Functional Characterization of Glycoprotein H Chimeras Composed of Conserved Domains of the Pseudorabies Virus and Herpes Simplex Virus 1 Homologs

Abstract

Membrane fusion is indispensable for entry of enveloped viruses into host cells. The conserved core fusion machinery of the Herpesviridae consists of glycoprotein B (gB) and the gH/gL complex. Recently, crystal structures of gH/gL of herpes simplex virus 2 (HSV-2) and Epstein-Barr virus and of a core fragment of pseudorabies virus (PrV) gH identified four structurally conserved gH domains. To investigate functional conservation, chimeric genes encoding combinations of individual domains of PrV and herpes simplex virus 1 (HSV-1) gH were expressed in rabbit kidney cells, and their processing and transport to the cell surface, as well as activity in fusion assays including gB, gD, and gL of PrV or HSV-1, were analyzed. Chimeric gH containing domain I of HSV-1 and domains II to IV of PrV exhibited limited fusion activity in the presence of PrV gB and gD and HSV-1 gL, but not of PrV gL. More strikingly, chimeric gH consisting of PrV domains I to III and HSV-1 domain IV exhibited considerable fusion activity together with PrV gB, gD, and gL. Replacing PrV gB with the HSV-1 protein significantly enhanced this activity. A cell line stably expressing this chimeric gH supported replication of gH-deleted PrV. Our results confirm the specificity of domain I for gL binding, demonstrate functional conservation of domain IV in two alphaherpesviruses from different genera, and indicate species-specific interactions of this domain with gB. They also suggest that gH domains II and III might form a structural and functional unit which does not tolerate major substitutions. IMPORTANCEEnvelope glycoprotein H (gH) is essential for herpesvirus-induced membrane fusion, which is required for host cell entry and viral spread. Although gH is structurally conserved within the Herpesviridae, its precise role and its interactions with other components of the viral fusion machinery are not fully understood. Chimeric proteins containing domains of gH proteins from different herpesviruses can serve as tools to elucidate the molecular basis of gH function. The present study shows that the C-terminal part of human herpesvirus 1 (herpes simplex virus 1) gH can functionally substitute for the corresponding part of suid herpesvirus 1 (pseudorabies virus) gH, whereas other tested combinations proved to be nonfunctional. Interestingly, the exchangeable fragment included the membrane-proximal end of the gH ectodomain (domain IV), which is most conserved in sequence and structure and might be capable of transient membrane interaction during fusion. M embrane fusion is an essential process in pro-and eukaryotic organisms and occurs, e.g., during cell division, autophagy, and endo-and exocytosis. For entry into host cells, enveloped viruses depend on fusion of their envelopes with cellular membranes. Fusion can occur at the plasma membrane either immediately after attachment or after endosomal uptake of the enveloped virus particles. Members of the Herpesviridae have been shown to utilize both pathways, but herpes simplex viruses 1 and 2 (HSV-1 an...

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

“…In contrast, the HSV-gH TMD efficiently substituted for the TMD in PrV gH in both assays. This is consistent with previous results showing that the complete HSV-1 gH C-terminal part comprising the conserved domain IV, as well as TMD and CD, could functionally substitute for the corresponding part in PrV gH, pointing to functional conservation (53).…”

“…2A). In contrast to HSV gH, which depends on the presence of gL for correct maturation (52), gL is dispensable for maturation of PrV gH (30,53). Thus, absence of gL did not influence processing of any of the gH proteins (data not shown).…”

“…For constructs with the TMD from HSV-1 gH but comprising the CD from PrV gH, the 3=-terminal part of the PrV gH ORF was amplified from pcDNA-gH using the desired forward OE primers in combination with the reverse primer DHrev1 ( Table 1). The 3=-terminal parts of the HSV-1 gH or PrV gD ORF were amplified from pcDNA-H1FgH (53) or pcDNA-gD (32), respectively, with the appropriate forward OE primers and the reverse primer HHV1 gH-R or US6-rev, respectively ( Table 1). The PCR products were purified, and 10 ng of each of the matching products, which overlapped by Ն15 bp, was mixed and amplified in a final PCR using only the primers for the termini.…”

“…Expression plasmids. Construction of expression plasmids for PrV-Ka and HSV-1 F gB, gH, and gL has been described previously (37,53). Expression plasmids for the truncated versions of gH and soluble gH were generated by PCR, using pcDNA-gH as the template, DH-3 as forward primer, and the corresponding reverse primers gH Δ668-686, gH Δ670-686, gH Δ678-686, gH Δ682-686, and gH ECD R (Table 1).…”

Functional Relevance of the N-Terminal Domain of Pseudorabies Virus Envelope Glycoprotein H and Its Interaction with Glycoprotein L

“…Functional conservation of this domain in PrV and HSV-1 could be demonstrated by analysis of chimeric gH consisting of PrV domains I to III and HSV-1 domain IV. This chimeric protein was not only capable of inducing membrane fusion when expressed together with HSV-1 or PrV gB and with PrV gD and gL but also supported replication of gH-deleted PrV (28). The less conserved domain III (H2) is composed of eight alpha-helices and contains a highly conserved amino acid stretch (serine-proline-cysteine) important for regulation of membrane fusion (29).…”