Comparative Analysis of Glycoprotein B (gB) of Equine Herpesvirus Type 1 and Type 4 (EHV-1 and EHV-4) in Cellular Tropism and Cell-to-Cell Transmission

Abstract

Glycoprotein B (gB) plays an important role in alphaherpesvirus cellular entry and acts in concert with gD and the gH/gL complex. To evaluate whether functional differences exist between gB1 and gB4, the corresponding genes were exchanged between the two viruses. The gB4-containing-EHV-1 (EHV-1_gB4) recombinant virus was analyzed for growth in culture, cell tropism, and cell entry rivaling no significant differences when compared to parental virus. We also disrupted a potential integrin-binding motif, which did not affect the function of gB in culture. In contrast, a significant reduction of plaque sizes and growth kinetics of gB1-containing-EHV-4 (EHV-4_gB1) was evident when compared to parental EHV-4 and revertant viruses. The reduction in virus growth may be attributable to the loss of functional interaction between gB and the other envelope proteins involved in virus entry, including gD and gH/gL. Alternatively, gB4 might have an additional function, required for EHV-4 replication, which is not fulfilled by gB1. In conclusion, our results show that the exchange of gB between EHV-1 and EHV-4 is possible, but results in a significant attenuation of virus growth in the case of EHV-4_gB1. The generation of stable recombinant viruses is a valuable tool to address viral entry in a comparative fashion and investigate this aspect of virus replication further.

Role of gB and pUS3 in Equine Herpesvirus 1 Transfer between Peripheral Blood Mononuclear Cells and Endothelial Cells: a DynamicIn VitroModel

“…It also supports our hypothesis that EHV-1 and EHV-4 go through distinctly different intracellular processes and differentially manipulate PBMC after entry. The exchange of gB in EHV-1 with its EHV-4 counterpart had a marked effect on viral transfer between infected PBMC and EC, despite the exchange having no significant effect on viral growth in vitro (20). EHV1_gB4 showed a significantly attenuated ability to transfer to the EC monolayer, indicating an important role of EHV-1 gB in this process of virus transfer and PBMC aggregation in vitro.…”

“…All viruses used in the study were recovered from infectious bacterial artificial chromosome (BAC) clones. Those were BACs of EHV-1 strain Ab4 (33) and EHV-4 strain TH20p (34), as well as modified BACs EHV-1_gB4, EHV-4_gB1, revertant EHV-1_gB1r (20), EHV1_gD4, EHV-4_gD1 (35), EHV-1⌬US3, and EHV-1 that contained US3 of EHV-4 (EHV-1_US3_4) in lieu of authentic US3, fully rescuing the parental EHV-1 phenotype and functioning as revertant for this study (A. Proft and W. Azab, unpublished data). monomeric red fluorescent protein (mRFP1)-labeled EHV-1 was previously constructed by inserting mRFP1 into VP26 of EHV-1 strain RacL11 (36).…”

“…There are certain viral proteins that likely play key roles during the transfer from PBMC to EC. The viral glycoprotein B (gB) is one of these candidates, as it is known to be essential for direct cell-to-cell spread of EHV-1 and EHV-4 by facilitating membrane fusion between neighboring cells (19)(20)(21). We posit that it is very likely that gB may take on a similar role in biologically relevant cells by mediating virus transfer from PBMC to EC.…”

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

“…Herpesvirus gB homologues are highly conserved and are essential for replication (Cai et al, 1988;Herrold et al, 1996;Neubauer et al, 1997;Peeters et al, 1992;Rauh & Mettenleiter, 1991;Spiesschaert et al, 2015). Across most of the members of the herpesvirus subfamilies, gB seems to possess a conserved cleavage site Arg-X-Lys/ Arg-Arg targeted by the cellular protease furin (Backovic et al, 2007;Glauser et al, 2013;Okazaki, 2007;Oliver et al, 2009;Sorem & Longnecker, 2009).…”

“…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 ).…”