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Pseudorabies virus glycoprotein M inhibits membrane fusion
B G Klupp1, R Nixdorf, T C Mettenleiter
1Institute of Molecular Biology, Friedrich-Loeffler-Institutes, Federal Research Centre for Virus Diseases of Animals, D-17498 Insel Riems, Germany.
Abstract:
A transient transfection-fusion assay was established to investigate membrane fusion mediated by pseudorabies virus (PrV) glycoproteins. Plasmids expressing PrV glycoproteins under control of the immediate-early 1 promoter-enhancer of human cytomegalovirus were transfected into rabbit kidney cells, and the extent of cell fusion was quantitated 27 to 42 h after transfection. Cotransfection of plasmids encoding PrV glycoproteins B (gB), gD, gH, and gL resulted in formation of polykaryocytes, as has been shown for homologous proteins of herpes simplex virus type 1 (HSV-1) (A. Turner, B. Bruun, T. Minson, and H. Browne, J. Virol. 72:873-875, 1998). However, in contrast to HSV-1, fusion was also observed when the gD-encoding plasmid was omitted, which indicates that PrV gB, gH, and gL are sufficient to mediate fusion. Fusogenic activity was enhanced when a carboxy-terminally truncated version of gB (gB-008) lacking the C-terminal 29 amino acids was used instead of wild-type gB. With gB-008, only gH was required in addition for fusion. A very rapid and extended fusion was observed after cotransfection of plasmids encoding gB-008 and gDH, a hybrid protein consisting of the N-terminal 271 amino acids of gD fused to the 590 C-terminal amino acids of gH. This protein has been shown to substitute for gH, gD, and gL function in the respective viral mutants (B. G. Klupp and T. C. Mettenleiter, J. Virol. 73:3014-3022, 1999). Cotransfection of plasmids encoding PrV gC, gE, gI, gK, and UL20 with gB-008 and gDH had no effect on fusion. However, inclusion of a gM-expressing plasmid strongly reduced the extent of fusion. An inhibitory effect was also observed after inclusion of plasmids encoding gM homologs of equine herpesvirus 1 or infectious laryngotracheitis virus but only in conjunction with expression of the gM complex partner, the gN homolog. Inhibition by PrV gM was not limited to PrV glycoprotein-mediated fusion but also affected fusion induced by the F protein of bovine respiratory syncytial virus, indicating a general mechanism of fusion inhibition by gM.
Insights
Pseudorabies virus (PrV) glycoproteins B, H, and L are sufficient for membrane fusion, with truncated gB enhancing this activity. PrV glycoprotein M inhibits fusion mediated by various viral proteins.
Area of Science:
- Virology
- Molecular Biology
- Cell Biology
Background:
- Membrane fusion is critical for viral entry and spread.
- Pseudorabies virus (PrV) glycoproteins play a role in cell-cell fusion.
- Understanding the specific roles of PrV glycoproteins in fusion is essential.
Purpose of the Study:
- To establish a transient transfection-fusion assay for studying PrV-mediated membrane fusion.
- To identify the minimal set of PrV glycoproteins required for cell fusion.
- To investigate the role of specific PrV glycoproteins, including truncated versions and hybrid proteins, in fusion.
Main Methods:
- Transient transfection of rabbit kidney cells with plasmids encoding PrV glycoproteins.
- Quantitation of cell fusion (polykaryocyte formation) at 27-42 hours post-transfection.
- Use of wild-type and truncated glycoproteins (gB-008), and a hybrid protein (gDH).
Main Results:
- PrV glycoproteins B (gB), gH, and gL were sufficient for cell fusion, unlike homologous HSV-1 proteins that require gD.
- A truncated gB (gB-008) enhanced fusion, requiring only gH in addition.
- PrV glycoprotein M (gM) significantly inhibited fusion, including fusion mediated by other viral proteins, suggesting a general inhibitory mechanism.
Conclusions:
- PrV gB, gH, and gL are sufficient for mediating membrane fusion.
- Truncated gB (gB-008) and a gD-gH hybrid protein (gDH) enhance fusogenic activity.
- PrV gM acts as a general inhibitor of viral glycoprotein-mediated membrane fusion.