Equine herpesvirus 1
(EHV-1), a member of the Alphaherpesvirus
subfamily, is the causative agent of respiratory distress, abortion, and neurological disease in horses (1
). Although many aspects of the virus life cycle have been very well characterized (15
), the early events in virus attachment and entry have not been systematically investigated. In particular, while entry receptors for several other alphaherpesviruses, including herpes simplex virus type 1 and 2 (HSV-1 and HSV-2), pseudorabies virus (PRV), and bovine herpesvirus 1 (BHV-1) have been identified, the ability of EHV-1 to use the same or different entry receptors is unknown.
HveA is a member of the tumor necrosis factor (TNF) receptor family and mediates entry of HSV-1 and HSV-2, but not PRV or BHV-1 (11
). HveC (nectin 1) belongs to the immunoglobulin superfamily and mediates entry of all of these alphaherpesviruses (6
). HveB, another immunoglobulin superfamily member, is also a receptor for PRV, HSV-2, and the Rid1 variant of HSV-1 (22
). In addition to HveA, HveB, and HveC, a modified form of heparan sulfate, 3-O
-sulfated heparan sulfate, can mediate the entry of HSV-1 (17
). All of the receptors are recognized by glycoprotein D (gD) that in turn initiates the entry process (3
While a specific entry receptor has yet to be characterized for EHV-1, studies have shown that EHV-1 uses the same subset of viral glycoproteins utilized by other alphaherpesviruses for binding and entry into permissive cells. Similar to HSV-1, positively charged residues in EHV-1 gB and gC bind to heparan sulfate (HS) moieties on the cell surface (16
). After initial attachment, EHV-1 gD is required for virus entry, but no description of its putative cognate receptor has been reported. In addition to gD, gB is also essential for virus penetration, growth, and cell-to-cell spread (13
). gH and gL are essential for HSV-1 and HSV-2 entry (12
), but whether these glycoproteins play a similar role in the EHV-1 entry pathway is unknown.
The aim of this study was to ascertain whether EHV-1 utilizes the same or novel entry receptor(s) compared with other well-characterized alphaherpesviruses. To this end, the infectivity of EHV-1 on a panel of cell lines that express different virus receptors was compared to the known infectivity patterns of other alphaherpesviruses. These cell lines included HSV-1- permissive cells CHO-A (HveA), CHO-C (HveC), and Vero, EHV-1-permissive rabbit kidney cell line RK13, and HSV-1-resistant cell lines CHO-K1 and J1.1-2.
CHO-K1, CHO-A, CHO-C, RK13, and Vero cells were infected with a recombinant EHV-1 (L11ΔgIΔgE), derived from the RacL11 strain, that expresses lacZ
), and the cells were stained with 5-bromo-4-chloro-3-indolyl-β-d-
galactopyranoside (X-Gal) 7 h postinfection (p.i.) to assess entry. L11ΔgIΔgE efficiently entered RK13, CHO-K1, CHO-A, and CHO-C cells but exhibited severely impaired entry into Vero cells (Fig. ). In contrast, an HSV-1 recombinant that expresses lacZ
) did not infect CHO-K1 cells but efficiently entered all other cell lines that express at least one of the HSV-1 entry receptors (Fig. ). At the same multiplicity of infection (MOI), L11ΔgIΔgE was further shown to enter another HSV-resistant cell line, J1.1-2 (Fig. ), as well as HSV-1-resistant CHO-B (HveB) cells (data not shown). An EHV-1 recombinant derived from the KyA strain, KyAΔgp2 (21
), also efficiently entered CHO-K1 and J1.1-2 cells (data not shown). These data showed that EHV-1 entry into cells is independent of any of the known alphaherpesvirus receptors. In addition, the ability of two EHV-1 strains to efficiently enter two different HSV-resistant cell lines, CHO-K1 and J1.1-2, suggests a common mechanism for EHV-1 entry into these cells which is not strain specific. However, from these data, it is unclear whether EHV-1 is utilizing one common entry receptor or two different receptors on these cells.
FIG. 1. EHV-1 entry into cells lacking known alphaherpesvirus receptors. CHO-K1, CHO-A, CHO-C, RK13, and Vero cells were infected with EHV-1 strain L11ΔgIΔgE (A) or HSV-1 strain QOZHG (B) at an MOI of 1. J1.1-2 cells were infected with EHV-1 strain (more ...)
Previous studies by Csellner et al. showed that EHV-1 gD is essential for EHV-1 entry into permissive RK13 cells (4
). To examine whether EHV-1 gD is required for entry into HSV receptor-negative cells, CHO-K1 cells were infected with a gD-null virus EHV-1ΔgD (derived from the HVS25A strain) (4
). Complemented EHV-1ΔgD was passed through noncomplementing RK13 or EHV-1 gD-expressing RK13 (RKgD) cells, and clarified infected-cell lysates were used to infect CHO-K1, Vero, or RK13 cells. As shown in Fig. , gD-complemented virus from RKgD cells [EHV-1ΔgD(gD+
)] was able to efficiently enter CHO-K1 and RK13 cells, while only a few lacZ+
cells were observed on infection with noncomplemented virus [EHV-1ΔgD(gD−
)]. These data supported the conclusion that gD is required for efficient entry of EHV-1 into HSV receptor-negative cells.
FIG. 2. EHV-1 entry into CHO cells is dependent upon gD. CHO-K1, Vero, and RK13 cells were mock infected or infected with a gD-null virus [EHV-1ΔgD(gD−)] or a gD-complemented virus [EHV-1ΔgD(gD+)] for 24 h. Cells were stained with (more ...)
In addition to gD, efficient EHV-1 entry also required the expression of glycosaminoglycans (GAGs), since CHO-K1 cells deficient in GAG synthesis (pgsA745) (10
) were poorly infected with EHV-1 after a 2-h attachment phase (Fig. ). However, the presence of cell surface GAGs was not sufficient for entry, since GAG-expressing Vero cells were highly resistant to EHV-1 infection. Entry of EHV-1 into CHO-K1 cells occurred rapidly, and the kinetics of EHV-1 entry into CHO-K1 cells were similar to that of EHV-1 on the standard permissive host cell line, RK13 (Fig. ). These data corroborate previous work showing that GAG receptor binding is essential for efficient alphaherpesvirus entry (16
FIG. 3. EHV-1 entry kinetics. CHO-K1, pgsA745, RK13, and Vero cells were infected with L11ΔgIΔgE at an MOI of 5. At the indicated time points (shown in minutes p.i. [m.p.i.]), input virus was inactivated by washing cells with glycine buffer. Cells (more ...)
To assay the ability of EHV-1 to replicate in CHO cells, a one-step growth assay was performed. CHO-K1, CHO-A, CHO-C, Vero, or RK13 cells were infected with L11ΔgIΔgE at an MOI of 1 (Fig. ). Cells were washed with acidic buffer at 2 h p.i., and the titers of supernatants harvested at various time points were determined in RK13 cells. At 8 h p.i., the virus titers were similar for CHO-K1, CHO-A, and CHO-C cells, while the titers in Vero cells were reduced 533-fold compared to those in CHO-K1 cells. The reduced titers in Vero cells at early time points corresponded with the decreased entry kinetics of EHV-1 in Vero cells (Fig. ); however, there was no postentry block to replication after EHV-1 entry in these cells. EHV-1 titers in RK13 cells reached a maximum of 3.27 × 107 PFU/ml at 48 h p.i. Compared to EHV-1 titers in RK13 cells, EHV-1 titers in CHO-K1 cells were reduced 72-fold, reaching a maximum of 4.53 × 105 PFU/ml at 48 h p.i. The maximum titers in Vero cells were reduced 744-fold compared to titers in RK13 cells and were reduced 10-fold compared to titers in CHO-K1 cells. These data show that EHV-1 can productively replicate and produce progeny virus in CHO-K1 cells.
FIG. 4. EHV-1 replication in CHO cells. CHO-K1, CHO-A, CHO-C, RK13, and Vero cells were infected with L11ΔgIΔgE at an MOI of 1. Two hours p.i., cells were washed with glycine buffer, and the medium was replaced. Supernatants from infected cells (more ...)
Interestingly, PRV and HSV-2 were previously shown to enter CHO-K1 cells (14
). However, entry into these cells was greatly enhanced when one of the alphaherpesvirus receptors was expressed (6
). Future studies will examine whether the entry receptor utilized by EHV-1 on CHO-K1 cells is similar or distinct from the receptor(s) recognized by HSV-2 and PRV.
In summary, EHV-1 entry is mediated by an uncharacterized receptor that is distinct from all of the previously identified alphaherpesvirus receptors. EHV-1 entry into HSV-resistant cells is dependent upon gD expression, and entry is greatly enhanced in cells that express GAGs. Current experiments are aimed at identifying the EHV-1 entry receptor or receptors.