WACCNES CONTAINING BOVINE HERPE SVIRUS 1 ATTENUATED BY MUTATION IN LATENCY-RELATED GENE

Vaccines for pathogenic Strains of bovine herpesvirus 1 (BHV-1) which are based on attenuated BHV-1 having a mutation in the latency-related gene are provided. Live, attenuated vaccines are also provided which express anti gens from other viral or bacterial pathogens and thus form the basis of a variety of vaccines

WACCNES CONTAINING BOVINE HERPE SVIRUS 1 ATTENUATED BY MUTATION IN LATENCY-RELATED GENE

Vaccines for pathogenic Strains of bovine herpesvirus 1 (BHV-1) which are based on attenuated BHV-1 having a mutation in the latency-related gene are provided. Live, attenuated vaccines are also provided which express anti gens from other viral or bacterial pathogens and thus form the basis of a variety of vaccines

The Bovine Herpesvirus 1 Immediate-Early Protein (bICP0) Associates with Histone Deacetylase 1 To Activate Transcription

Infected-cell protein 0 encoded by bovine herpesvirus 1 (BHV-1) (bICP0) is necessary for efficient productive infection, in large part, because it activates all 3 classes of BHV-1 genes (U. V. Wirth, C. Fraefel, B. Vogt, C. Vlcek, V. Paces, and M. Schwyzer, J. Virol. 66:2763–2772, 1992). Although bICP0 is believed to be a functional homologue of herpes simplex virus type 1-encoded ICP0, the only well-conserved domain between the proteins is a zinc ring finger located near the amino terminus of both proteins. Our previous studies demonstrated that bICP0 is toxic to transfected cells but does not appear to directly induce apoptosis (Inman, M., Y. Zhang, V. Geiser, and C. Jones, J. Gen. Virol. 82:483–492, 2001). C-terminal sequences in the last 320 amino acids of bICP0 mediate subcellular localization. Mutagenesis of the zinc ring finger within bICP0 revealed that this domain was important for transcriptional activation. In this study, we demonstrate that bICP0 interacts with histone deacetylase 1 (HDAC1), which results in activation of a simple promoter containing four consensus Myc-Max binding sites. The interaction between bICP0 and HDAC1 correlated with inhibition of Mad-dependent transcriptional repression. In resting CV-1 cells, bICP0 relieved HDAC1-mediated transcriptional repression. The zinc ring finger was required for relieving HDAC1-induced repression but not for interacting with HDAC1. In fetal bovine lung cells but not in a human epithelial cell line, bICP0 expression correlated with reduced steadystate levels of HDAC1 in crude cytoplasmic extracts. We hypothesize that the ability of bICP0 to overcome HDAC1-induced repression plays a role in promoting productive infection in highly differentiated cell types

HSV Latency-Associated Transcript and Neuronal Apoptosis

Thompson and Sawtell report that the Promega Anti-PARP p85 antibody did not recognize cleaved PARP in mouse or rabbit cells in their experiments, and conclude that the results reported with this antibody by Perng et al. (1) are an artifact. The Promega antibody was generated against a peptide based on the sequence of human p85. Although the corresponding bovine sequence differs by two amino acids, the antibody reacts with both human and bovine p85 (2). The mouse and rat sequences for this region of p85 differ from the human sequence by a single amino acid that corresponds to one of the bovine amino acid differences. External testers have successfully stained mouse and rat p85 using Promega Anti-PARP p85 (2). Thus, the negative mouse results reported by Thompson and Sawtell are surprising, and call into question the validity of their negative rabbit results. Extracts that we prepared (Fig. 1) from rabbit skin cells induced to undergo apoptosis by staurosporin (lane RS-S) contained a band of approximately 85 kD that was recognized by Anti-PARP p85, and that comigrated with the p85 band induced in human Jurkat cells by staurosporin (lane Jurkat-S) or anti-Fas antibody (lane Jurkat-F). Clearly, then, the Promega antibody recognizes the rabbit cleaved PARP p85 protein, and the argument to the contrary by Thompson and Sawtell has no merit. Their negative mouse and rabbit results apparently stemmed from technical problems, a bad batch of antibody, or some other unknown factor

Activation of Caspases and p53 by Bovine Herpesvirus 1 Infection Results in Programmed Cell Death and Efficient Virus Release

Programmed cell death (PCD), or apoptosis, is initiated in response to various stimuli, including virus infection. Bovine herpesvirus 1 (BHV-1) induces PCD in peripheral blood mononuclear cells at the G0/G1 phase of the cell cycle (E. Hanon, S. Hoornaert, F. Dequiedt, A. Vanderplasschen, J. Lyaku, L. Willems, and P.-P. Pastoret, Virology 232:351–358, 1997). However, penetration of virus particles is not required for PCD (E. Hanon, G. Meyer, A. Vanderplasschen, C. Dessy-Doize, E. Thiry, and P. P. Pastoret, J. Virol. 72:7638–7641, 1998). The mechanism by which BHV-1 induces PCD in peripheral blood mononuclear cells is not understood, nor is it clear whether nonlymphoid cells undergo PCD following infection. This study demonstrates that infection of bovine kidney (MDBK) cells with BHV-1 leads to PCD, as judged by terminal deoxynucleotidyltransferase- mediated dUTP-biotin nick end labeling, DNA laddering, and chromatin condensation. p53 appears to be important in this process, because p53 levels and promoter activity increased after infection. Expression of proteins that are stimulated by p53 (p21Waf1 and Bax) is also activated after infection. Cleavage of Bcl-xL, a protein that inhibits PCD, occurred after infection, suggesting that caspases (interleukin-1β- converting enzyme-like proteases) were activated. Other caspase substrates [poly(ADP-ribose) polymerase and actin] are also cleaved during the late stages of infection. Inhibition of caspase activity delayed cytotoxic activity and virus release but increased the overall virus yield. Taken together, these results indicate that nonlymphoid cells undergo PCD near the end of productive infection and further suggest that caspases enhance virus release

Identification of a Novel Bovine Herpesvirus 1 Transcript Containing a Small Open Reading Frame That Is Expressed in Trigeminal Ganglia of Latently Infected Cattle

Bovine herpesvirus 1 (BHV-1), like other Alphaherpesvirinae subfamily members, establishes latency in sensory neurons. The latency-related (LR) RNA is abundantly expressed during latency, and expression of an LR protein is required for the latency reactivation cycle in cattle. Within LR promoter sequences, a 135-aminoacid open reading frame (ORF) was identified, ORF-E, that is antisense to the LR RNA. ORF-E is also downstream of the gene encoding the major viral transcriptional activator, bICP0. Strand-specific reverse transcription-PCR demonstrated that a transcript containing ORF-E was consistently expressed in trigeminal ganglia (TG) of latently infected calves, productively infected cultured cells, and acutely infected calves. As expected, a late transcript encoding glycoprotein C was not detected in TG of latently infected calves. The ORF-E transcript is polyadenylated and is expressed early when cultured bovine cells are productively infected. Protein coding sequences containing ORF-E were fused to green fluorescent protein (GFP) to examine the cellular localization of the putative protein. In transiently transfected mouse neuroblastoma (neuro-2A) and human neuroblastoma (SK-N-SH) cells, the ORF-E/GFP fusion protein was detected in discreet domains within the nucleus. In contrast, the ORF-E/GFP fusion protein was detected in the cytoplasm and nucleus of rabbit skin cells and bovine kidney cells. As expected, the GFP protein was expressed in the cytoplasm and nucleus of transfected cells. These studies indicate that the ORF-E transcript is consistently expressed during latency. We suggest that the ORF-E gene regulates some aspect of the latency reactivation cycle

The interaction between KSHV RTA and cellular RBP-Jκ and their subsequent DNA binding are not sufficient for activation of RBP-Jκ

Kaposi’s sarcoma-associated herpesvirus (KSHV) replication and transcription activator (RTA) is necessary and sufficient for the switch from KSHV latency to lytic replication. RTA activates promoters by several mechanisms. RTA can bind to sequences in viral promoters and activate transcription. In addition, RTA interacts with the cellular recombination signal sequence-binding protein-J kappa (RBP- Jκ), a transcriptional repressor, converts the repressor into an activator and activates viral promoters via RBP- Jκ. Because RBP- Jκ is required for RTA to activate lytic replication, it is important to understand how RTA cooperates with RBP- Jκ protein to activate KSHV lytic replication program. Previously, we identified an RTA mutant, RTA-K152E, which has a defect in its direct DNA-binding activity. In this report, the effect of the mutant RTA on KSHV activation via RBP- Jκ protein is examined. We demonstrate that RTA-K152E interacts with RBP- Jκ physically and the mutant RTA and RBP-Jκ complex binds to target DNA properly in vivo and in vitro. However, the complex of RTA-K152E and RBP- Jκ does not activate transcription. Furthermore, the RTA mutant (RTA-K12E) inhibits cellular Notch-mediated RBP- Jκ activation. These data collectively suggest that the complex between KSHV RTA and cellular RBP- Jκ and the subsequent DNA binding by the complex are not sufficient for the activation of RBP- Jκ protein. Other factor(s) whether additional cofactor(s) in the complex or the intrinsic conformation of RTA, are predicted to be required for the activation of RBP- Jκ protein by RTA

The interaction between KSHV RTA and cellular RBP-Jκ and their subsequent DNA binding are not sufficient for activation of RBP-Jκ

Kaposi’s sarcoma-associated herpesvirus (KSHV) replication and transcription activator (RTA) is necessary and sufficient for the switch from KSHV latency to lytic replication. RTA activates promoters by several mechanisms. RTA can bind to sequences in viral promoters and activate transcription. In addition, RTA interacts with the cellular recombination signal sequence-binding protein-J kappa (RBP- Jκ), a transcriptional repressor, converts the repressor into an activator and activates viral promoters via RBP- Jκ. Because RBP- Jκ is required for RTA to activate lytic replication, it is important to understand how RTA cooperates with RBP- Jκ protein to activate KSHV lytic replication program. Previously, we identified an RTA mutant, RTA-K152E, which has a defect in its direct DNA-binding activity. In this report, the effect of the mutant RTA on KSHV activation via RBP- Jκ protein is examined. We demonstrate that RTA-K152E interacts with RBP- Jκ physically and the mutant RTA and RBP-Jκ complex binds to target DNA properly in vivo and in vitro. However, the complex of RTA-K152E and RBP- Jκ does not activate transcription. Furthermore, the RTA mutant (RTA-K12E) inhibits cellular Notch-mediated RBP- Jκ activation. These data collectively suggest that the complex between KSHV RTA and cellular RBP- Jκ and the subsequent DNA binding by the complex are not sufficient for the activation of RBP- Jκ protein. Other factor(s) whether additional cofactor(s) in the complex or the intrinsic conformation of RTA, are predicted to be required for the activation of RBP- Jκ protein by RTA

Bovine herpesvirus 1 immediate-early protein (bICP0) interacts with the histone acetyltransferase p300, which stimulates productive infection and gC promoter activity

The immediate-early protein, bICP0, of Bovine herpesvirus 1 (BHV-1) transactivates viral promoters and stimulates productive infection. bICP0 is expressed constitutively during productive infection, as its gene contains an immediate- early and an early promoter. Like other ICP0 homologues encoded by members of the subfamily Alphaherpesvirinae, bICP0 contains a zinc RING finger located near its N terminus. Mutations that disrupt the bICP0 zinc RING finger impair its ability to activate transcription, stimulate productive infection, inhibit interferon-dependent transcription in certain cell types and regulate subnuclear localization. bICP0 also interacts with a cellular chromatin-remodeling enzyme, histone deacetylase 1 (HDAC1), and can relieve HDAC1-mediated transcriptional repression, suggesting that bICP0 inhibits silencing of the viral genome. In this study, it was shown that bICP0 interacted with the histone acetyltransferase p300 during productive infection and in transiently transfected cells. In addition, p300 enhanced BHV-1 productive infection and transactivated a late viral promoter (gC). In contrast, a CH3-domain deletion mutant of p300, which is a dominant-negative mutant, did not activate the gC promoter. bICP0 and p300 cooperated to activate the gC promoter, suggesting that there is a synergistic effect on promoter activation. As p300 can activate certain antiviral signaling pathways (for example, interferon), it was hypothesized that interactions between p300 and bICP0 may dampen the antiviral response following infection

Premature Expression of the Latency-Related RNA Encoded by Bovine Herpesvirus Type 1 Correlates With Higher Levels of Beta Interferon RNA Expression in Productively Infected Cells

Bovine herpesvirus type 1 (BHV-1) is an important pathogen that can initiate bovine respiratory disease complex. Like other members of the subfamily Alphaherpesvirinae, BHV-1 establishes latency in sensory neurons. The latency-related (LR) gene expresses a family of alternatively spliced transcripts in infected sensory neurons that have the potential to encode several LR proteins. An LR mutant virus that contains three stop codons near the 5’ terminus of the first open reading frame in the LR gene does not express two LR proteins or reactivate from latency. In addition, the LR mutant virus induces higher levels of apoptosis in trigeminal ganglionic neurons and grows less efficiently in certain tissues of infected calves. In spite of the reduced pathogenesis, the LR mutant virus, wild-type BHV-1, and the LR rescued virus exhibit identical growth properties in cultured bovine cells. In this study, we demonstrated that during early phases of productive infection the LR mutant virus expressed higher levels of LR-RNA relative to the LR rescued virus or wt BHV-1. Bovine kidney cells infected with the LR mutant virus also induced higher levels of beta interferon RNA and interferon response genes. These results suggest that inappropriate expression of LR-RNA, in the absence of LR protein expression, may influence the latency-reactivation cycle and pathogenic potential of BHV-1