Two novel single amino acid syncytial mutations in the carboxy terminus of glycoprotein B of herpes simplex virus type 1 confer a unique pathogenic phenotype.

We previously reported on a variant of the herpes simplex type 1 (HSV-1) strain 17 syn+, named 17 hep syn, capable of forming giant polykaryocytes (syncytia) in tissue culture and which induced a striking alteration in the pathogenesis of infection in vivo. Following footpad inoculation of mice, 17 hep syn infection resulted in a marked clinicopathologic acute inflammatory response of the inoculated limb and mice died without antecedant limb paralysis typical of the wild-type 17 syn+ infection. The syncytial and pathogenic phenotypes were mapped to a cloned 670-base pair Kpnl-Pstl (0.345-0.351 map units) DNA fragment encoding the carboxy terminal portion of the glycoprotein B (gB). In this report, we focus on the genetics of the region of the 17 hep syn gB gene that conferred both the syncytial and pathogenic phenotypes to 17 syn+. Five 17 syn+ x 17 hep syn syncytial recombinant viruses, R1-R5, generated in marker transfer experiments with cloned 17 hep syn fragments containing gB sequences, produced 17 hep syn-like disease in mice. Sequence analysis of the Kpnl-Pstl fragment of 17 hep syn revealed a single base pair change when compared to the 17 syn+ sequence, predicting an alanine (GCC codon) to valine (GTC codon) amino acid substitution at residue 825 of the mature gB protein, plus loss of an Ncol restriction endonuclease site. Southern blot analysis of Ncol digests of viral DNAs showed that all of the recombinants except R4 contained the same mutation as 17 hep syn. The syncytial phenotype of R4 was, however, mapped to the same region as 17 hep syn and the other recombinants, and the DNA sequence of the 670-base pair Kpnl-Pstl clone of R4 revealed another single base pair change predicting a leucine (CTC codon) to histadine (CAC codon) amino acid substitution at residue 787 of gB. The mutant gBs did not effect viral growth as all of the recombinant viruses had similar in vitro replication kinetics to wild-type HSV-1. These data provide direct evidence that at least two mutations can exist in the carboxy terminus of gB of HSV-1 that promote syncytial formation in vitro and effect pathogenesis in vivo.