A bovine herpesvirus 1 protein expressed in latently infected neurons (ORF2) promotes neurite sprouting in the presence of activated Notch1 or Notch3

Abstract

Bovine herpesvirus 1 (BHV-1) infection induces clinical symptoms in the upper respiratory tract, inhibits immune responses, and can lead to life-threatening secondary bacterial infections. Following acute infection, BHV-1 establishes latency in sensory neurons within trigeminal ganglia, but stress can induce reactivation from latency. The latency-related (LR) RNA is the only viral transcript abundantly expressed in latently infected sensory neurons. An LR mutant virus with stop codons at the amino terminus of the first open reading frame (ORF) in the LR gene (ORF2) is not reactivated from latency, in part because it induces higher levels of apoptosis in infected neurons. ORF2 inhibits apoptosis in transiently transfected cells, suggesting that it plays a crucial role in the latency-reactivation cycle. ORF2 also interacts with Notch1 or Notch3 and inhibits its ability to trans activate certain viral promoters. Notch3 RNA and protein levels are increased during reactivation from latency, suggesting that Notch may promote reactivation. Activated Notch signaling interferes with neuronal differentiation, in part because neurite and axon generation is blocked. In this study, we demonstrated that ORF2 promotes neurite formation in mouse neuroblastoma cells overexpressing Notch1 or Notch3. ORF2 also interfered with Notch-mediated trans activation of the promoter that regulates the expression of Hairy Enhancer of Split 5, an inhibitor of neurite formation. Additional studies provided evidence that ORF2 promotes the degradation of Notch3, but not that of Notch1, in a proteasome-dependent manner. In summary, these studies suggest that ORF2 promotes a mature neuronal phenotype that enhances the survival of infected neurons and consequently increases the pool of latently infected neurons.

Fig 1

Expression of Notch and ORF2 in TG. TG were collected from calves that were mock infected, latently infected (at least 60 days postinfection), or latently infected and treated with DEX at the indicated times (hours [H]) after DEX treatment to induce reactivation from latency. Thin sections were cut and subjected to immunohistochemistry analysis with antibodies that specifically recognize Notch3 (A), Notch1 (B), or ORF2 (C) as described in Materials and Methods. Biotinylated goat anti-rabbit IgG was used as a secondary antibody. Thin sections from mock-infected calves were used as a negative control. Arrows indicate positive neurons.

Fig 2

ORF2 antagonizes Notch inhibition of neurite formation. (A) Neuro-2A cells were cotransfected with a plasmid expressing the Notch1 or Notch3 ICD, a plasmid expressing ORF2, and a plasmid expressing the LacZ gene (transfection control). To induce neurite sprouting, 24 h after transfection, cells were seeded into new plates at a low density (2,000/cm²) and then starved in medium with 0.5% serum for 3 days. Cells were then fixed, and β-Gal⁺ cells were identified as described in Materials and Methods. (B) The percentage of β-Gal⁺ cells containing neurites was calculated by dividing the number of β-Gal⁺ cells with a neurite length at least twice the diameter of the cell by the total number of β-Gal⁺ cells. The average of at least three independent experiments is shown with the respective standard deviation. An asterisk denotes significant differences (P < 0.05) in β-Gal⁺ Neuro-2A cells containing neurites following cotransfection with the ORF2 reporter and a Notch1 or Notch3 expression plasmid relative to β-Gal⁺ Neuro-2A cells with neurites following transfection with a plasmid expressing Notch1 or Notch3 plus the empty vector, as determined by the Student t test.

Fig 3

Schematics of the ORF2 and mutant ORF2 constructs used for this study. (A) Amino acid sequence of ORF2. The nuclear localization signal (underlined), 15 putative phosphorylation sites (gray-shaded amino acids) and five consensus protein kinase A and/or protein kinase C phosphorylation sites (gray-shaded amino acids with white lettering) are shown. The plus signs denote every 10th amino acid in ORF2. (B) ORF2 coding sequences (BamHI-SalI) were cloned into the pUC57 vector, and transposon linker insertion reactions were performed as previously described (44). Vertical lines with the respective numbers indicate the nucleotide position of the respective transposon insertions. The relative position of the consensus nuclear localization signal (NLS) is denoted by the white rectangle.

Fig 4

Localization of ORF2 sequences necessary to inhibit neurite formation in the presence of Notch1 or Notch3. Neuro-2A cells were cotransfected with a plasmid expressing the Notch1 (A), or Notch3 (B) ICD, a plasmid expressing WT ORF2 or the designated mutant ORF2 construct, and a plasmid expressing LacZ. Neurite formation was measured as described in the legend to Fig. 2 after serum starvation and the identification of β-Gal⁺ Neuro-2A cells. The results represent the average of at least three independent experiments with the respective standard deviation. An asterisk denotes significant differences (P < 0.05) in β-Gal⁺ Neuro-2A cells containing neurites that were cotransfected with the ORF2 reporter and a Notch1 or Notch3 expression plasmid relative to β-Gal⁺ Neuro-2A cells containing neurites after transfection with a plasmid expressing Notch1 or Notch3 plus the empty vector, as determined by the Student t test.

Fig 5

ORF2 interferes with Notch-mediated trans activation of the HES5 promoter. Neuro-2A cells were cotransfected with a plasmid containing the firefly luciferase gene downstream of the HES5 promoter, a plasmid expressing the Notch1 (A, C) or Notch3 (B, D) ICD, and increasing amounts of a plasmid expressing ORF2 (C, D). Promoter activity was measured by using a dual-luciferase assay. A plasmid expressing Renilla luciferase under the control of a minimal herpesvirus TK promoter was used as an internal control. The results are the average of four independent experiments, and the error bars denote the standard deviation. An asterisk denotes significant differences (P < 0.05) in HES5 promoter activity in Neuro-2A cells following cotransfection with the ORF2 reporter and a Notch1 or Notch3 expression plasmid relative to HES5 promoter activity following transfection with a plasmid expressing Notch1 or Notch3 plus the empty vector, as determined by the Student t test.

Fig 6

Localization of ORF2 sequences important for Notch-mediated trans activation of the HES5 promoter. Neuro-2A cells were cotransfected with a plasmid expressing the Notch1 or Notch3 ICD, a plasmid expressing WT or mutant ORF2, and a plasmid containing the firefly luciferase gene downstream of the HES5 promoter. Luciferase assay was performed as described in the legend to Fig. 5. The results are the average of three independent experiments, and the error bar denotes the standard deviation. Asterisks denote significant differences (P < 0.05) in HES5 promoter activity in Neuro-2A cells following cotransfection with the ORF2 reporter and a Notch1 or Notch3 expression plasmid relative to HES5 promoter activity following transfection with a plasmid expressing Notch1 or Notch3 plus the empty vector, as determined by the Student t test.

Fig 7

ORF2 reduces the steady-state levels of Notch3. (A) Neuro-2A cells were transfected with the designated plasmids and then collected and processed for Western blot analysis. Cultures were treated with 100 μM CHX for 1 or 2 h at 40 h after transfection. To inhibit proteasome activity, cells were treated with lactacystin (15 μM) 10 h prior to CHX treatment. (B) The 2A/ORF2 plasmid contains ORF2 cloned such that there is a one-nucleotide frameshift downstream of the N-terminal Flag epitope. The 2B/ORF2 plasmid contains ORF2 in frame with the N-terminal Flag epitope. The Western blots were probed with a Flag-specific monoclonal antibody or an ORF2-specific peptide antibody. (C, D) The effects of the respective mutant ORF2 constructs on steady-state Notch3 protein levels were examined. Neuro-2A cells were cotransfected with ORF2 or the designated mutant ORF2 constructs and a plasmid expressing Notch3. Cultures were treated with CHX as described above. A mouse anti-Flag antibody was used to detect WT and mutant ORF2, while Notch3 was detected by using a rabbit anti-Notch3 antibody. β-Actin protein levels were determined to confirm that equal amounts of protein were loaded in each lane. The molecular weights (10³) of the respective bands are shown to the left of each Western blot.