Two Separate Tyrosine-Based YXXL/Φ Motifs within the Glycoprotein E Cytoplasmic Tail of Bovine Herpesvirus 1 Contribute in Virus Anterograde Neuronal Transport

Abstract

Bovine herpesvirus 1 (BHV-1) causes respiratory infection and abortion in cattle. Following a primary infection, BHV-1 establishes lifelong latency in the trigeminal ganglia (TG). Periodic reactivation of the latent virus in TG neurons results in anterograde virus transport to nerve endings in the nasal mucosa and nasal virus shedding. The BHV-1 glycoprotein E cytoplasmic tail (gE-CT) is necessary for virus cell-to-cell spread in epithelial cells and neuronal anterograde transport. Recently, we identified two tyrosine residues, Y467 and Y563, within the tyrosine-based motifs ₄₆₇YTSL₄₇₀ and ₅₆₃YTVV₅₆₆, which, together, account for the gE CT-mediated efficient cell-to-cell spread of BHV-1 in epithelial cells. Here, we determined that in primary neuron cultures in vitro, the individual alanine exchange Y467A or Y563A mutants had significantly diminished anterograde axonal spread. Remarkably, the double-alanine-exchanged Y467A/Y563A mutant virus was not transported anterogradely. Following intranasal infection of rabbits, both wild-type (wt) and the Y467A/Y563A mutant viruses established latency in the TG. Upon dexamethasone-induced reactivation, both wt and the mutant viruses reactivated and replicated equally efficiently in the TG. However, upon reactivation, only the wt, not the mutant, was isolated from nasal swabs. Therefore, the gE-CT tyrosine residues Y467 and Y563 together are required for gE CT-mediated anterograde neuronal transport.

Keywords: BHV-1; YXXL/Φ sorting motifs; anterograde neuronal traffic; compartmentalized primary neurons; gE CT domain; glycoprotein E; microfluidic chambers; reactivation from latency.

Figure 1

(A) Schematic diagram showing the BHV-1 gE protein and its subdomains. The gE extracellular (Ecto), transmembrane (TM), cytoplasmic tail, and acidic (AD) domains are marked. (B) Predicted amino acid sequence spanning the gE CT region of BHV-1 (residue 451–575) showing the two tyrosine (Y)-based YXXL/YXXΦ motifs (₄₆₇YTSL₄₇₀ and ₅₆₃YTVV₅₆₆), serine (S) residues, and an acidic residue cluster (aa 483–502) shown in three sequential segments, AD1, AD2, and AD3, that correspond to the deletion mutants in Figure 2. The gE and its surrounding genes of various gE CT mutants used in this study to elucidate the functional role of gE CT motifs in BHV-1 anterograde neuronal transport were analyzed by sequencing. Further, the mutants were characterized with respect to cell-to-cell spread property, gE subcellular localization, and plaque sizes in non-neuronal cells [17].

Figure 2

Anterograde spread of BHV-1 wt and various gE CT mutant viruses in compartmentalized primary neuronal culture. DRG neurons seeded in the soma compartment of microfluidic chambers were allowed to grow for two weeks, by which time the axons extended across the grooves to the neurite chamber and formed an extensive network there. MDBK cells were plated in the neurite chamber two days before the virus infection of the neurons in the soma chamber. Reconstituted BAC containing (*) vBHV-1WT*, gE CT-null*, gE ΔAD1*, gE ΔAD2*, gE ΔAD3*, gE ₅₃₅SS_536*, gEΔ ₄₅₇YDIL₄₆₀*, gE Y467A, gE Y563A, and gE Y467A/Y563A viruses at an MOI of 50 (approximately 50 PFUs/neuron) were used for each virus infections. Representative fluorescent microscopic images shown were taken at 74 hpi. (Scale bar: 200 μm).

Figure 3

Nasal virus shedding of BHV-1 lox (wt) or BHV-1 Y467A/Y563A in infected rabbits. Quantification of BHV-1 wt and BHV-1 Y467A/Y563A viruses present in nasal swabs of rabbits during the primary infection, latency, and dexamethasone-induced latency reactivation. The viruses were isolated at the indicated time points and titrated by plaque assay on MDBK cells. The data represent averages and standard deviations for each group. Dex refers to the onset of dexamethasone injection. The p-values are indicated as * p < 0.05, ** p < 0.01. and nonsignificant (ns) for p > 0.05. Note that on Dex 7* day, nasal swabs were collected only from the rabbits infected with Y467A/Y563A double mutant virus.

Figure 4

RT-PCR amplification of BHV-1 VP5 (251-bp)-specific mRNA sequence from TGs of rabbits following reactivation. (A) Amplification of 251 bp VP5 transcript sequence from the TGs of BHV-1 lox wt- and gE Y467A/Y563A-infected rabbits following the latency reactivation. The positive control is the VP5 transcript amplified from wt virus-infected MDBK cells, and the negative control is uninfected MDBK cellular RNA. (B) As an internal control, the GAPDH (841-bp) transcript sequence was amplified using the primer pairs 5′-tgttccagtatgattccaccc-3′ (forward) and 5′-tccaccaccctgttgctgta-3′ (reverse) from each respective TG RNA sample. (C) Verification of the virus genomic DNA contamination: The RT-PCR reaction was carried out as in (A) but without the reverse transcription step using the VP5 specific primer pairs 5′-tgcggtctgcgagttcatc-3′ (forward) and 5′-cgccgctcatgttgtactg-3′ (reverse). (D) VP5-specific mRNA levels in TG of wt and mutant-infected rabbits were normalized to the intracellular GAPDH gene transcription levels using the Quantity One software (Bio-Rad laboratories). (E) Statistical analysis of VP-5 transcription in TGs of rabbits infected with BHV1 wt versus gE Y467A/Y563A mutant following latency-reactivation. Data represent the mean ± SEM. The p values, p > 0.05 = nonsignificant (ns).