Cellular transcription factors induced in trigeminal ganglia during dexamethasone-induced reactivation from latency stimulate bovine herpesvirus 1 productive infection and certain viral promoters.

Bovine herpesvirus 1 (BHV-1), an alphaherpesvirinae subfamily member, establishes latency in sensory neurons. Elevated corticosteroid levels, due to stress, reproducibly triggers reactivation from latency in the field. A single intravenous injection of the synthetic corticosteroid dexamethasone (DEX) to latently infected calves consistently induces reactivation from latency. Lytic cycle viral gene expression is detected in sensory neurons within 6 h after DEX treatment of latently infected calves. These observations suggested that DEX stimulated expression of cellular genes leads to lytic cycle viral gene expression and productive infection. In this study, a commercially available assay-Bovine Gene Chip-was used to compare cellular gene expression in the trigeminal ganglia (TG) of calves latently infected with BHV-1 versus DEX-treated animals. Relative to TG prepared from latently infected calves, 11 cellular genes were induced more than 10-fold 3 h after DEX treatment. Pentraxin three, a regulator of innate immunity and neurodegeneration, was stimulated 35- to 63-fold after 3 or 6 h of DEX treatment. Two transcription factors, promyelocytic leukemia zinc finger (PLZF) and Slug were induced more than 15-fold 3 h after DEX treatment. PLZF or Slug stimulated productive infection 20- or 5-fold, respectively, and Slug stimulated the late glycoprotein C promoter more than 10-fold. Additional DEX-induced transcription factors also stimulated productive infection and certain viral promoters. These studies suggest that DEX-inducible cellular transcription factors and/or signaling pathways stimulate lytic cycle viral gene expression, which subsequently leads to successful reactivation from latency in a small subset of latently infected neurons.

Regulation of the latency-reactivation cycle by products encoded by the bovine herpesvirus 1 (BHV-1) latency-related gene.

Like other α-herpesvirinae subfamily members, the primary site for bovine herpesvirus 1 (BHV-1) latency is ganglionic sensory neurons. Periodically BHV-1 reactivates from latency, virus is shed, and consequently virus transmission occurs. Transcription from the latency-related (LR) gene is readily detected in neurons of trigeminal ganglia (TG) of calves or rabbits latently infected with BHV-1. Two micro-RNAs and a transcript encompassing a small open reading frame (ORF-E) located within the LR promoter can also be detected in TG of latently infected calves. A BHV-1 mutant that contains stop codons near the beginning of the first open reading frame (ORF2) within the major LR transcript (LR mutant virus) has been characterized. The LR mutant virus does not express ORF2, a reading frame that lacks an initiating ATG (reading frame B), and has reduced expression of ORF1 during productive infection. The LR mutant virus does not reactivate from latency following dexamethasone treatment suggesting that LR protein expression regulates the latency-reactivation cycle. Higher levels of apoptosis occur in TG neurons of calves infected with the LR mutant viruses when compared to wild-type BHV-1 indicating that the anti-apoptotic properties of the LR gene is necessary for the latency-reactivation cycle. ORF2 inhibits apoptosis and regulates certain viral promoters, in part, because it interacts with three cellular transcription factors (C/EBP-alpha, Notch1, and Notch3). Although ORF2 is important for the latency-reactivation cycle, we predict that other LR gene products play a supportive role during life-long latency in cattle.

Small non-coding RNAs encoded within the herpes simplex virus type 1 latency associated transcript (LAT) cooperate with the retinoic acid inducible gene I (RIG-I) to induce beta-interferon promoter activity and promote cell survival.

The herpes simplex virus type 1 (HSV-1) latency-associated transcript (LAT) is abundantly expressed in latently infected trigeminal ganglionic sensory neurons. Expression of the first 1.5 kb of LAT coding sequences restores wild type reactivation to a LAT null HSV-1 mutant. The anti-apoptosis functions of the first 1.5 kb of LAT coding sequences are important for wild type levels of reactivation from latency. Two small non-coding RNAs (sncRNAs) contained within the first 1.5 kb of LAT coding sequences are expressed in trigeminal ganglia of latently infected mice, they cooperate to inhibit apoptosis, and reduce the efficiency of productive infection. In this study, we demonstrated that LAT sncRNA1 cooperates with the RNA sensor, retinoic acid inducible gene I (RIG-I), to stimulate IFN-ß promoter activity and NF-κB dependent transcription in human or mouse cells. LAT sncRNA2 stimulated RIG-I induction of NF-κB dependent transcription in mouse neuroblastoma cells (Neuro-2A) but not human 293 cells. Since it is well established that NF-κB interferes with apoptosis, we tested whether the sncRNAs cooperated with RIG-I to inhibit apoptosis. In Neuro-2A cells, both sncRNAs cooperated with RIG-I to inhibit cold-shock induced apoptosis. Double stranded RNA (PolyI:C) stimulates RIG-I dependent signaling; but enhanced cold-shock induced apoptosis. PolyI:C, but not LAT sncRNAs, interfered with protein synthesis when cotransfected with RIG-I, which correlated with increased levels of cold-shock induced apoptosis. LAT sncRNA1 appeared to interact with RIG-I in transiently transfected cells suggesting this interaction stimulates RIG-I.

ICP27 protein encoded by bovine herpesvirus type 1 (bICP27) interferes with promoter activity of the bovine genes encoding beta interferon 1 (IFN-ß1) and IFN-ß3.

Bovine herpes virus 1 (BHV-1) infection leads to upper respiratory tract infections, conjunctivitis, and the infection predisposes cattle to secondary bacterial infections. The infected cell protein 0 (bICP0) encoded by BHV-1 suppresses antiviral innate immune signaling by interfering with expression of interferon beta (IFN-ß). In contrast to humans or mice, cattle contain three IFN-ß genes that have distinct transcriptional promoters. We previously cloned and characterized all three bovine IFN-ß promoters. In this study, we provide evidence that bICP27; a viral early protein that shuttles between the nucleus and cytoplasm inhibits transcriptional activity of two bovine IFN-ß gene promoters (IFN-ß1 and IFN-ß3). Conversely, the BHV-1 infected cell protein 0 (bICP0) early promoter was not inhibited by bICP27. C-terminal mutants lacking the bICP27 zinc RING finger-like motif did not efficiently inhibit IFN-ß3 promoter activity but inhibited IFN-ß1 promoter activity as efficiently as wild type bICP27. An N-terminal mutant lacking the nuclear localization signal (NLS) and nucleolar localization signal (NoLS) was localized to the cytoplasm and this mutant had no effect on IFN-ß promoter activity. In summary, these studies provided evidence that bICP27 inhibited IFN-ß1 and IFN-ß3 promoter activity in transiently transfected cells.

Infection of cultured bovine cells with bovine herpesvirus 1 (BHV-1) or Sendai virus induces different beta interferon subtypes.

In contrast to mice or humans, cattle contain three beta interferon (IFN-ß) genes with distinct transcriptional promoters suggesting IFN-ß gene expression is not stimulated the same by different viruses. To test this hypothesis, we compared expression of the three IFN-ß subtypes after infection with a RNA virus, Sendai, versus a large DNA virus, bovine herpesvirus 1 (BHV-1). Infection of low passage bovine kidney (BK) or established bovine kidney cells (CRIB) with Sendai virus has consistently led to high levels of IFN-ß1 RNA. Conversely, infection of CRIB cells, but not BK cells, with BHV-1 increased IFN-ß3 RNA levels and to a lesser extent the other two IFN-ß subtypes. Inhibition of de novo protein synthesis with cycloheximide resulted in higher levels of IFN-ß1 and IFN-ß2 RNA levels after BHV-1 infection. Further studies demonstrated that BHV-1 immediate early and/or early genes were primarily responsible for inhibiting the IFN response in BK cells. The three bovine IFN-ß promoters were cloned upstream of a reporter gene construct, and their properties analyzed in transient transfection assays. Only the IFN-ß3 promoter was trans-activated by IRF3 (interferon responsive factor 3). IRF7 and double stranded RNA (polyI:C) stimulated IFN-ß1 and IFN-ß3 promoter activity, but not IFN-ß2. Relative to the human IFN-ß promoter, the IFN-ß3 promoter contained fewer nucleotide differences in the positive regulatory domain III (PRD III), PRD IV, and PRD I compared to the IFN-ß1 and IFN-ß2 promoter. Collectively, these studies provide evidence that virus infection differentially stimulates expression of the three bovine IFN-ß genes.

Cytoplasmic localized infected cell protein 0 (bICP0) encoded by bovine herpesvirus 1 inhibits ß interferon promoter activity and reduces IRF3 (interferon response factor 3) protein levels.

Bovine herpesvirus 1 (BHV-1), an alpha-herpesvirinae subfamily member, establishes a life-long latent infection in sensory neurons. Periodically, BHV-1 reactivates from latency, infectious virus is spread, and consequently virus transmission occurs. BHV-1 acute infection causes upper respiratory track infections and conjunctivitis in infected cattle. As a result of transient immune-suppression, BHV-1 infections can also lead to life-threatening secondary bacterial pneumonia that is referred to as bovine respiratory disease. The infected cell protein 0 (bICP0) encoded by BHV-1 reduces human ß-interferon (IFN-ß) promoter activity, in part, by inducing degradation of interferon response factor 3 (IRF3) and interacting with IRF7. In contrast to humans, cattle contain three IFN-ß genes. All three bovine IFN-ß proteins have anti-viral activity: but each IFN-ß gene has a distinct transcriptional promoter. We have recently cloned and characterized the three bovine IFN-ß promoters. Relative to the human IFN-ß promoter, each of the three IFN-ß promoters contain differences in the four positive regulatory domains that are required for virus-induced activity. In this study, we demonstrate that bICP0 effectively inhibits bovine IFN-ß promoter activity following transfection of low passage bovine cells with interferon response factor 3 (IRF3) or IRF7. A bICP0 mutant that localizes to the cytoplasm inhibits bovine IFN-ß promoter activity as efficiently as wt bICP0. The cytoplasmic localized bICP0 protein also induced IRF3 degradation with similar efficiency as wt bICP0. In summary, these studies suggested that cytoplasmic localized bICP0 plays a role in inhibiting the IFN-ß response during productive infection.