Porcine Complement Regulatory Protein CD46 Is a Major Receptor for Atypical Porcine Pestivirus but Not for Classical Swine Fever Virus

Abstract

Pestiviruses such as bovine viral diarrhea virus (BVDV) and classical swine fever virus (CSFV) belong to the family Flaviviridae and represent pathogens of outstanding veterinary relevance. Pestiviruses enter cells via receptor-mediated endocytosis. For entry in bovine cells, complement regulatory protein CD46bov serves as cellular receptor for BVDV. In this study, the role of porcine CD46pig in cellular entry was investigated for the recently discovered atypical porcine pestivirus (APPV), CSFV, and Bungowannah virus (BuPV) in order to elucidate the observed differences in host cell tropism. A cell culture adapted APPV variant, which shows enhanced viral replication in vitro, was generated and demonstrated a strict tropism of APPV for porcine cells. One of the porcine cell lines displayed areas of CD46pig expressing and areas of non-expressing cells and one single cell line revealed not to express any CD46pig, respectively. The CD46pig deficient porcine lymphoma cells, known to facilitate CSFV replication, was the only porcine cell line non-permissive to APPV, indicating a significant difference in the entry mechanism of APPV and CSFV. Infection experiments with a set of genetically engineered CD46pig knockout cells confirmed that CD46pig is a major receptor of APPV as CD46bov is for BVDV. In contrast, it is apparently not an essential determinant in host cell entry of other porcine pestiviruses such as CSFV and BuPV. Existence of a CD46pig independent entry mechanism illustrates that the pestiviral entry process is more diverse than previously recognized. IMPORTANCE Pestiviruses comprise animal pathogens such as classical swine fever virus (CSFV) and bovine viral diarrhea virus (BVDV) that cause notifiable diseases with great economic impact. Several additional pestivirus species affecting animal health were recently identified, including atypical porcine pestivirus (APPV). APPV is associated with health problems in piglets and highly abundant in pig populations worldwide. Complement control protein CD46 serves as a receptor for diverse bacterial and viral pathogens, including particular adenoviruses, herpesviruses, measles virus (MeV), and BVDV. Porcine CD46 (CD46pig) was suggested to be a major receptor for CSFV. Here, we identified remarkable differences in relevance of CD46pig during entry of porcine pestiviruses. Resembling BVDV, efficient APPV infection in cell culture depends on CD46pig, while other porcine pestiviruses can efficiently enter and infect cells in absence of CD46pig. Thus, the study provides insights into the entry process of these pathogens and may help to understand differences in their biology.

A β-Hairpin Motif in the Envelope Protein E2 Mediates Receptor Binding of Bovine Viral Diarrhea Virus

“…Further supporting this alternative mechanism of entry, generation of stable CD46-knockout MDBK cells showed that BVDV was still able to enter into knockout cells, albeit with markedly reduced growth [ 14 ]. In turn, the role of CD46 as a receptor for different pestiviruses of swine is controversial [ 13 , 15 ]. Porcine cell lines that do not express CD46 were shown to be non-permissive to APPV infection, while porcine pestiviruses such as CSFV and BuPV efficiently infected these cells, indicating a CD46-independent mechanism of entry [ 15 ].…”

“…In turn, the role of CD46 as a receptor for different pestiviruses of swine is controversial [ 13 , 15 ]. Porcine cell lines that do not express CD46 were shown to be non-permissive to APPV infection, while porcine pestiviruses such as CSFV and BuPV efficiently infected these cells, indicating a CD46-independent mechanism of entry [ 15 ]. Additional binding partners of CSFV E2 have been identified and mapping of amino acids involved in these interactions has been achieved using yeast-two hybrid system [ 32 , 33 , 34 ].…”

“…In spite of increasing virus attachment in heterologous cell lines, overexpression of bovine CD46 is not sufficient to turn cells susceptible to infection [ 13 ]. In turn, CSFV is still able to enter different cell lines deficient of porcine CD46 [ 15 ]. Thus, the use of co-receptors appears as a common theme in pestivirus entry.…”

Atypical Porcine Pestiviruses: Relationships and Conserved Structural Features

“…Bovine CD46 has been demonstrated to act as a receptor for BVDV [73,74] and its porcine counterpart has also been proposed as a receptor for CSFV [75]. However, recent results employing knock-out cell lines demonstrate that porcine CD46 is not a receptor for CSFV, but instead for APPV [76]. Given the differences between BVDV and APPV E2 already at the sequence level, this result is highly intriguing, as CD46 s structure is considered quite conserved across different species.…”

Vaccination with virus-like particles of atypical porcine pestivirus inhibits virus replication in tissues of BALB/c mice

“…It also acts as an IFN antagonist, blocking IFN synthesis and thereby facilitating virus replication [ 18 ]. The E2 protein specifically binds to its receptor, the cell-surface complement regulatory protein CD46, to mediate entry of the virus into the cell [ 20 ]. The envelope glycoprotein E2 is the main immunogenic protein of APPV, and an E2 subunit vaccine has been shown to induce a Th2-type immune response in mice [ 21 ].…”