Restoration of function of carboxy-terminally truncated pseudorabies virusglycoprotein B by point mutations in the ectodomain

Glycoprotein B (gB) of pseudorabies virus (PrV) is essential for virus entr y into target cells and direct viral cell-to-cell spread. Recently, we desc ribed a carboxy-terminally truncated derivative of PrV gB, gB-007, which wa s inefficiently incorporated into virions, was unable to complement infecti vity, but was fully capable of restoring direct viral cell-to-cell spread o f gB-negative PrV (R. Nixdorf, B. G. Klupp, and T. C. Mettenleiter, J. Viro l. 74:7137-7145, 2000). Since recombinant PrV-007, which expresses gB-007 i nstead of wild-type gB, was able to spread directly from cell to cell, we a ttempted to obtain compensatory mutations leading to restoration of the ent ry defect by performing serial passages in cell culture. This procedure has previously been used to successfully restore entry defects in gD- or gL-de ficient PrV mutants. From an initial titer of 100 PFU per ml in the superna tant, titers increased, reaching wild-type levels of up to 10(7) PFU after ca. 20 passages. One single-plaque isolate of the passaged mutant, designat ed PrV-007Pass, was further characterized. PrV-007Pass gB was efficiently i ncorporated into the viral envelope and restored infectivity to a gB-negati ve PrV mutant, PrV-gB(-). Interestingly, localization of PrV-007Pass gB in the plasma membrane was similar to that of PrV-007. In contrast, wild-type gB is mainly found in intracellular vesicles. Marker rescue experiments and transcomplementation assays demonstrated the presence of compensatory muta tions within the gB gene of PrV-007Pass. DNA sequencing revealed two point mutations in the gB open reading frame of PrV-007Pass, resulting in amino a cid substitutions at positions 305 and 744 of gB, both of which are require d for compensation of the defect in PrV-007. Our data again demonstrate the power of reversion analysis of herpesviruses and suggest that cytosolic an d ectodomains play a role in incorporation of gB into virions.