Permissive cells and viral entry

PRRS virus has a very strict cellular tropism being pulmonary alveolar macrophages (PAMs) and macrophages of lymphoid tissues and placenta the predominant differentiated cells supporting virus replication in vivo (Duan et al., 1997a, 1997b;

Wensvoort et al., 1991). Conversely, monocyte-derived cells such as peripheral blood monocytes, peritoneal macrophages and bone marrow progenitor cells are resistant to viral infection (Duan et al., 1997a, 1997b). Recently it has been described that PRRSV is also able to infect and replicated in bone marrow (Bm) and monocyte-derived (Mo) dendritic cells (DC) (BmDC, MoDC) while pulmonary DCs (Charerntantanakul et al., 2006; Flores-Mendoza et al., 2008; Loving et al., 2007; Silva-Campa et al., 2010;

Wang et al., 2007) and plasmacytoid DCs are not permissive to the virus (Calzada-Nova et al., 2011).

In vitro, PRRSV was first isolated on primary cultures of PAMs (Wensvoort et al., 1991) and so far, these cells remain the only non-genetically modified porcine cells that can be used for viral propagation. On the other hand, the continuous cell lines MARC-145 and CL2621 (subclones of MA-104, an African green monkey kidney cell line) permit the complete replication cycle of several PRRSV isolates (Benfield et al., 1992; Collins et al., 1992; Kim et al., 1993) and are routinely used for in vitro propagation of the virus and for the large scale production of PRRSV vaccine strains.

Although it was thought that European and American strains replicated mostly in MAPs and continuous cell lines, respectively, it has been shown that several genotype 1 strains can replicate also in MARC-145 and that most of genotype 2 isolates growth in both MAPs and continuous cell lines. Moreover, some genotype 2 strain grow better in MAPs than in continuous cell lines (Fuentes de Abin et al., 2009).

Figure 1. Interactions between PRRSV and host cell receptors during the establishment of the infection.

The tropism of PRRSV is determined by the interaction of viral surface proteins with specific membrane receptors in the permissive cells. At least 6 molecules have been described as potential cellular receptors for PRRSV, which include heparan sulphate, vimentin, CD151, CD163 (cysteine-rich scavenger receptor), porcine sialohadesin (PoSn, also named Siglec-1 or CD169) and DC-SIGN (dendritic cell-specific

GP 3 GP 3 GP3

PoSn (CD169)

Heparan sulphate

CD163

GP4

GP3

GP2

E M

GP5

intracellular adhesion molecule-3-grabbing non integrin, also known as CD209) (Zhang and Yoo, 2015). Among these, PoSn and CD163 are expressed exclusively in macrophages and have been considered as the most important putative receptors for PRRSV.

Early studies showed that the infection of susceptible cells starts with a low affinity interaction between the GP5/M heterodimer of the virus and the heparan sulphate of PAMs, followed by high affinity interaction between sialic acid residues of GP5 and the PoSn (Delputte et al., 2002; Delputte and Nauwynck, 2004; Delputte et al., 2004;

Van Breedam et al., 2010a; Van Gorp et al., 2010) (Figure 1). Thus, the multimeric complex formed by GP2a, GP3 and GP4, specifically interact with the CD163 mediating the viral internalization into macrophages (Das et al., 2010) through clathrin-mediated endocytosis (Nauwynck et al., 1999). Once inside the endosoma, a pH drop along with the activity of the E catepsin, lead to the uncoating and release of the viral RNA into the cytoplasm and the replication process starts (Calvert et al., 2007; Lee et al., 2010; Misinzo et al., 2008; Van Breedam et al., 2010b; Van Gorp et al., 2008; Van Gorp et al., 2009).

Although this could be considered as the most common mechanism of PRRSV entry into permissive cells, several studies indicated that alternative processes of infection involving different cellular receptors, and consequently a great range of permissive cells, may also exist (Frydas et al., 2013; Frydas et al., 2015; Huang et al., 2009;

Shanmukhappa et al., 2007). For example, PRRSV is able to infect Marc-145 cells very efficiently, but this cell line does not express PoSn. Therefore, vimentin expressed in Marc-145 has been described to interact with other cytoskeletal filaments to mediate the transport of the virus in the cytosol (Kim et al., 2006). Prather et al.

(2013) also showed that PoSn gene-knockout piglets challenged with a genotype 2 strain, developed viremia and antibody responses similar to those of control pigs, clearly demonstrating that PoSn is not required for the infection with PRRSV.

Moreover, two recently studies by Frydas et al. (2013, 2015) showed that Lena strain and an highly pathogenic genotype 1, subtype 1 isolate are able to replicated more efficiently in nasal mucosa than LV or LV-like strains and that they have the capacity

to infect a broader range of cell types, including CD163⁺PoSn^- and to a lesser extent CD163^-PoSn^- cells. Those cells seem to have specific functions in antigen capturing and presentation but their characterization is still lacking. These studies further confirm that: 1) the presence of PoSn is not required for the PRRSV infection; 2) alternative receptors can be used for the viral entry and, 3) PRRSV tropism may be dependent of the viral strain.

The evidence of infection in CD163^-PoSn^- cells of the nasal mucosa suggests that even CD163 is dispensable for the virus entry. In this sense, CD151 was proposed as alternative receptor for PRRSV in cells lacking CD163. Shanmukhappa et al. (2007) demonstrated that BHK-21 cells, that are CD163^-Sn^- and not susceptible for PRRSV infection, became susceptible after over-expression of CD151 by gene transfection. In addition, the PRRSV infection in MARC-145 is completely blocked by anti-CD151 antibody, indicating that this receptor is one of the key molecules facilitating or allowing PRRSV infection. As a matter of fact, CD151 was found to interact specifically with the PRRSV 3’-UTR and was proposed to be involved in the fusion between the viral envelope and the endosome as well as in the re-localization of the ribonucleoprotein complexes to promote viral replication (Shanmukhappa et al., 2007). Lastly, porcine DC-SIGN is expressed on myeloid-derived DCs and macrophages present in skin, lymphoid tissues and placenta (Subramaniam et al., 2014). This molecule can be involved in the infection of cells as shown in transfected BHK-21 cells (Huang et al., 2009). In summary, the utilization of different receptors by PRRSV is still controversial and debatable. Nevertheless, CD163 remains the core receptor of PRRSV and the main determinant on susceptibility of cells (Zhang and Yoo, 2015).