ANALYSIS OF THE CONTRIBUTIONS OF THE EQUINE HERPESVIRUS-1 GLYCOPROTEIN GB HOMOLOG TO VIRUS ENTRY AND DIRECT CELL-TO-CELL SPREAD

Experiments to analyze the functions of the equine herpesvirus 1 (EHV- 1) glycoprotein gB were performed. Cell lines which stably expressed e ither the full-length EHV-1 gB or only the extracellular portion of gB (amino acids 1 to 844) were constructed and were termed TCgBf and TCg B Delta, respectively. Using the cell line TCgBf, a gB-negative viral mutant, L11 Delta gB, was generated by replacing a 2.1-kb Bg/II-Nrul f ragment in the EHV-1 strain RacL11 gB with the Escherichia coil LacZ g ene. EHV-1 strain RacL11, the modified live vaccine strain RacH, and L 11 Delta gB were used for functional studies. It was shown that: (i) E HV-1 gB is essential for virus growth in vitro since gB-negative L11 D elta gB exhibited titers of <10 PFU/ml when grown and titrated on nonc omplementing cells. (ii) The cell line expressing truncated gB (TCgB D elta) did not complement for the growth of L11 Delta gB, but the RacH virus grew to titers comparable to those of RacL11 in all cell lines t ested. Since RacH had amino acids 944-980 of gB replaced by 7 missense amino acids as determined by nucleotide sequence analysis, the extrem e carboxyterminus but not a domain between amino acid residues 845 and 943, probably the transmembrane domain, of EHV-1 gB is dispensable fo r virus growth in cultured cells. (iii) Single infected cells but no p laque formation were observed after infection of noncomplementing cell s with L11 Delta gB, demonstrating the requirement of EHV-1 gB for dir ect cell-to-cell spread of infection. (iv) The attachment of gB-negati ve L11 Delta gB virions to target cells was similar to both phenotypic ally complemented L11 Delta gB and parent RacL11 virus. (v) L11 Delta gB viral titers could be enhanced by using the fusogen polyethylene gl ycol (PEG). The increase of L11 Delta gB titers by PEG treatment, howe ver, was considerably lower compared to gB-negative pseudorabies virus , suggesting that EHV-1 gB might not be as Stringently required for vi rus penetration as are its homologs in other Alphaherpesvirinae. (C) 1 997 Academic Press