In the current study, we provide evidence that CD163 is a putative PRRSV receptor, closely associated with infection. Transient transfections of CD163 cDNAs from six cell types (encompassing five animal species) are capable of rendering nonpermissive cell lines permissive to PRRSV infection. Stable transfection with a CD163 cDNA was necessary and sufficient to generate fully permissive recombinant cell lines from established porcine kidney, feline kidney, and hamster kidney cell lines. Some of these cell lines retained the PRRSV-permissive phenotype for at least 80 cell passages, supported at least 60 serial virus passages, and produced in excess of 105 TCID50/ml of progeny virus.
CD163 is a scavenger receptor (SR) protein. The SR proteins comprise a large number of cell surface and soluble glycoproteins involved in the recognition of various ligands, including proteins, polyribonucleotides, polysaccharides, and lipids (28
). These proteins are therefore capable of binding a wide range of host molecules and pathogens. Sequencing revealed that SR genes could be further divided into different types based on the commonality of motifs or domains. The SRCR domain has been found in more than 25 different secreted and/or membrane-anchored proteins. Many SRCR proteins are expressed by leukocytes and are involved in the development of the immune system and in regulation of the immune response (28
The domain that defines the SRCR family of proteins consists of 100 to 110 amino acid residues. Molecules with SRCR domains are further divided into two groups based on the location and number of cysteine residues. Members of group A have six cysteine residues, and those of group B have eight cysteine residues. CD163, which was originally identified as a specific differentiation protein of macrophages and monocytes, is now known to be a group B SRCR protein containing eight cysteine residues per domain (7
CD163 is a type 1 membrane protein. The extracellular domain of CD163 consists of nine SRCR tandem repeats, followed by a transmembrane segment and an intracellular cytoplasmic tail. Several variants of CD163 with different cytoplasmic domains have been described and are the result of alternative splicing of the CD163 primary transcript. Expression of CD163 is low in undifferentiated cells (10
) and generally increases following stimulation and activation of macrophages. Human CD163 lacking the transmembrane domain sheds into the bloodstream and exhibits cytokine-like functions (9
). One well-characterized function of CD163 involves scavenging of hemoglobin, which is mediated by endocytosis of haptoglobin-hemoglobin complexes (15
SusCD163v1 is missing the entire first and second SRCR repeats, yet it still confers permissivity to PRRSV infection, indicating that these two domains are not required for binding of PRRSV. Of six Vero cell splice variants isolated, only the two that lack the hydrophobic transmembrane domain (Vero CD163v4 and v5) failed to function as PRRSV receptors when transfected into BHK-21 cells. This suggests that CD163 in the type 1 membrane protein configuration (the form that scavenges haptoglobin-hemoglobin complexes) is preferred over soluble forms as the active PRRSV receptor. All other CD163 cDNAs tested in this study, from human, pig, mouse, dog, or African green monkey (MARC-145 and Vero) cells, contained the transmembrane domain and functioned as PRRSV receptors. Among these are alternative splice patterns that encode cytoplasmic tails in different reading frames, as has been reported for human CD163 transcripts (24
). Therefore, sequence variations within the cytoplasmic domain do not appear to determine PRRSV receptor function.
Sánchez-Torres et al. (27
) reported that CD163 is involved in the uptake of another porcine virus, African swine fever virus (ASFV), and that expression of CD163 on porcine macrophages and/or monocytes correlates with susceptibility to ASFV. When CD163+
cells were separated, susceptibility to ASFV was associated with the CD163+
cell population. The authors demonstrated an association between CD163 expression and ASFV infection but did not provide evidence that CD163 is capable of converting nonsusceptible cells to ASFV susceptibility. Our study is the first to firmly establish a role for CD163 in viral entry.
Given the strict tropism of PRRSV for pigs, it is somewhat unexpected that CD163 homologs from divergent mammalian species (human, monkey, dog, and mouse) can functionally replace porcine CD163 in several cell lines. It would appear that the species specificity of PRRSV infection might not be based on receptor binding alone but also on downstream cellular processes in the replication cycle. For example, canine DH82 cells support PRRSV internalization and gene expression but do not yield progeny virus, indicating a block in one or more late stages of viral replication (data not shown). Consistent with a role in the early stages of viral infection, CD163 cDNA from DH82 cells is sufficient to convert nonsusceptible BHK-21 cells to PRRSV susceptibility.
Our results do not exclude the possibility of an unidentified cofactor, which is present in some established CD163− cell lines (such as PK032495, NLFK, and BHK-21) but absent in some CD163+ cells (such as primary mouse peritoneal macrophages and differentiated human U937 cells). Hence, the presence of both CD163 and cofactor(s) may be required for efficient attachment, entry, and uncoating.
A previous study suggested that sialoadhesin is a binding factor that is capable of mediating attachment and internalization of PRRSV (32
). It is possible that binding to sialoadhesin is a necessary first step in a pathway that also includes CD163, leading to uncoating and release of viral RNA into the cytoplasm. However, the PK-15 cell line used in that study did not express detectable levels of sialoadhesin (32
). Our RT-PCR results further confirmed that sialoadhesin mRNA was not detectable in PK-15 or PK032495 cells under conditions that readily amplified sialoadhesin mRNA from PAMs. In spite of this, introduction of a CD163 gene into PK032495 cells was sufficient to render them fully permissive to PRRSV infection, arguing against a requirement for sialoadhesin in CD163-mediated initiation of infection. Additional studies are needed to identify possible cofactor(s) and to further dissect the interactions between CD163 and PRRSV.