Integrin β3, a RACK1 interacting protein, is critical for porcine reproductive and respiratory syndrome virus infection and NF-κB activation in Marc-145 cells

Highlights

• ITGB3 was identified as a RACK1 interacting protein.

• Abrogation of ITGB3 inhibited PRRSV replication and NF-κB activation.

• Overexpression of ITGB3 enhanced PRRSV replication and NF-κB activation.

• Inhibition of ITGB3 alleviated cytopathic effects and reduced the viral titer.

• RACK1 and ITGB3 are NF-κB target genes, while indirectly regulating each other.

Abstract

Porcine reproductive and respiratory syndrome virus (PRRSV) is the pathogen of porcine reproductive and respiratory syndrome (PRRS), which is one of the most economically harmful diseases in modern pig production worldwide. Receptor of activated protein C kinase 1 (RACK1) was previously shown to be indispensable for the PRRSV replication and NF-κB activation in Marc-145 cells. Here we identified a membrane protein, integrin β3 (ITGB3), as a RACK1-interacting protein. PRRSV infection in Marc-145 cells upregulated the ITGB3 expression. Abrogation of ITGB3 by siRNA knockdown or antibody blocking inhibited PRRSV infection and NF-κB activation, while on the other hand, overexpression of ITGB3 enhanced PRRSV infection and NF-κB activation. Furthermore, inhibition of ITGB3 alleviated the cytopathic effects and reduced the TCID₅₀ titer in Marc-145 cells. We also showed that RACK1 and ITGB3 were NF-κB target genes during PRRSV infection, and that they regulated each other. Our data indicated that ITGB3, presumably as a co-receptor, played an imperative role during PRRSV infection and NF-κB activation in Marc-145 cells. PRRSV infection activates a positive feedback loop involving the activation of NF-κB and upregulation of ITGB3 and RACK1 in Marc-145 cells. The findings would advance our elaborated understanding of the molecular host–pathogen interaction mechanisms underlying PRRSV infection in swine and suggest ITGB3 and NF-κB signaling pathway as potential therapeutic targets for PRRS control.

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.