Glycoproteins D of Equine Herpesvirus Type 1 (EHV-1) and EHV-4 Determine Cellular Tropism Independently of Integrins

Abstract

Equine herpesvirus type 1 (EHV-1) and EHV-4 are genetically and antigenically very similar, but their pathogenic potentials are strikingly different. The differences in pathogenicity between both viruses seem to be reflected in cellular host range: EHV-1 can readily be propagated in many cell types of multiple species, while EHV-4 entry and replication appear to be restricted mainly to equine cells. The clear difference in cellular tropism may well be associated with differences in the gene products involved in virus entry and/or spread from cell to cell. Here we show that (i) most of the EHV-1 permissive cell lines became resistant to EHV-1 expressing EHV-4 glycoprotein D (gD4) and the opposite was observed for EHV-4 harboring EHV-1 gD (gD1). (ii) The absence of integrins did not inhibit entry into and replication of EHV-1 in CHO-K1 or peripheral blood mononuclear cells (PBMC). Furthermore, integrin-negative K562 cells did not acquire the ability to bind to gD1 when ␣V␤3 integrin was overexpressed. (iii) PBMC could be infected with similar efficiencies by both EHV-1 and EHV-4 in vitro. (iv) In contrast to results for equine fibroblasts and cells of endothelial or epithelial origin, we were unable to block entry of EHV-1 or EHV-4 into PBMC with antibodies directed against major histocompatibility complex class I (MHC-I), a result that indicates that these viruses utilize a different receptor(s) to infect PBMC. Cumulatively, we provide evidence that efficient EHV-1 and EHV-4 entry is dependent mainly on gD, which can bind to multiple cell surface receptors, and that gD has a defining role with respect to cellular host range of EHV-1 and EHV-4.

Glycoprotein B of equine herpesvirus type 1 has two recognition sites for subtilisin-like proteases that are cleaved by furin

“…The generated BACs were maintained in Escherichia coli GS1783 (a kind gift from Dr Greg Smith, NorthWestern University, Chicago, IL, USA), which harbours the recombination system of phage l under the control of a temperature-sensitive repressor (Lee et al, 2001). Deletion or replacement of the furin cleavage motif(s) was facilitated by two-step recombination as described previously (Azab & Osterrieder, 2012;Spiesschaert et al, 2015;Tischer et al, 2006). Briefly, mutagenesis primers (Table 1) were used to generate homology arms (50 nt) by PCR enabling the deletion or substitution of the furin motif and insertion of the kanamycin resistance gene (Kan R ).…”

Glycoprotein H and α4β1 Integrins Determine the Entry Pathway of Alphaherpesviruses

“…EHV-1 strain L11⌬gp2 (36); EHV-4 recovered from an infectious bacterial artificial chromosome (BAC) clone (37); EHV-1 mutants harboring gD4 (EHV-1gD4) (20), gH4 (EHV-1gH4) or gH S440A (EHV1gH S440A ) (33); EHV-4 harboring gD1 (EHV-4gD1) (20) or gH1 (EHV4gH1) (33) were used in this study. All of these viruses express enhanced green fluorescent protein for rapid identification of infected cells.…”

“…Positive clones were subjected to a second round of Red recombination to obtain the final constructs after excision of the gene for Kan r . Finally, all viruses were reconstituted as described before (20).…”

“…Although the two viruses are highly similar in terms of genetic and antigenic structure, differences in cell tropism, host range, and clinical disease are well known (20)(21)(22). As is the case with HSV-1, EHV-1 can enter some cells, such as rabbit kidney (RK13) and equine dermal (ED) cells, through direct fusion with the plasma membrane at neutral pH, a process that is mediated by gC, gD, gB, and the gH/gL complex (23)(24)(25).…”

“…Several viruses, including some herpesviruses, utilize integrins for entry into cells, and examples include Epstein-Barr virus (EBV) (30), human cytomegalovirus (HCMV) (31), and Kaposi's sarcomaassociated herpesvirus (KSHV) (32). Recently, we showed that different integrins, including ␣V␤3, ␣V␤5, ␣4␤1, and ␣4␤7, have no measurable effect on EHV-1 or EHV-4 infection (20,33). Integrin interaction with extracellular matrix proteins lead to a series of signaling events that involve the activation of focal adhesion kinase, c-Src kinase, phosphatidylinositol 3-kinase, and cytoskeletal proteins such as paxillin (26,29,34,35).…”