title: Functional Relevance of the N-Terminal Domain of Pseudorabies Virus Envelope Glycoprotein H and Its Interaction with Glycoprotein L.
abstract: Several envelope glycoproteins are involved in herpesvirus entry into cells, direct cell-to-cell spread, and induction of cell fusion. The membrane fusion protein glycoprotein B (gB) and the presumably gB-activating heterodimer gH/gL are essential for these processes and conserved throughout theHerpesviridaeHowever, 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 and 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 inin vitrofusion 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 vitroactivity of mutated gH32/98was restored when it was coexpressed 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 gBB4.1incis, as well as intrans, independent of gL. Thus, PrV gL and the gL-interacting domain of gH are not strictly required for function.IMPORTANCEMembrane fusion is crucial for infectious entry and spread of enveloped viruses. While many enveloped viruses require only one or two proteins for receptor binding and membrane fusion, herpesvirus infection depends on several envelope glycoproteins. Besides subfamily-specific receptor binding proteins, the core fusion machinery consists of the conserved fusion protein gB and the gH/gL complex. The role of the latter is unclear, but it is hypothesized to interact with gB for fusion activation. Using isogenic virus recombinants, we demonstrate here that gL and the gL-binding domain of PrV gH are not strictly required for membrane fusion during virus entry and spread when concomitantly mutations in gB are present which increase its fusogenicity. Thus, our results strongly support the notion of a functional gB-gH interaction during the fusion process.
authors.affiliation: Institute of Molecular Virology and Cell Biology, Friedrich-Loeffler-Institut, Greifswald-Insel Riems, Germany.
authors.forename: Melina
authors.initials: M
authors.lastname: Vallbracht
authors.affiliation: Institute of Molecular Virology and Cell Biology, Friedrich-Loeffler-Institut, Greifswald-Insel Riems, Germany.
authors.forename: Sascha
authors.initials: S
authors.lastname: Rehwaldt
authors.affiliation: Institute of Molecular Virology and Cell Biology, Friedrich-Loeffler-Institut, Greifswald-Insel Riems, Germany.
authors.forename: Barbara G
authors.initials: BG
authors.lastname: Klupp
authors.affiliation: Institute of Molecular Virology and Cell Biology, Friedrich-Loeffler-Institut, Greifswald-Insel Riems, Germany thomas.mettenleiter@fli.de.
authors.forename: Thomas C
authors.initials: TC
authors.lastname: Mettenleiter
authors.affiliation: Institute of Molecular Virology and Cell Biology, Friedrich-Loeffler-Institut, Greifswald-Insel Riems, Germany.
authors.forename: Walter
authors.initials: W
authors.lastname: Fuchs
mesh_terms: D000818:Animals; D002460:Cell Line; D011558:Herpesvirus 1, Suid / Q000235:genetics* / Q000378:metabolism; D008561:Membrane Fusion / Q000235:genetics*; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D011817:Rabbits; D017384:Sequence Deletion / Q000235:genetics; D014759:Viral Envelope Proteins / Q000235:genetics* / Q000378:metabolism*; D053585:Virus Attachment; D053586:Virus Internalization; D014779:Virus Replication / Q000235:genetics
chemical_list: D014759:Viral Envelope Proteins; C068731:glycoprotein gH, pseudorabies virus
keywords: glycoprotein B; glycoproteins gH/gL; herpesvirus; membrane fusion; pseudorabies virus; virus entry