Restoration of function of carboxy-terminally truncated pseudorabies virus glycoprotein B by point mutations in the ectodomain.

Glycoprotein B (gB) of pseudorabies virus (PrV) is essential for virus entry into target cells and direct viral cell-to-cell spread. Recently, we described a carboxy-terminally truncated derivative of PrV gB, gB-007, which was inefficiently incorporated into virions, was unable to complement infectivity, but was fully capable of restoring direct viral cell-to-cell spread of gB-negative PrV (R. Nixdorf, B. G. Klupp, and T. C. Mettenleiter, J. Virol. 74:7137-7145, 2000). Since recombinant PrV-007, which expresses gB-007 instead of wild-type gB, was able to spread directly from cell to cell, we attempted to obtain compensatory mutations leading to restoration of the entry defect by performing serial passages in cell culture. This procedure has previously been used to successfully restore entry defects in gD- or gL-deficient PrV mutants. From an initial titer of 100 PFU per ml in the supernatant, titers increased, reaching wild-type levels of up to 10(7) PFU after ca. 20 passages. One single-plaque isolate of the passaged mutant, designated PrV-007Pass, was further characterized. PrV-007Pass gB was efficiently incorporated into the viral envelope and restored infectivity to a gB-negative PrV mutant, PrV-gB(-). Interestingly, localization of PrV-007Pass gB in the plasma membrane was similar to that of PrV-007. In contrast, wild-type gB is mainly found in intracellular vesicles. Marker rescue experiments and trans-complementation assays demonstrated the presence of compensatory mutations within the gB gene of PrV-007Pass. DNA sequencing revealed two point mutations in the gB open reading frame of PrV-007Pass, resulting in amino acid substitutions at positions 305 and 744 of gB, both of which are required for compensation of the defect in PrV-007. Our data again demonstrate the power of reversion analysis of herpesviruses and suggest that cytosolic and ectodomains play a role in incorporation of gB into virions.

Pseudorabies virus glycoprotein M inhibits membrane fusion.

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.

Effects of truncation of the carboxy terminus of pseudorabies virus glycoprotein B on infectivity.

Glycoproteins homologous to the type I membrane glycoprotein B (gB) of herpes simplex virus 1 (HSV-1) are the most highly conserved glycoproteins within the family Herpesviridae and are present in members of each herpesvirus subfamily. In the alphaherpesvirus pseudorabies virus (PrV), gB is required for entry into target cells and for direct viral cell-to-cell spread. These processes, though related, appear to be distinct, and thus it was interesting to analyze whether they require different functions of gB. To this end, we established cell lines stably expressing different carboxy-terminally truncated versions of PrV gB by deleting either (i) one predicted intracytoplasmic alpha-helical domain encompassing putative YQRL and dileucine internalization signals, (ii) two predicted intracytoplasmic alpha-helical domains, (iii) the complete intracytoplasmic domain, or (iv) the intracytoplasmic domain and the transmembrane anchor region. Confocal laser scanning microscopy showed that gB derivatives lacking at least the last 29 amino acids (aa) localize close to the plasma membrane, while the full-length protein accumulates in intracellular aggregations. Trans-complementation studies with a gB-deleted PrV (PrV-gB(-)) demonstrated that the 29-aa truncated form lacking the putative internalization signals and the C-terminal alpha-helical domain (gB-008) was efficiently incorporated into PrV-gB(-) virions and efficiently complemented infectivity and cell-to-cell spread. Moreover, gB-008 exhibited an enhanced fusogenic activity. In contrast, gB proteins lacking both alpha-helical domains (gB-007), the complete intracytoplasmic domain, or the intracytoplasmic domain and transmembrane anchor were only inefficiently or not at all incorporated into PrV-gB(-) virions and did not complement infectivity. However, gB-007 was able to mediate cell-to-cell spread of PrV-gB(-). Similar phenotypes were observed when virus recombinants expressing gB-008 or gB-007, respectively, instead of wild-type gB were isolated and analyzed. Thus, our data show that internalization of gB is not required for gB incorporation into virions nor for its function in either entry or cell-to-cell spread. Moreover, they indicate different requirements for gB in these membrane fusion processes.

Infection of Chinese hamster ovary cells by pseudorabies virus.

Chinese hamster ovary (CHO) cells have recently been used for identification of receptors for several alphaherpesviruses, including pseudorabies virus (PrV) (R. J. Geraghty, C. Krummenacher, G. H. Cohen, R. J. Eisenberg, and P. G. Spear, Science 280:1618-1620, 1998). The experiments were based on the fact that CHO cells are inefficient target cells for PrV. However, a detailed analysis of the interaction between PrV and CHO wild-type and recombinant PrV-receptor bearing cells has not been performed. We show here that PrV has a growth defect on CHO cells which leads to a ca. 100-fold reduction in plating efficiency, strongly delayed penetration kinetics, and a 10(4)-fold reduction in one-step growth. Entry of PrV into CHO cells is significantly delayed but is not affected by inhibitors of endocytosis, suggesting that the mechanism of penetration resembles that on permissive cells. The defects in plating efficiency and penetration could be corrected by expression of herpesvirus entry mediators B (HveB), HveC, or HveD, with HveC being the most effective. However, the defects in one-step growth and plaque formation were not corrected by expression of PrV receptors, indicating an additional restriction in viral replication after entry. Surprisingly, PrV infection of CHO cells was sensitive to neutralization by a gB-specific monoclonal antibody, which does not inhibit PrV infection of other host cells. Moreover, the same monoclonal antibody neutralized PrV infectivity on cells displaying the interference phenomenon by overexpression of gD and subsequent intracellular sequestration of gD receptors. Thus, absence of gD receptors on two different host cells leads to an increased sensitivity of PrV toward gB neutralization. We hypothesize that this is due to the increased requirement for interaction of gB with a cellular surface protein in the absence of the gD-gD receptor interaction. As expected, CHO cells are as susceptible as other host cells to infection by PrV gD(-) Pass, an infectious gD-negative PrV mutant. However, PrV gD(-) Pass was also not able to form plaques on CHO cells.