Structure-function analysis of herpes simplex virus type 1 gD and gH-gL: clues from gDgH chimeras.
In alphaherpesviruses, glycoprotein B (gB), gD, gH, and gL are essential for virus entry. A replication-competent gL-null pseudorabies virus (PrV) (B. G. Klupp and T. C. Mettenleiter, J. Virol. 73:3014-3022, 1999) was shown to express a gDgH hybrid protein that could replace gD, gH, and gL in cell-cell fusion and null virus complementation assays. To study this phenomenon in herpes simplex virus type 1 (HSV-1), we constructed four gDgH chimeras, joining the first 308 gD amino acids to various gH N-terminal truncations. The chimeras were named for the first amino acid of gH at which each was truncated: 22, 259, 388, and 432. All chimeras were immunoprecipitated with both gD and gH antibodies to conformational epitopes. Normally, transport of gH to the cell surface requires gH-gL complex formation. Chimera 22 contains full-length gH fused to gD308. Unlike PrV gDgH, chimera 22 required gL for transport to the surface of transfected Vero cells. Interestingly, although chimera 259 failed to reach the cell surface, chimeras 388 and 432 exhibited gL-independent transport. To examine gD and gH domain function, each chimera was tested in cell-cell fusion and null virus complementation assays. Unlike PrV gDgH, none of the HSV-1 chimeras substituted for gL for fusion. Only chimera 22 was able to replace gH for fusion and could also replace either gH or gD in the complementation assay. Surprisingly, this chimera performed very poorly as a substitute for gD in the fusion assay despite its ability to complement gD-null virus and bind HSV entry receptors (HveA and nectin-1). Chimeras 388 and 432, which contain the same portion of gD as that in chimera 22, substituted for gD for fusion at 25 to 50% of wild-type levels. However, these chimeras functioned poorly in gD-null virus complementation assays. The results highlight the fact that these two functional assays are measuring two related but distinct processes.
Cairns
Tina M
TM
Department of Microbiology, School of Dental Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA. tmcairns@biochem.dental.upenn.edu
Milne
Richard S B
RS
Ponce-de-Leon
Manuel
M
Tobin
Deanna K
DK
Cohen
Gary H
GH
Eisenberg
Roselyn J
RJ
D000818:Animals; D015815:Cell Adhesion Molecules / Q000378:metabolism; D002459:Cell Fusion; D002460:Cell Line; D002522:Chlorocebus aethiops; D018259:Herpesvirus 1, Human / Q000235:genetics / Q000378:metabolism / Q000472:pathogenicity*; D006801:Humans; D000075983:Nectins; D011233:Precipitin Tests; D018124:Receptors, Tumor Necrosis Factor / Q000378:metabolism; D053240:Receptors, Tumor Necrosis Factor, Member 14; D011991:Receptors, Virus / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014709:Vero Cells; D014759:Viral Envelope Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D015815:Cell Adhesion Molecules; C000619010:NECTIN1 protein, human; D000075983:Nectins; D018124:Receptors, Tumor Necrosis Factor; D053240:Receptors, Tumor Necrosis Factor, Member 14; D011991:Receptors, Virus; D011993:Recombinant Fusion Proteins; C506028:TNFRSF14 protein, human; D014759:Viral Envelope Proteins; C039447:glycoprotein D, Human herpesvirus 1; C044200:glycoprotein H, herpes simplex virus type 1; C077407:glycoprotein L, Human herpesvirus 1