Integrin β3, a RACK1 interacting protein, is critical for porcine reproductive and respiratory syndrome virus infection and NF-κB activation in Marc-145 cells
Introduction
Porcine reproductive and respiratory syndrome (PRRS) is characterized by respiratory disorders in piglets and reproductive failure in sows (Pejsak and Markowska-Daniel, 1997). The contributing pathogen, porcine reproductive and respiratory syndrome virus (PRRSV), is a swine-specific enveloped single-stranded positive-sense RNA virus that belongs to the Betaarterivirus genus of the Arteriviridae family (Kuhn et al., 2016). Since the first PRRS outbreak in the United States in 1987, PRRSV infections have been nowadays detected in almost all swine-producing countries, causing one of the highest economic loss in modern pig production worldwide (Lunney et al., 2010). Several cell surface receptors that are involved in PRRSV infection in porcine alveolar macrophages (PAMs) has been identified, including heparan sulphate (HS) (Delputte et al., 2002), sialoadhesin (Sn) (Vanderheijden et al., 2003) and CD163 (Calvert et al., 2007; Van Gorp et al., 2008).
Integrins are a family of heterodimer transmembrane receptors consisting of eighteen α-subunits and eight β-subunits that play pivotal roles in the binding of cells to extracellular matrix (ECM) (Plow et al., 2000; van der Flier and Sonnenberg, 2001), transmembrane signal transduction (Hynes, 2002; Miranti and Brugge, 2002) and immune responses (Hamidi and Ivaska, 2018). Cellular integrins are common receptors exploited by diverse viral pathogens for cell entry and infection (Stewart and Nemerow, 2007), such as Epstein-Barr virus (EBV) (Tugizov et al., 2003), human immunodeficiency virus 1 (HIV-1) (Arthos et al., 2008; Cicala et al., 2009) and Simian virus 40 (Stergiou et al., 2013). The signalling functions of integrins are achieved through the formation of signalling complexes at the cytoplasmic side of the plasma membrane with its cofactors, for example focal adhesion kinase (FAK) (Ruoslahti and Reed, 1994) and receptor of activated protein C kinase 1 (RACK1) (Cox et al., 2003; Trerotola et al., 2012).
RACK1, a 36-kDa protein comprising of seven WD-40 repeats, is well known as a scaffolder protein (Adams et al., 2011) and plays its unique functions in various cancers (Zhou et al., 2017; Liu et al., 2017; Hu et al., 2019) and infections (Hu et al., 2019; Lee et al., 2019; Bi et al., 2018; Majzoub et al., 2014; Liu et al., 2019). RACK1 serves as an adaptor molecule for the binding of key signaling molecules (Cox et al., 2003; Zhou et al., 2017; Liu et al., 2017; Hu et al., 2019; Bolger, 2017; Erbil et al., 2016; Liliental and Chang, 1998), with elaborate involvement in the regulation of multiple signal pathways (Hu et al., 2019; Erbil et al., 2016; Vomastek et al., 2007). RACK1 is reported to interact with β integrins to stabilize the focal adhesion through cell-ECM interaction with the entanglement of integrin-induced FAK autophosphoryation (Cox et al., 2003; Liliental and Chang, 1998; Buensuceso et al., 2001).
Nuclear factor kappa B (NF-κB), whose activation by a variety of signals regulates the expression of many target genes, is a key regulator of cellular events. NF-κB signaling pathway modulates a broad range of biological processes including immune response, inflammation, cell proliferation, tumorigenesis and apoptosis. Binding of the viral particle to its receptors and the accumulation of viral products, including dsRNA and viral proteins, activate NF‐κB signaling cascades through various processes. Multiple viruses, including HIV (Manches et al., 2012; Dufrasne et al., 2018), herpesviruses (Havemeier et al., 2014), and hepatitis C virus (HCV) (Kanda et al., 2006), have in turn evolved sophisticated strategies to alert NF-κB signaling.
Our previous studies (Bi et al., 2018; Liu et al., 2019) showed that RACK1 is indispensable for PRRSV replication and NF-κB activation in Marc-145 cells. As a build-up of that concept, the current study identified integrin β3 (ITGB3) as an interacting factor of RACK1 during the PRRSV infection in Marc-145 cells. Our data indicated that PRRSV infection upregulates the expression of RACK1-interacting ITGB3, which participated in the activation of the NF-κB signaling pathway and in turn promotes PRRSV infection, presumably through regulation of RACK1 and ITGB3. The findings can advance our elaborated understanding of the molecular host–pathogen interaction mechanisms of PRRSV and suggest ITGB3 and NF-κB signaling pathway as potential therapeutic targets for PRRS control.
Section snippets
Material and methods
Most of the materials and methods used in this study were described with details in our previous studies (Bi et al., 2018; Liu et al., 2019; Zhu et al., 2018; Zheng et al., 2015).
ITGB3 was identified as a RACK1 interacting protein
Venny diagram analysis (https://bioinfogp.cnb.csic.es/tools/venny/) of the four datasets from the pulldown-MS assay (Supplementary Table 1) revealed that there were 179 RACK1-interacting proteins shared within all the four infection time points (Fig. 1A). Further gene ontology (http://amigo.geneontology.org/amigo/landing) pathway analysis (Fig. 1B) of the 179 overlapping proteins showed that integrin signaling pathway (including ITGB3 and its related factors such as ILK, ACTN1, PIK3C2A, MAPK3
Discussion
RACK1 is well known to interact with the membrane proximal region of the cytoplasmic tail of integrins β1 (Trerotola et al., 2012; Hu et al., 2019), β2 (Feng et al., 2012), β3 (Besson et al., 2002) and β5 (Liliental and Chang, 1998). The first identification of integrin β3 (ITGB3) as a RACK1-interacting protein by pulldown and mass spectrometry in Marc-145 cell in this study is consistent with previous discoveries in other species. Besides, some other RACK1 interacting and ITGB3 related
Author statement
siRNA transfection, treatment with ITGB3 antibody and NF-κB inhibitor, overexpression of ITGB3 in Marc-145 cells, Western blot analysis, Indirect immunofluorescence staining, TCID50 measurement: Chao Yang, Rui Lan; Total RNA isolation and RT-qPCR analysis: Junlong Bi; Pulldown and mass spectrometry: Xiaochun Wang, Qian Zhao; analyses of MS data: Xidan Li; cell culture and virus infection: Guishu Yang; Writing up: Jing Wang, Yingbo Lin and Jianping Liu; Project administration, Conceptualization
Author contributions
Chao Yang and Rui Lan performed all the western blots related experiments and overexpression of ITGB3. Xiaochun Wang and Qian Zhao performed the overexpression of RACK1 and subsequent pulldown. Xidan Li and Junlong Bi performed all the data analysis. Jing Wang and Guishu Yang performed the RT-qPCR and immunofluorescence staining. Yingbo Lin generated the mechanistic hypothesis and modified the manuscript. Jianping Liu and Gefen Yin supervised this study and wrote the manuscript.
Declaration of Competing Interest
None.
Acknowledgements
This work was supported by the National Natural Science Foundation of China (grant no. 31560705 and 31960701), by the Key Projects of Yunnan Provincial Natural Science Foundation (No. 2016FA018), by Program for Innovative Research Team (in Science and Technology) in University of Yunnan Province (IRTSTYN) and by Pig Disease Research Center, Yunnan Agricultural University. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for
References (60)
- et al.
The anchoring protein RACK1 links protein kinase Cepsilon to integrin beta chains. Requirements for adhesion and motility
J. Biol. Chem.
(2002) The RNA-binding protein SERBP1 interacts selectively with the signaling protein RACK1
Cell. Signal.
(2017)Porcine reproductive and respiratory syndrome virus induces interleukin-1beta through MyD88/ERK/AP-1 and NLRP3 inflammasome in microglia
Vet. Microbiol.
(2018)- et al.
Interaction of West Nile virus with alpha v beta 3 integrin mediates virus entry into cells
J. Biol. Chem.
(2004) Modulation of the NF-kappa B signaling pathway by the HIV-2 envelope glycoprotein and its incomplete BST-2 antagonism
Virology
(2018)RACK1 is an interaction partner of ATG5 and a novel regulator of autophagy
J. Biol. Chem.
(2016)Integrin alphavbeta3 promotes infection by Japanese encephalitis virus
Res. Vet. Sci.
(2017)Kindlin-3 mediates integrin alphaLbeta2 outside-in signaling, and it interacts with scaffold protein receptor for activated-C kinase 1 (RACK1)
J. Biol. Chem.
(2012)LiCl inhibits PRRSV infection by enhancing Wnt/beta-catenin pathway and suppressing inflammatory responses
Antiviral Res.
(2015)Downregulation of tumor suppressor RACK1 by Helicobacter pylori infection promotes gastric carcinogenesis through the integrin beta-1/NF-kappaB signaling pathway
Cancer Lett.
(2019)
Integrins: bidirectional, allosteric signaling machines
Cell
State of hepatitis C viral replication enhances activation of NF-kB- and AP-1-signaling induced by hepatitis B virus X
Cancer Lett.
Activation of Integrin-RACK1/PKCalpha signalling in human articular chondrocyte mechanotransduction
Osteoarthr. Cartil.
Rack1, a receptor for activated protein kinase C, interacts with integrin beta subunit
J. Biol. Chem.
Overexpression of RACK1 enhanced the replication of porcine reproductive and respiratory syndrome virus in Marc-145 cells and promoted the NF-kappaB activation via upregulating the expression and phosphorylation of TRAF2
Gene
Porcine reproductive and respiratory syndrome virus: an update on an emerging and re-emerging viral disease of swine
Virus Res.
RACK1 controls IRES-mediated translation of viruses
Cell
Losses due to porcine reproductive and respiratory syndrome in a large swine farm
Comp. Immunol. Microbiol.
Ligand binding to integrins
J. Biol. Chem.
Anchorage Dependence, Integrins, and Apoptosis
Cell
Cell integrins: commonly used receptors for diverse viral pathogens
Trends Microbiol.
Up-regulation of integrin beta3 expression in porcine vascular endothelial cells cultured in vitro by classical swine fever virus
Vet. Immunol. Immunopathol.
Up-regulated expression of beta3 integrin induced by dengue virus serotype 2 infection associated with virus entry into human dermal microvascular endothelial cells
Biochem. Biophys. Res. Commun.
RACK1, A multifaceted scaffolding protein: dtructure and function
Cell Commun. Signal
HIV-1 envelope protein binds to and signals through integrin alpha(4)beta(7), the gut mucosal homing receptor for peripheral T cells
Nat. Immunol.
RACK1 is indispensable for porcine reproductive and respiratory syndrome virus replication and NF-kappaB activation in Marc-145 cells
Sci. Rep.
Highly pathogenic porcine reproductive and respiratory syndrome virus induces prostaglandin E2 production through cyclooxygenase 1, which is dependent on the ERK1/2-p-C/EBP-beta pathway
J. Virol.
The WD protein Rack1 mediates protein kinase C and integrin-dependent cell migration
J. Cell. Sci.
CD163 expression confers susceptibility to porcine reproductive and respiratory syndrome viruses
J. Virol.
The mouse RACK1 gene is regulated by nuclear factor-kappa B and contributes to cell survival
Mol. Pharmacol.
Cited by (10)
Classical swine fever virus infection suppresses claudin-1 expression to facilitate its replication in PK-15 cells
2021, Microbial PathogenesisCitation Excerpt :The sequences of siRNAs used in this study are listed in Table 1 and the oligonucleotides were synthesized by Sangon Biotech (Shanghai, China). The flowing processes were based on the standard molecular biological protocol and similar to our previous study [44]. In brief, the full length claudin-1 cDNA was reverse transcribed from the total RNA of PK-15 cells and amplified using the primer pair CLDN1-Over-F and CLDN1-Over-R (sequences listed in Table 1, oligonucleotides purchased from Tsingke Biotech, Kunming, China) designed according to NM_001244539.1 for Sus scrofa claudin-1 mRNA.
Small molecule screening identified cepharanthine as an inhibitor of porcine reproductive and respiratory syndrome virus infection in vitro by suppressing integrins/ILK/RACK1/PKCα/NF-κB signalling axis
2021, Veterinary MicrobiologyCitation Excerpt :Cell number was counted and the 50 % tissue culture infected dose (TCID50) was determined by Reed-Muench method. According to our previous studies (Bi et al., 2018; Liu et al., 2019, 2020; Yang et al., 2020), total proteins containing viral and cellular proteins were isolated from Marc-145 cells and PAMs from all treatments at indicated time points (2, 12, 24, 36, 48 and 60 hpi). The collected protein samples were resolved under denaturing conditions using 8 % Bis-Tris NovexNuPAGE gels, transferred to nitrocellulose membranes and blocked in TBST containing 5 % (w/v) dehydrated milk and 0.05 % Tween 20 with shaking at room temperature for 1 h. Viral N protein or the selected cellular proteins were probed by incubation with the primary antibodies (Table 1) at 4 °C overnight with rocking, followed by incubation with the corresponding goat anti-mouse or goat anti-rabbit horse radish peroxidase (HRP) conjugated antibody (Table 1) with rocking at room temperature for 1 h. Subsequently, the detected protein bands were visualized with chemiluminescent ECL Plus substrate (Pierce, Rockford, IL) and imaged using chemiluminescent film (Kodak, Rochester, New York).
Inhibition of porcine reproductive and respiratory syndrome virus by PKC inhibitor dequalinium chloride in vitro
2020, Veterinary MicrobiologyCitation Excerpt :No.RP1001, BioTeke Corporation, Beijing) from Marc-145 cells and PAM cells at the different time points (2, 12, 24, 36 and 48 h post infection for both cell types, with additional 60 hpi for Marc-145 cells). RT-qPCR was performed to quantify the expression level of viral ORF7 gene and relative exptression level of cellular RACK1 gene with cellular GAPDH served as an internal control (primer sequences in Table 1), according to our previous reports (Bi et al., 2018; Liu et al., 2019; Yang et al., 2020). In order to perform the absolution quantification of viral ORF7 gene, according to the standard molecular biological protocols, in brief, the viral RNA was isolated, reverse transcribed, amplified, gel purified and cloned into vector pMD-18 T, followed by transformation into competent DH5α cells.
- 1
These authors contributed equally to this work.