A glycine-rich bovine herpesvirus 5 (BHV-5) gE-specific epitope within the ectodomain is important for BHV-5 neurovirulence

Abstract

The bovine herpesvirus 5 (BHV-5) gE ectodomain contains a glycine-rich epitope coding region (gE5 epitope), residues 204 to 218, that is significantly different from the corresponding gE region of BHV-1. Deletion of the gE epitope significantly reduced the neurovirulence of BHV-5 in rabbits. Pulse-chase analyses revealed that the epitope-deleted and wild-type gE were synthesized as N-glycosylated endoglycosidase H-sensitive precursors with approximate molecular masses of 85 kDa and 86 kDa, respectively. Like the wild-type gE, epitope-deleted gE complexed with gI and was readily transported from the endoplasmic reticulum. Concomitantly, the epitope-deleted and wild-type gE acquired posttranslational modifications in the Golgi leading to an increased apparent molecular mass of 93-kDa (epitope-deleted gE) and 94-kDa (wild-type gE). The kinetics of mutant and wild-type gE processing were similar, and both mature proteins were resistant to endoglycosidase H but sensitive to glycopeptidase F. The gE epitope-deleted BHV-5 formed wild-type-sized plaques in MDBK cells, and the epitope-deleted gE was expressed on the cell surface. However, rabbits infected intranasally with gE epitope-deleted BHV-5 did not develop seizures, and only 20% of the infected rabbits showed mild neurological signs. The epitope-deleted virus replicated efficiently in the olfactory epithelium. However, within the brains of these rabbits there was a 10- to 20-fold reduction in infected neurons compared with the number of infected neurons within the brains of rabbits infected with the gE5 epitope-reverted and wild-type BHV-5. In comparison, 70 to 80% of the rabbits exhibited severe neurological signs when infected with the gE5 epitope-reverted and wild-type BHV-5. These results indicated that anterograde transport of the gE epitope-deleted virus from the olfactory receptor neurons to the olfactory bulb is defective and that, within the central nervous system, the gE5 epitope-coding region was required for expression of the full virulence potential of BHV-5.

FIG. 1.

Schematic illustration of the construction of a glycine-rich gE epitope-deleted BHV-5. (A) Schematic of the BHV-5 gE gene with the locations of the epitope coding region (residues 204 to 218), cysteine clusters (regions C1 and C2), N-linked glycosylation sites (N), transmembrane domain (TM), acidic domain (AD), and YXXL motifs shown. The positions of primer pairs P1-P2 and P3-P4 are indicated. (B) Scheme of the construction of the gE epitope deletion plasmid. In plasmid pBHV-5 gEΔ204-218/temp, restriction sites for BamHI and KpnI created by primers P2 and P3, respectively, flank the deleted epitope coding region (15 amino acids). As described in Materials and Methods, the reading frame after the deletion point was changed. However, the reading frame was restored by digesting the plasmid DNA with KpnI and BamHI, followed by blunt ending with mung bean nuclease and religation. (C) Primer P1 and P4 sequences, used with P2 and P3, respectively, are shown with the restricted sites created.

FIG. 2.

Immunoblotting analysis of BHV-5 epitope-deleted (BHV-5 gE epitopeΔ) and BHV-5 epitope-reverted (BHV-5 gE epitopeR) viruses. (A) Identification of gE protein in wild-type BHV-5, BHV-5gE epitopeΔ, and BHV-5gE epitopeR viruses by immunoblotting with BHV-5 gE epitope-specific (residues 204 to 218) rabbit polyclonal antibody. (B) Immunoblotting of an identical blot with a rabbit polyclonal antibody specific for the carboxy-terminal 210 amino acids of BHV-1 gE that cross-react with BHV-5 gE (11, 41).

FIG. 3.

Autoradiograph showing pulse-chase analysis of gE in BHV-5 and BHV-5 gE epitopeΔ virus-infected cell lysates. Infected MDBK cells were pulse-labeled for 30 min in 100 μCi of [³⁵S]cysteine-methionine per ml beginning at 6 h postinfection. Cell monolayers were then washed twice with serum-free DMEM and incubated in complete growth medium without labeled cysteine and methionine. Detergent extracts were prepared from identical samples harvested at times ranging from 0 to 240 min following the labeling period. Extracts were then subjected to immunoprecipitation with gE-specific rabbit antiserum (11, 41), and the precipitated proteins were separated by SDS-PAGE (10% gel). Mock, BHV-5, and BHV-5gE epitopeΔ (leftmost three lanes) represent cell lysates after steady-state labeling for 10 h beginning at 6 h postinfection. Note that in the case of gE epitope-deleted BHV-5, both the mature (93 kDa) and precursor forms (85 kDa) of gE are slightly smaller (approximately 1 kDa), which corresponds to the predicted molecular sizes of the deleted epitope coding region. The 62-, 46-, and 45-kDa proteins representing mature gI, gI precursor (pgI), and a proteolytic cleavage fragment of mature gI (gI^C), respectively, are indicated.

FIG. 4.

EndoH digestion of gE and gI in BHV-5gE epitopeΔ and wild-type BHV-5. Infected MDBK cells were steady-state labeled or pulse-labeled for 30 min and chased for 0, 15, or 30 min as in Fig. 3. Lysates of labeled cells were immunoprecipitated with gE-specific polyclonal rabbit serum and adjusted to 0.5% SDS. Samples were boiled for 10 min and digested with EndoH (+) as described in Materials and Methods. SDS-PAGE of the samples was performed as described for Fig. 3. For a control, untreated samples (−) are shown. Note that the 46-kDa precursor gI was sensitive to EndoH after 0, 15, and 30 min of chase (yielding a faster-migrating 44.5-kDa band), while the 45-kDa gI^C is EndoH resistant in steady-state samples.

FIG. 5.

Surface expression of gE in cells infected with wild-type and BHV-5 gE epitopeΔ viruses. MDBK cells were infected with wild-type and BHV-5 gE epitopeΔ at 5 PFU per cell. At 8 h postinfection, the cells were fixed, permeabilized, reacted with anti-gE polyclonal antibody, and stained with Cy2-conjugated donkey anti-rabbit immunoglobulin G. In the case of the Cy2 control (data not shown), the slides were incubated with PBS prior to staining with Cy2. The anti-gE antibody used for surface labeling is specific for the carboxy-terminal 210 amino acids of BHV-1 gE (41). This antibody is not suitable for surface labeling without permeabilization. Samples were examined with a Bio-Rad MRC1024Es confocal laser scanning microscope. Confocal images were collected with a 100× objective with a 488=nm laser line for excitation and 522-nm emission filter, with the phase contrast mode of the Bio-Rad LaserSharp imaging program. Arrows point to surface labeling. Bar, 20 μm.

FIG. 6.

Localization of viral antigen in brain sections. Animals were inoculated intranasally with BHV-5gE epitopeΔ, BHV-5 gE-deleted (gEΔ), BHV-5 gE epitope R, or BHV-5 wild-type (WT) viruses as described in Materials and Methods. The animals were euthanized on days 6, 8, 10, and 12 postinfection for BHV-5 gE epitopeΔ, 10 and 12 days postinfection for BHV-5gEΔ, or when they showed neurological signs for BHV-5 gE epitopeR and wild-type BHV-5 (8 to 10 days postinfection). Their brains were processed for immunohistochemical analysis as described in Materials and Methods. Sections for BHV-5 gE epitopeΔ and BHV-5gEΔ are from day 12 postinfection. BHV-5gE epitopeR and wild-type virus-infected rabbit brain sections are from day 10 postinfection. Representative sections of the anterior olfactory nucleus (AON), piriform cortex (PIR), and amygdala (AMYG) are shown. Bar in anterior olfactory nucleus and piriform cortex, 1,000 μm: bar in amygdala, 500 μm.

FIG. 7.

Localization of viral antigen in representative sections showing the hippocampus (HIPPO), lateral dorsal tegmentum (LDT), and cingulate cortex (CG). In this assay, wild-type and gE BHV-5 epitopeR spread to the hippocampus, lateral dorsal tegmentum, and cingulate cortex. However, no labeling was found in these areas for gE epitopeΔ and gEΔ BHV-5. Arrows point to infected neurons. Bar in hippocampus, 1,000 μm; bar in lateral dorsal tegmentum and cingulate cortex, 500 μm.

FIG. 8.

Localization of virus-specific antigens by immunofluorescence-confocal microscopy in the processes of olfactory receptor neurons of rabbits infected with BHV-5 gE epitopeR, BHV-5 gE epitopeΔ, and BHV-5 gEΔ viruses. Viral antigen (red fluorescence) as detected by BHV-5-specific bovine polyclonal antibody is seen in the processes of olfactory receptor neuronal cells (marked by arrows). The processes are labeled with neurofilament-specific antibodies (green fluorescence). Merged images show colocalization of viral and neurofilament antigen. Bar, 30 μm.