Glycoprotein gL-independent infectivity of pseudorabies virus is mediated by a gD-gH fusion protein

Envelope glycoproteins gH and gL, which form a complex, are conserved throu ghout the family Herpesviridae. The gH-gL complex is essential for the fusi on between the virion envelope and the cellular cytoplasmic membrane during penetration and is also required for direct viral cell-to-cell spread from infected to adjacent noninfected cells. It has been proposed for several h erpesviruses that gL is required for proper folding, intracellular transpor t, and virion localization of gH. In pseudorabies virus (PrV), glycoprotein gL is necessary for infectivity but is dispensable for virion localization of gH. A virus mutant lacking gL, PrV-gL beta, is defective in entry into target cells, and direct cell-to-cell spread is drastically reduced, result ing in only single or small foci of infected cells (B. G, Klupp, W, Fuchs, E. Weiland, and T. C. Mettenleiter, J. Virol. 71:7687-7695, 1997), We used this limited cell-to-cell spreading ability of PrV-Delta gL beta for serial passaging of cells infected with transcomplemented virus by coseeding with noninfected cells. After repeated passaging, plaque formation was restored and infectivity in the supernatant was observed. One single-plaque isolate , designated PrV-Delta gLPass, was further characterized, To identify the m utation leading to this gL-independent infectious phenotype, Southern and W estern blot analyses, radioimmunoprecipitations, and DNA sequencing were pe rformed. The results showed that rearrangement of a genomic region comprisi ng part of the gH gene into a duplicated copy of part of the unique short r egion resulted in a fusion fragment predicted to encode a protein consistin g of the N-terminal 271 amino acids of go fused to the C-terminal 590 resid ues of gH. Western blotting and radioimmunoprecipitation with gD- and gH-sp ecific antibodies verified the presence of a gDH fusion protein, To prove t hat this fusion protein mediates infectivity of PrV-Delta gLPass, cotransfe ction of PrV-Delta gL beta DNA with the cloned fusion fragment was performe d, and a cell line, Nde-67, carrying the fusion gene was established, After cotransfection, infectious gL-negative PrV was recovered, and propagation of PrV-Delta gL beta on Nde-67 cells produced infectious virions. Thus, a g DH fusion polypeptide can compensate for function of the essential gL in en try and cell-to-cell spread of PrV.