Localization of sequences in a protein (ORF2) encoded by the latency-related gene of bovine herpesvirus 1 that inhibits apoptosis and interferes with Notch1-mediated trans-activation of the bICP0 promoter

Bovine herpesvirus 1 (BHV-1) infection induces clinical symptoms in the upper respiratory tract, inhibits immune responses, and can result in life-threatening secondary bacterial infections. Following acute infection, BHV-1 establishes latency in sensory neurons within trigeminal ganglia. Periodically, reactivation from latency occurs, resulting in virus transmission. The latency-related (LR) RNA is abundantly expressed in latently infected sensory neurons, suggesting that LR gene products regulate the latency-reactivation cycle. An LR mutant virus with stop codons at the amino terminus of the first open reading frame (ORF) in the LR gene (ORF2) does not reactivate 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 an important role in the latency-reactivation cycle. ORF2 also interacts with Notch1 or Notch3 and consequently inhibits their ability to trans-activate the bICP0 early and glycoprotein C promoters. In this study, we identified ORF2 sequences that were necessary for inhibiting cold shock-induced apoptosis or Notch1-mediated trans-activation of the bICP0 early promoter and stimulation of productive infection. Relative to ORF2 sequences necessary for inhibiting apoptosis, distinct domains in ORF2 were important for interfering with Notch1-mediated trans-activation. Five consensus protein kinase A and/or protein kinase C phosphorylation sites within ORF2 regulate the steady-state levels of ORF2 in transfected cells. A nuclear localization signal in ORF2 was necessary for inhibiting Notch1-mediated trans-activation but not apoptosis. In summary, ORF2 has more than one functional domain that regulates its stability and functional properties.     

Small Noncoding RNAs Encoded within the Bovine Herpesvirus 1 Latency-Related Gene Can Reduce Steady-State Levels of Infected Cell Protein 0 (bICP0)

Following acute infection in mucosal epithelium, bovine herpes virus 1 (BHV-1) establishes lifelong latency in sensory neurons within trigeminal ganglia. The latency-related RNA (LR-RNA) is abundantly expressed in sensory neurons of latently infected calves. Expression of LR proteins is necessary for the latency reactivation cycle because a mutant virus that does not express LR proteins is unable to reactivate from latency after dexamethasone treatment. LR-RNA sequences also inhibit bICP0 expression, productive infection, and cell growth. However, it is unclear how LR-RNA mediates these functions. In this study, we identified a 463-bp region within the LR gene (the XbaI-PstI [XP] fragment) that inhibited bICP0 protein and RNA expression in transiently transfected mouse neuroblastoma cells. Small noncoding RNAs (sncRNAs) encoded within the XP fragment (20 to 90 nucleotides in length) were detected in transiently transfected mouse neuroblastoma cells. Two families of sncRNAs were cloned from this region, and each family was predicted to contain a mature microRNA (miRNA). Both miRNAs were predicted to base pair with bICP0 mRNA sequences, suggesting that they reduce bICP0 levels. To test this prediction, sequences encompassing the respective sncRNAs and mature miRNAs were synthesized and cloned into a small interfering RNA expression vector. Both sncRNA families and their respective miRNAs inhibited bICP0 protein expression in mouse neuroblastoma cells and productive infection in bovine cells. In trigeminal ganglia of latently infected calves, an sncRNA that migrated between nucleotides 20 and 25 hybridized to the XP fragment. During dexamethasone-induced reactivation from latency, XP-specific sncRNA levels were reduced, suggesting that these sncRNAs support the establishment and maintenance of lifelong latency in cattle.Bovine herpes virus 1 (BHV-1) infection leads to respiratory and genital disorders, abortion, conjunctivitis, and/or multisystemic infection in small calves (19-21, 23). Consequently, BHV-1 infections are a significant economic loss to the cattle industry. As with other Alphaherpesvirinae subfamily members, the primary site for a BHV-1 latent infection is sensory ganglionic neurons (19, 20, 23). Virus reactivation from latency can occur after stress, suggesting that corticosteroids play a role in this process.During latency, viral gene expression is restricted to the latency-related (LR) gene and open reading frame E (ORF-E) (13, 23, 35, 36). The LR gene contains two open reading frames (ORF1 and ORF2) and two reading frames (RF-B and RF-C) (24). A fraction of LR-RNA is polyadenylated and alternatively spliced in trigeminal ganglia (TG), suggesting that more than one protein is expressed (4, 5, 12). A peptide antibody directed against ORF2 recognizes a protein encoded by the LR gene (12, 17, 18). LR protein expression is necessary for the latency reactivation cycle because a mutant BHV-1 strain with three stop codons at the N terminus of ORF2 does not reactivate from latency (14, 33). Furthermore, the LR mutant virus has diminished clinical symptoms and reduced shedding of infectious virus from the eye, TG, and tonsil (14, 15, 33). Finally, the LR mutant virus induces higher levels of apoptosis in TG neurons, in part because a protein encoded by the LR gene (ORF2) inhibits apoptosis (3, 14, 15, 26, 40). Three LR proteins, including ORF2, have reduced or no expression in cells infected with the LR mutant virus (18, 27).Although proteins encoded by the LR gene are necessary for the latency reactivation cycle, non-protein coding functions within LR-RNA have also been identified. For example, the intact LR gene inhibits the ability of bICP0 to stimulate productive infection in a dose-dependent manner (1, 9). Insertion of three in-frame stop codons at the amino terminus of the first ORF within the LR gene (ORF2) inhibited bICP0 repression with an efficiency similar to that of the wild-type (wt) LR gene, suggesting that expression of an LR protein is not required (9). Since the LR gene is antisense to bICP0 coding sequences, we assumed that LR-RNA hybridized to bICP0 RNA sequences and interfered with bICP0 expression. However, we were unable to obtain data suggesting that antisense repression was the major reason why the LR gene inhibited bICP0 expression. LR gene products also inhibit mammalian cell growth (8, 38), and the cell growth-inhibitory function of the LR gene maps to a 463-bp XbaI-PstI (XP) fragment (8). Sequences within the XP region have the potential to form stem-loop secondary structures, suggesting that there are small noncoding RNAs (sncRNAs) expressed from the XP region.In this study, we demonstrated that the XP fragment efficiently inhibits bICP0 protein levels and, to a lesser extent, bICP0 RNA levels. Northern blot analysis using the XP fragment as a probe detected sncRNAs migrating between 20 and 90 nucleotides (nt). Two families of sncRNAs with the same 5′ terminus but different 3′ termini were cloned from this region. Members of these two families of sncRNAs inhibited bICP0 expression with an efficiency similar to that of the XP fragment. Each family of sncRNAs has the potential to generate a mature microRNA (miRNA). Sequences encompassing the mature miRNA also inhibited bICP0 expression in transiently transfected cells. Although the miRNA sequences have the potential to base pair with bICP0 mRNA, the miRNA sequences do not overlap bICP0 RNA sequences. Finally, LR-specific sncRNAs and miRNAs inhibited productive infection approximately 2-fold, suggesting that LR-specific sncRNAs support the establishment and maintenance of lifelong latency in cattle.     

A mutation in the latency-related gene of bovine herpesvirus 1 inhibits protein expression from open reading frame 2 and an adjacent reading frame during productive infection

The latency-related (LR) gene of bovine herpesvirus 1 (BHV-1) is abundantly expressed during latency. A mutant BHV-1 strain that contains three stop codons at the 5′ terminus of the LR gene (LR mutant) does not reactivate from latency. This study demonstrates that the LR mutant does not express open reading frame 2 or an adjacent reading frame that lacks an initiating ATG (reading frame C). Since the LR mutant and wild-type BHV-1 express similar levels of LR RNA, we conclude that LR protein expression plays an important role in regulating the latency reactivation cycle in cattle.     

The latency-related gene of bovine herpesvirus 1 encodes a product which inhibits cell cycle progression.

Bovine herpesvirus 1 (BHV-1) establishes a latent infection in the sensory ganglionic neurons of cattle. The exclusive viral RNA expressed in a latent infection is the latency-related (LR) RNA, suggesting that it regulates some aspect of a latent infection. During the course of a productive infection, alphaherpesviruses induce certain events which occur during cell cycle progression. Consequently, we hypothesized that a BHV-1 infection might induce events in neurons which occur during cell cycle progression. In agreement with this hypothesis, cyclin A was detected in neurons of trigeminal ganglia when rabbits were infected. Neuronal cell cycle progression or inappropriate expression of cyclin A leads to apoptosis, suggesting that a viral factor inhibits the deleterious effects of cyclin A expression. The BHV-1 LR gene inhibited cell cycle progression and proliferation of human osteosarcoma cells. Antibodies directed against cyclin A or the LR protein coprecipitated the LR protein or cyclin A, respectively, suggesting that the two proteins interact with each other. We conclude that LR gene products inhibit cell cycle progression and hypothesize that this activity enhances the survival of infected neurons.