA mAb. GAPDH expression was used as an internal loading control.
Figure 5
Figure 5
Induction of GRP78 protein expression in NDV infected-HeLa cells and DF-1 cells. (A) HeLa Cells were infected with NDV at an MOI of 1, and the GRP78 and HN protein levels were assessed by Western blotting with GRP78 and HN antibodies at 24 hpi and 36 hpi. β-actin expression served as the internal loading control. (B, C) Relative expression levels of GRP78 mRNA were determined by RT-qPCR at 24 hpi and 36 hpi, respectively, in HeLa (B) and DF-1 cells (C). (D) HeLa cells were infected with NDV at an MOI of 0.5, 1 and 1.5, respectively, and the GRP78 and HN protein levels were assessed by Western blotting at 24 hpi. (E, F) GRP78 mRNA levels in HeLa (E) and DF-1 (F) cells infected with NDV at an MOI of 0.5, 1 and 1.5, respectively, were determined by RT-qPCR.All results are presented as the mean ± SD of data from three independent experiments. **p < 0.01.
Figure 6
Figure 6
Knockdown of GRP78 inhibits NDV infection and replication. (A) Cytotoxicity assay of cells transfected with siRNA targeting GRP78. HeLa and DF-1 cells were transfected with siRNAs targeting GRP78 and siRNA-NC for 36 h, and the cell viability was assessed using the CCK-8 kit. (B) HeLa cells were transfected with GRP78 siRNAs for 36 h. GRP78 protein expression was analyzed by Western blotting. (C) HeLa cells and DF-1 cells were transfected with GRP78 siRNAs for 36 h. GRP78 mRNA levels were analyzed by RT-qPCR. (D) HeLa cells were transfected with either siRNA GRP78 1# or siNC, and DF-1 cells were transfected with either siRNA GRP78 3# or siNC for 36 h, followed by infection with NDV (MOI=1). The expression of GRP78 mRNA was quantitated by real-time PCR. (E) HeLa cells were transfected with either siRNA GRP78 1# or siNC for 36 h, and infected with NDV (MOI=1). The protein expression of GRP78 was analyzed by Western blotting. (F) The virus titers in the culture supernatant were determined by TCID50 assay. (G) The copy number of NDV was analyzed by real-time PCR. All results are presented as the mean ± SD of data from three independent experiments. **p < 0.01; ***p < 0.001; ns, p > 0.05.
Figure 7
Figure 7
Overexpression of GRP78 promotes NDV replication. (A) HeLa and DF-1 cells were transfected with pXJ40-Flag-C-GRP78 or pXJ40 vector for 24 h and infected with NDV (MOI=1) for 24 h. The mRNA levels of GRP78 were analyzed by RT-qPCR. (B) The protein levels of GRP78 were detected by Western blotting in HeLa cells. (C) The copy number of NDV was analyzed by real-time PCR. (D) The virus titers in the culture supernatant were determined by TCID50 assay. All results are presented as the mean ± SD of data from three independent experiments. **p < 0.01; ***p < 0.001.
Figure 8
Figure 8
Functional involvement of GRP78 in the attachment of NDV to cells. (A, B) Effects of GRP78 knockdown and overexpression on the attachment of NDV to cells. HeLa and DF-1 cells were transfected with either siGRP78 for 36 h (A) or pXJ40-Flag-C-GRP78 for 24 h (B), incubated with NDV (MOI=1) at 4°C for 1 h, and the GRP78 mRNA levels were determined by RT-qPCR. (C, D) HeLa and DF-1 cells were transfected with either siGRP78 for 36 h (C) or pXJ40-Flag-C-GRP78 for 24 h (D), incubated with NDV (MOI=1) at 4°C for 1 h, and the NDV mRNA levels were determined by RT-qPCR. All results are presented as the mean ± SD of data from three independent experiments. **p < 0.01; ***p < 0.001. (E) Co-localization of GRP78 and NDV in HeLa cells during the attachment stage. HeLa cells were incubated with NDV (MOI=10) at 4°C for 1 h, followed by immunostaining with rabbit anti-GRP78 pAbs (red) and rabbit anti-NDV pAb (green), respectively, and examined by confocal microscopy. Nuclei were stained with DAPI (blue).
Figure 9
Figure 9
Effects of GRP78-knockdown and overexpression on the entry of NDV. (A, B) HeLa cells and DF-1 cells were transfected with either siGRP78 for 36 h (A) or pXJ40-Flag-C-GRP78 for 24 h (B), incubated with NDV (MOI=1) at 4°C for 1 h, followed by shifting to 37°C for another 1 h after thorough washing. GRP78 mRNA levels were determined by RT-qPCR. (C, D) HeLa cells and DF-1 cells were transfected with either siGRP78 for 36 h (C) or pXJ40-Flag-C-GRP78 for 24 h (D), incubated with NDV (MOI=1) at 4°C for 1 h, followed by shifting to 37°C for another 1 h after thorough washing. NDV mRNA levels were determined by RT-qPCR.All results are presented as the mean ± SD of data from three independent experiments. **p < 0.01; ***p < 0.001.
Figure 10
Figure 10
GRP78 antibody inhibits NDV infection in HeLa cells. (A, B) Inhibition of NDV infection by GRP78 pAbs. HeLa cells were incubated with varying concentrations of GRP78 pAbs or rabbit IgG for 1 h at 37°C, and incubated with NDV (MOI=1) for 1 h at 4°C. After 24 hpi, cells and supernatants were harvested separately, and the levels of NDV mRNA were determined using RT-qPCR (A), and the virus titers in the supernatants were determined using TCID50 assay (B). All results are presented as the mean ± SD of data from three independent experiments. **, p < 0.01. (C) NDV infectivity was determined by immunofluorescence assay (IFA) using rabbit anti-HN pAbs (green). Nuclei were stained with DAPI (blue).
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The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This research was funded by Guangdong Provincial Scientific Research Institutions Key Areas R&D Plans (grant number 2021B0707010009 and 2022B1111040001), National Natural Science Foundation of China grants (31972660 and 32170152) and Zhaoqing Xijiang Innovative Team Foundation of China (grant number P20211154-0202).
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