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 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 and gH 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 (gH 32/98 ). 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-gH 32/98 K), which was incapable of entry and spread. Interestingly, in vitro activity of mutated gH 32/98 was restored when it was coexpressed with hyperfusogenic gB B4.1 , obtained from a passaged gL deletion mutant of PrV. Moreover, the entry and spread defects of pPrV-gH 32/98 K were compensated by the mutations in gB B4.1 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. IMPORTANCE Membrane 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. Editor Richard M. Longnecker, Northwestern University

Functional Role of N-Linked Glycosylation in Pseudorabies Virus Glycoprotein gH

“…The oligonucleotide primers used for mutagenesis leading to inactivation of the potential N-glycosylation sites are indicated in Table 1. The resulting expression plasmids were digested with DrdI, and a 3,461-bp fragment containing the modified gH gene together with a downstream kanamycin resistance (Kan r ) gene was purified from an agarose gel and used for Red-mediated mutagenesis of pPrV-ΔgH in E. coli as described previously (30,31,33). Desired BAC recombinants were selected on LB agar plates supplemented with kanamycin (50 g/ml) and chloramphenicol (30 g/ml).…”

“…In vitro fusion assays. The fusogenic properties of the gH mutants were analyzed using a transienttransfection-based cell fusion assay (30,51). Briefly, approximately 3 ϫ 10 5 RK13 cells per well were seeded onto 12-well cell culture plates and after 20 h at 37°C transfected with 400 ng each of expression plasmids for EGFP (pEGFP-N1; Clontech) and PrV-Ka glycoproteins gB (or C-terminally truncated gB-008), gD, gL, and wild-type or mutagenized gH (29,31,50,52) in 100 l Opti-MEM using 2 l Lipofectamine 2000 as described above.…”

“…5B). N77 is located in the structurally uncharacterized gH domain I, which is important for binding of gL (30). Removal of the carbohydrates from this site might affect optimal interaction of gH with gL and thus interfere with efficient membrane fusion.…”

“…However, in PrV, Bovine herpesvirus 4, and Murid herpesvirus 4, gL is not required for correct folding, transport, or virion incorporation of gH (22)(23)(24)(25)(26)(27). Moreover, infection by PrV can occur in the absence of gL and the gL-binding domain of gH when compensatory mutations in other glycoproteins are present (28)(29)(30). In addition, the absence of gL obviously facilitates maturation of certain N-glycans of PrV gH, which are possibly masked during wild-type (WT) replication (25).…”

Functional Relevance of the Transmembrane Domain and Cytoplasmic Tail of the Pseudorabies Virus Glycoprotein H for Membrane Fusion

“…In contrast to that of HSV (49), PrV gL is not necessary for correct transport, processing, or virion incorporation of gH and, moreover, the gL-binding domain in PrV gH is dispensable for the fusion process in the presence of compensatory mutations in gH and/or gB (31,33). Reports on HSV-1 indicated that soluble gH/gL, lacking the membrane anchor, is sufficient to mediate low levels of membrane fusion in transient assays (9).…”