Functional relevance of the N-terminal domain of pseudorabies virus envelope glycoprotein H and its interaction with glycoprotein L

Several envelope glycoproteins are involved in herpesvirus entry into cells, direct cell-to-cell spread, and induction of cell fusion. The membrane fusion protein gB and the presumably gB-activating heterodimer gH/gL are essential for these processes and conserved throughout the Herpesviridae. However, after extended cell culture passage of gL-negative mutants of the alphaherpesvirus pseudorabies virus (PrV) phenotypic revertants could be isolated which had acquired spontaneous mutations affecting the gL-interacting N-terminal part of the gH ectodomain (gDH, gHB4.1) (B. G. Klupp and T. C. Mettenleiter, J Virol 73:3014 –3022, 1999; C. Schröter, M. Vallbracht, J. Altenschmidt, S. Kargoll, W. Fuchs, B. G. Klupp and T. C. Mettenleiter, J Virol 90:2264-2272, 2016). To investigate the functional relevance of this part of gH in more detail, we introduced an in-frame deletion of 66 codons at the 5’ end of the plasmid-cloned gH gene (gH32/98). The N-terminal signal peptide was retained and the deletion did not affect expression or processing of gH, but abrogated its function in in vitro fusion assays. Insertion of the engineered gH gene into the PrV genome resulted in a defective mutant (pPrV-gH32/98K), which was incapable of entry and spread. Interestingly, in vitro activity of mutated gH32/98 was restored when co-expressed with hyperfusogenic gBB4.1, obtained from a passaged gL-deletion mutant of PrV. Moreover, the entry and spread defects of pPrV-gH32/98K were compensated by the mutations in gBB4.1 in cis, as well as in trans independent of gL. Thus, PrV gL and the gL-interacting domain of gH are not strictly required for function.