Research paperRecombinant MYH9 protein C-terminal domain blocks porcine reproductive and respiratory syndrome virus internalization by direct interaction with viral glycoprotein 5
Introduction
Porcine reproductive and respiratory syndrome virus (PRRSV) is a positive-sense, single-stranded enveloped RNA virus belonging to the family Arteriviridae (Lunney et al., 2016). The genome of PRRSV is around 15 kb and contains more than 10 open reading frames (ORFs) (Dokland, 2010; Snijder and Meulenberg, 1998). ORF1a and ORF1b of PRRSV account for three-fourths of its entire genome size and encode all non-structural proteins required for viral replication, while ORFs 2–7 encode structural protein components of the virion (Lunney et al., 2016). Genetically, there are two PRRSV genotypes: Type 1, European-like (prototype Lelystad), and Type 2, North American-like (prototype VR-2332) (Mardassi et al., 1994; Wensvoort et al., 1991). Recently, based on a new proposed classification scheme, PRRSV Type 1 and Type 2 have been classified into two species in the genus Porartevirus, designated PRRSV-1 and PRRSV-2, respectively, under the new taxonomy (Adams et al., 2016; Kuhn et al., 2016).
PRRSV-1 and PRRSV-2 strains share approximately 60% nucleotide sequence identity and exhibit serotype difference (Forsberg, 2005; van Woensel et al., 1998). PRRSV infection is highly restricted to cells of the monocyte-macrophage lineage, such as porcine alveolar macrophages (PAMs) (Albina et al., 1998; Morgan et al., 2016). PRRSV propagation in vitro is generally conducted in MARC-145 cells, a subclone of the MA-104 epithelial cell line originating from monkey kidney (Kim et al., 1993). Numerous studies demonstrate that PRRSV infection is mediated by various cellular receptors or factors (Shi et al., 2015) such as heparin sulfate (HS) (Delputte et al., 2002), vimentin (Kim et al., 2006), CD151 (Wu et al., 2014), porcine CD163 (CD163) (Guo et al., 2014), sialoadhesin (CD169) (Delputte et al., 2007), DC-SIGN (CD209) (Pineyro et al., 2016), and MYH9 (Gao et al., 2016). However, only CD163 and MYH9 have been shown to be indispensable for PRRSV infection (Burkard et al., 2017; Gao et al., 2016).
Current strategies of PRRS control are inadequate despite substantial efforts by numerous researchers. Since its emergence in 1987, PRRS remains the major challenge to the swine industry globally, even though vaccines against PRRS have been commercially available for decades (Butler et al., 2014; Rowland et al., 2012; Tian et al., 2007). This lack of success underscores the urgent need for novel methods for PRRSV control and prevention.
Recently, by probing PRRSV permissive cells with an anti-idiotypic monoclonal antibody (Mab2-5G2), we identified MYH9 as the novel cellular host factor that binds to PRRSV major glycoprotein GP5 (Gao et al., 2016; Zhou et al., 2008). Subsequently, additional investigations demonstrated that MYH9 serves as a host factor for PRRSV and plays an indispensable role in PRRSV internalization by host cells (Gao et al., 2016). MYH9 is a member of the large myosin superfamily and is an endogenous non-muscle myosin II heterohexamer that is composed of two essential light chains (to stabilize the heavy chain structure), two regulatory light chains (that bind to the heavy chains to connect the head and rod domains), and two MYH9 heavy chains held together by interactions between coiled-coil rod domains (Chen et al., 2003; Fang et al., 2010). Generally, MYH9 functions as a motor protein involved in cell migration, integrin-mediated adhesion, epithelial cell polarization, cell-cell adhesion, and morphogenesis (Vicente-Manzanares et al., 2009).
MYH9 (also known as non-muscle myosin heavy chain 9) and its associated gene MYH9 have been heavily investigated for their associations with certain genetic diseases, such as MYH9-related platelet disorders and chronic kidney disease caused by mutations of MYH9 (Althaus and Greinacher, 2009; Althaus et al., 2011; Singh et al., 2009). The amino-terminal head domain of MYH9 possesses an ATPase activity and contains binding sites for both actin and the light chains of myosin (Obungu et al., 2003; Sellers, 2000). However, the roles that MYH9 plays during viral infection or other infectious diseases have not yet been thoroughly investigated. In recent years, data from other groups have demonstrated that MYH9 functions as a cellular factor for several viruses, including herpes simplex virus-1 (HSV-1), severe fever with thrombocytopenia syndrome virus (SFTSV), and Epstein-Barr virus (EBV) (Arii et al., 2010; Sun et al., 2014; Xiong et al., 2015), which suggest a novel role of MYH9 in virus infections. Meanwhile, our research has recently demonstrated that the C-terminal portion of MYH9 interacts with Mab2-5G2 (Gao et al., 2016).
The objective of this study was to investigate whether the C-terminal portion of MYH9 (hereafter referred to as PRA) is able to inhibit PRRSV replication in vitro. The recombinant PRA protein was expressed and purified from a prokaryotic expression system. Our results showed that PRA exhibited broad-spectrum anti-PRRSV activity in both susceptible monkey cells (MARC-145) and swine cells (PK-15CD163, CRL2843CD163, and PAMs). Further analysis revealed that PRA was able to capture PRRSV virions via GP5 interaction. Taken together, our data showed that PRA holds promise as a novel broad-spectrum inhibitor of infection by heterogeneous PRRSV isolates.
Section snippets
Cells, viruses, and chemicals
Porcine alveolar macrophages (PAMs) were collected from a 4-week-old PRRSV-negative pig as previously described (Xiao et al., 2014). The immortalized PAM cell line 3D4/21 (ATCC®CRL-2843™) was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). PRRSV-permissive CRL2843CD163 cells were generated by introducing porcine CD163 cDNA into ATCC®CRL-2843™ via lentiviral vector-mediated transduction under puromycin selection (Thermo Fisher Scientific, Waltham, MA, USA) at a
PRA inhibits PRRSV replication in vitro
We previously identified MYH9 as an indispensable factor for PRRSV infection in both MARC-145 and PAM cells (Gao et al., 2016). In that study, the C-terminal domain of MYH9 with aa 1651–1960 (designated PRA) (Fig. 1A) was found to bind Mab2-5G2 and was proposed to interact with PRRSV-GP5. Here, we hypothesize that PRA protein could serve as a novel blocker of PRRSV binding to permissive cells and subsequent infection. To test this hypothesis, recombinant PRA protein was produced by E. coli
Discussion
HSV-1 is the first virus reported to utilize MYH9 as its cellular receptor (Arii et al., 2010). Since then, several other viruses, including PRRSV, have been identified that enter host cells via MYH9 (Gao et al., 2016; Sun et al., 2014; Xiong et al., 2015). Moreover, interactions between MYH9 and viral proteins such as PRRSV GP5 or HSV-1 gB have been observed, as blocking of MYH9 with specific antibody inhibits virus infection in those systems as well (Arii et al., 2010; Gao et al., 2016).
Acknowledgement
We thank Dr. Ding Yu from School of Life Sciences, Fudan University for kindly providing sfGFP-TEV-His6Nd2 plasmid, Dr. Xiang-Jin Meng from Virginia Polytechnic Institute & State University for generously providing the pIR-VR2385-CA plasmid, Dr. Hanchun Yang from China Agriculture University and Dr. Kegong Tian from the National Research Center for Veterinary Medicine for kindly providing PRRSV-1 GZ11-G1 and PRRSV-1 P073-3 isolate, respectively. This study was financially supported by grants
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These authors contributed equally to this work.