Effects of truncation of the carboxy terminus of pseudorabies virus glycoprotein B on infectivity

Glycoproteins homologous to the type I membrane glycoprotein B (gB) of herp es simplex virus 1 (HSV-1) are the most highly conserved glycoproteins with in the family Herpesviridae and are present in members of each herpesvirus subfamily. In the alphaherpesvirus pseudorabies virus (PrV), gB is required for entry into target cells and for direct viral fell-to-cell spread. Thes e processes, though related, appear to be distinct, and thus it was interes ting to analyze whether they require different functions of gB, To this end , we established cell lines stably expressing different carboxy-terminally truncated versions of PrV gB by deleting either (i) one predicted intracyto plasmic alpha-helical domain encompassing putative YQRL and dileucine inter nalization signals, (ii) two predicted intracytoplasmic alpha-helical domai ns, (iii) the complete intracytoplasmic domain, or (iv) the intracytoplasmi c domain and the transmembrane anchor region. Confocal laser scanning micro scopy showed that gB derivatives lacking at least the last 29 amino acids ( aa) localize close to the plasma membrane, while the full-length protein ac cumulates in intracellular aggregations. Trans-complementation studies with a gB-deleted PrV (PrV-gB(-)) demonstrated that the 29-aa truncated form la cking the putative internalization signals and the C-terminal alpha-helical domain (gB-008) was efficiently incorporated into PrV-gB(-) virions and ef ficiently complemented infectivity and cell-to-cell spread, Moreover, gB-00 8 exhibited an enhanced fusogenic activity. In contrast, gB proteins lackin g both alpha-helical domains (gB-007), the complete intracytoplasmic domain , or the intracytoplasmic domain and transmembrane anchor were only ineffic iently or not at all incorporated into PrV-gB(-) virions and did not comple ment infectivity, However, gB-007 was able to mediate cell-to-cell spread o f PrV-gB(-). Similar phenotypes were observed when virus recombinants expre ssing gB-008 or gB-007, respectively, instead of wild-type gB were isolated and analyzed. Thus, our data show that internalization of gB is not requir ed for gB incorporation into virions nor for its function in either entry o r cell-to-cell spread. Moreover, they indicate different requirements for g B in these membrane fusion processes.