In this study, we demonstrated that the expression of ORF2 in Neuro-2A cells allowed neurite formation to occur in the presence of activated Notch1 or Notch3. As expected, Notch1 or Notch3 interferes with neurite formation in serum-arrested Neuro-2A cells (31
). Although previous studies demonstrated that ORF2 interfered with the ability of Notch1 and Notch3 to activate productive infection and trans
activate certain viral promoters (26
), they did not address whether ORF2 influences Notch-mediated signaling in the context of known cellular functions. It now seems clear that, in general, ORF2 interferes with Notch-mediated signaling. Activated Notch is required for the growth of certain human tumors (49
), suggesting that ORF2 may have therapeutic value with respect to inhibition of the growth of tumors that are dependent on Notch for proliferation.
Many other genes, in addition to Notch, negatively regulate neurite sprouting. These include A1 adenosine receptor, merlin, adenomatous polyposis coli protein, β-catenin, Cdc42-interacting protein 4, harmine, HES-1, HES-5, proline/serine-rich coiled-coil protein 1 (also known as DDA3), and Van Gogh 1 (47
). Conversely, a number of genes promote neurite sprouting. For example, a complex containing diacylglycerol kinase ζ, Rac1, and syntrophin promotes neurite outgrowth (60
). In addition, the Brn-3a transcription factor, CD47 (also referred to as integrin-associated protein), degenerin/epithelial Na+
channel protein, Dickkopf-1, insulin-like growth factors I and II, NF-κB, plasticity-related gene 5, the Prickle 1 or 2 gene, protruding, retina-derived growth factor, and Wnt-3a promote neurite outgrowth (62
). Several proteins that positively or negatively regulate neurite outgrowth belong to the Wnt signaling pathway (adenomatous polyposis coli protein, β-catenin, Wnt-3a, and Dickkopf-1). The Wnt and Notch signaling pathways have considerable cross talk, and thus, activation or repression of the Notch signaling pathway impacts Wnt signaling (72
). It is not clear whether ORF2 influences the Wnt signaling pathway and/or other genes that regulate neurite outgrowth.
In general, there was a correlation between the abilities of the respective mutant ORF2 constructs to inhibit neurite formation, the bICP0 early promoter (44
), BHV-1 productive infection (44
), and Notch-mediated trans
activation of the HES-5 promoter ( contains a summary of the results for the respective mutant constructs). The ability of four transposon mutant ORF2 constructs (ORF2-95, ORF2-134, ORF2-240, and ORF2-271) to enhance the degradation of Notch3 did not correlate with neurite formation, activation of productive BHV-1 infection, and activation of BHV-1 promoters, suggesting that sufficient levels of Notch3 were present to perform these functions. Because of the complexity of the regulation of neurite formation, it is also possible that ORF2 inhibits other cellular proteins that interfere with neurite formation. Interestingly, two phosphorylation mutant constructs (ORF2-P and ORF2-AP) did not effectively inhibit Notch-mediated trans
activation of the HES5 promoter, whereas the same mutant constructs promoted neurite formation and inhibited Notch1-mediated trans
activation of the bICP0 early promoter with WT efficiency (44
). For activated Notch to stimulate transcription, Notch, CSL, and MAML must form a complex at a consensus CSL binding site (reviewed in references 27
). Consequently, ORF2 may (i) interfere with the formation of the Notch-CSL-MAML complex, (ii) prevent the Notch-CSL-MAML complex from interacting with certain CSL consensus binding sites, or (iii) interfere with certain coactivators recruited to the Notch-CSL-MAML complex (27
). Reduction of the steady-state levels of Notch3 by ORF2 would also interfere with the trans
activation potential of Notch3. We speculate that ORF2 phosphorylation influences trans
activation of the bICP0 early promoter but not the HES5 promoter because Notch-mediated trans
activation requires the formation of different complexes on these two promoters. The ORF2 phosphorylation mutant constructs are more stable than ORF2 (44
), suggesting that the differential effects of inhibiting Notch-mediated trans
activation were not merely due to lower levels of mutant ORF2 in transfected cells. Considering the complexity of Notch-mediated transcriptional activation and neurite sprouting, it is not surprising that different mutant ORF2 constructs may affect one process but not others. On the basis of our present studies, we believe that ORF2 interference with Notch3 functions is mediated by the ability of ORF2 to interact with Notch3, sequester it to the rim of the nucleus (26
), and enhance Notch3 degradation.
Summary of the Notch-inhibiting functions of ORF2 and a panel of mutant ORF2 constructsa
ORF2, in the absence of other viral gene products, inhibits apoptosis (25
) in transiently transfected cells. The antiapoptosis functions of ORF2 are believed to be crucial for the latency-reactivation cycle because an LR mutant virus that contains stop codons at the amino terminus of ORF2 induces higher levels of apoptosis in TG neurons during the establishment of latency (22
) and is not reactivated from latency after DEX treatment (6
). ORF2 promotion of neurite formation in the presence of Notch may maintain normal neuronal functions, including neuronal survival, after infection. Productive BHV-1 infection induces Notch1 protein levels (26
), suggesting that during the establishment of latency, ORF2 maintains axonal projections in infected neurons by promoting neurite sprouting in the presence of activated Notch. In the absence of ORF2 (LR mutant virus, for example), we predict that certain infected neurons are more susceptible to loss of axonal projections because activated Notch is present. It is well established that neurons with damaged or cut axons can undergo Wallerian degeneration, a slow form of neuronal death (36
). The fact that Notch stimulates productive infection (26
) also favors virus-induced neuronal cell death. In neuronal progenitor cells, Notch activation induces apoptosis by a p53-dependent pathway (38
). Collectively, these observations suggest that the ability of ORF2 to restrain Notch functions complements the antiapoptosis functions of ORF2 to promote the establishment and life-long maintenance of latency.
During DEX-induced reactivation from latency, Notch3 RNA (26
), as well as protein, levels are increased in TG, and HES6 RNA levels are increased during reactivation from latency (43
). Examination of DEX-induced transcription in TG of calves latently infected with BHV-1 revealed that many genes activated by Notch signaling are induced (data not shown), suggesting that the Notch signaling pathway may enhance reactivation from latency. Since ORF2 protein expression is reduced during DEX-induced reactivation, we predict that activated Notch family members would not be restrained by ORF2, suggesting that activated Notch destabilizes normal neuronal functions and may induce neuronal cell death, as well as reactivation from latency.