Equus caballus Major Histocompatibility Complex Class I Is an Entry Receptor for Equine Herpesvirus Type 1

In this study, Equus caballus major histocompatibility complex class I (MHC-I) was identified as a cellular entry receptor for the alphaherpesvirus equine herpesvirus type 1 (EHV-1). This novel EHV-1 receptor was discovered using a cDNA library from equine macrophages. cDNAs from this EHV-1-susceptible cell type were inserted into EHV-1-resistant B78H1 murine melanoma cells, these cells were infected with an EHV-1 lacZ reporter virus, and cells that supported virus infection were identified by X-Gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside) staining. Positive cells were subjected to several rounds of purification to obtain homogeneous cell populations that were shown to be uniformly infected with EHV-1. cDNAs from these cell populations were amplified by PCR and then sequenced. The sequence data revealed that the EHV-1-susceptible cells had acquired an E. caballus MHC-I cDNA. Cell surface expression of this receptor was verified by confocal immunofluorescence microscopy. The MHC-I cDNA was cloned into a mammalian expression vector, and stable B78H1 cell lines were generated that express this receptor. These cell lines were susceptible to EHV-1 infection while the parental B78H1 cells remained resistant to infection. In addition, EHV-1 infection of the B78H1 MHC-I-expressing cell lines was inhibited in a dose-dependent manner by an anti-MHC-I antibody.Equine herpesvirus type 1 (EHV-1) is a major pathogen affecting horses worldwide. Clinical signs of infection range from initial respiratory distress, fever, inappetance, and malaise to more serious secondary conditions including paralysis in some cases and abortigenic disease in pregnant mares (2). The virus is readily spread via direct transmission from horse to horse or via contact with contaminated surroundings. Due to the latent program of the virus, there is a constant reservoir of EHV-1 within the equine population, and frequent reactivation events trigger outbreaks and expose naïve horses to the virus (35).At the cellular level, EHV-1 initially attaches to cells via an interaction between two of its glycoproteins, gC and gB, and cell surface heparin sulfate (36, 41). While these electrostatic interactions mediate virus binding, they do not trigger the entry of the virus into cells. For entry to proceed, a secondary triggering event mediated by gD must occur (10, 14). After entry is initiated, EHV-1 enters cells either by directly fusing with the plasma membrane or via endocytosis (17). After fusion between the viral envelope and a cellular membrane, viral capsids are released into the cytoplasm and then actively transported to the nucleus along microtubules (18).Previous studies showed that EHV-1 utilizes a cell receptor that is distinct from any of the known alphaherpesvirus entry receptors (14). The goal of the present study was to identify a functional EHV-1 entry receptor by screening an equine macrophage cDNA library. To identify a receptor, we transferred equine cDNAs (48) from an equine macrophage library into cells that are highly resistant to EHV-1. These cDNA-transduced cells were then screened for their ability to mediate EHV-1 infection. Using this approach, we successfully converted a set of highly resistant cells to a state of complete susceptibility to EHV-1. From this converted set of cells, we amplified and sequenced the incorporated equine cDNA. The sequencing results revealed that the equine cDNA isolated from our screen codes for Equus caballus major histocompatibility complex class I (MHC-I) protein, and further assays confirmed that this receptor is utilized by EHV-1 for entry into cells.