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

Biosynthesis of Selenocysteine on Its tRNA in Eukaryotes

Selenocysteine (Sec) is cotranslationally inserted into protein in response to UGA codons and is the 21st amino acid in the genetic code. However, the means by which Sec is synthesized in eukaryotes is not known. Herein, comparative genomics and experimental analyses revealed that the mammalian Sec synthase (SecS) is the previously identified pyridoxal phosphate-containing protein known as the soluble liver antigen. SecS required selenophosphate and O-phosphoseryl-tRNA([Ser]Sec) as substrates to generate selenocysteyl-tRNA([Ser]Sec). Moreover, it was found that Sec was synthesized on the tRNA scaffold from selenide, ATP, and serine using tRNA([Ser]Sec), seryl-tRNA synthetase, O-phosphoseryl-tRNA([Ser]Sec) kinase, selenophosphate synthetase, and SecS. By identifying the pathway of Sec biosynthesis in mammals, this study not only functionally characterized SecS but also assigned the function of the O-phosphoseryl-tRNA([Ser]Sec) kinase. In addition, we found that selenophosphate synthetase 2 could synthesize monoselenophosphate in vitro but selenophosphate synthetase 1 could not. Conservation of the overall pathway of Sec biosynthesis suggests that this pathway is also active in other eukaryotes and archaea that synthesize selenoproteins

Functional analysis of the bovine herpesvirus -1 gene encoding bICP0, a promiscuous trans-activator, that stimulates productive infection and inhibits interferon (IFN) signaling pathways

Virus-host interactions determine the outcome of the infection. Resistance to and recovery from a viral infection depends upon the efficiency by which the immune system inhibits viral spread. Most viruses encode factors to suppress the innate immune response. Bovine Herpesvirus-1 (BHV-1) infects cattle and similar to other alphaherpesvirinae subfamily members, establishes latency in sensory neurons. During acute infection and establishment of latency, BHV-1 viral genes interact with various cellular factors and repress the immune responses. One of the immediate early proteins, bICP0 has been identified as a viral component that suppresses innate immune responses. bICP0 can transactivate all viral promoters, and as such stimulates productive infection. Consequently, I have hypothesized that bICP0 has two functions that are crucial for stimulating productive infection: blocking of interferon signaling and activation of viral transcription. This hypothesis is the basis of my dissertation research. The zinc RING finger and transactivation domains within bICP0 are believed to contribute significantly to the functions of bICP0. I have demonstrated that the bICP0 C-terminus domains are required for inhibiting IFN-β promoter activation. The C-terminus domains and the zinc RING finger are required for degrading interferon regulatory factor 3 (IRF3), a transcription factor involved in Type I IFN expression. The studies also suggest that bICP0 binds to interferon regulatory factor 7 (IRF7) and thus prevents it from activating the IFN-β promoter. Collectively these studies indicate that bICP0 disarms the innate immune response by targeting the transcription factors IRF3 and IRF7. To understand the role of zinc RING finger in productive infection, I generated a bICP0 zinc RING finger mutant BHV-1 virus. The mutant virus was characterized in cultured bovine cells and calves. In cell culture, the mutant virus has delayed growth as compared to the wild type or rescued virus. Similarly in calves, the mutant virus did not replicate as efficiently as the rescued virus and the immune response was also reduced. I believe these studies provide valuable information with respect to understanding BHV-1 pathogenesis, and the role that the zinc RING finger plays in virus-host interactions

The Infected Cell Protein 0 Encoded by Bovine Herpesvirus 1 (bICP0) Induces Degradation of Interferon Response Factor 3 and, Consequently, Inhibits Beta Interferon Promoter Activity

The ICP0 protein (bICP0) encoded by bovine herpesvirus 1 is the major viral regulatory protein because it stimulates all viral promoters and, consequently, productive infection. Like other ICP0 analogues encoded by Alphaherpesvirinae subfamily members, bICP0 contains a zinc RING finger near its amino terminus that is necessary for activating transcription, regulating subcellular localization, and inhibiting interferon-dependent transcription. In this study, we discovered that sequences near the C terminus, and the zinc RING finger, are necessary for inhibiting the human beta interferon (IFN-β) promoter. In contrast to herpes simplex virus type 1-encoded ICP0, bICP0 reduces interferon response factor 3 (IRF3), but not IRF7, protein levels in transiently transfected cells. The zinc RING finger and sequences near the C terminus are necessary for bICP0-induced degradation of IRF3. A proteasome inhibitor, lactacystin, interfered with bICP0-induced degradation of IRF3, suggesting that bICP0, directly or indirectly, targets IRF3 for proteasome-dependent degradation. IRF3, but not IRF7, is not readily detectable in the nuclei of productively infected bovine cells during the late stages of infection. In the context of productive infection, IRF3 and IRF7 are detected in the nucleus at early times after infection. At late times after infection, IRF7, but not IRF3, is still detectable in the nuclei of infected cells. Collectively, these studies suggest that the ability of bICP0 to reduce IRF3 protein levels is important with respect to disarming the IFN response during productive infection

The Infected Cell Protein 0 Encoded by Bovine Herpesvirus 1 (bICP0) Associates with Interferon Regulatory Factor 7 and Consequently Inhibits Beta Interferon Promoter Activity▿

The bICP0 protein encoded by bovine herpesvirus 1 stimulates productive infection and viral gene expression but inhibits interferon (IFN)-dependent transcription. bICP0 inhibits beta IFN (IFN-β) promoter activity and induces degradation of IFN regulatory factor 3 (IRF3). Although bICP0 inhibits the trans-activation activity of IRF7, IRF7 protein levels are not reduced. In this study, we demonstrate that bICP0 is associated with IRF7. Furthermore, bICP0 inhibits the ability of IRF7 to trans-activate the IFN-β promoter in the absence of IRF3 expression. The interaction between bICP0 and IRF7 correlates with reduced trans-activation of the IFN-β promoter by IRF7

The Zinc RING Finger of Bovine Herpesvirus 1-Encoded bICP0 Protein Is Crucial for Viral Replication and Virulence▿

Bovine herpesvirus 1 (BHV-1) infected cell protein 0 (bICP0) stimulates productive infection, in part by activating viral gene expression. The C3HC4 zinc RING finger of bICP0 is crucial for activating viral transcription and productive infection. In this study, we used a bacterial artificial chromosome containing a wild-type (wt) virulent BHV-1 strain to generate a single amino acid mutation in the C3HC4 zinc RING finger of bICP0. This virus (the 51g mutant) contains a cysteine-to-glycine mutation (51st amino acid) in the C3HC4 zinc RING finger of bICP0. A plasmid expressing the 51g mutant protein did not transactivate viral promoter activity as efficiently as wt bICP0. The 51g mutant virus expressed higher levels of the bICP0 protein than did the 51g rescued virus (51gR) but yielded reduced virus titers following infection of permissive bovine cells. The 51g mutant virus, but not the 51gR virus, grew poorly in bovine cells pretreated with imiquimod to stimulate interferon production. During acute infection of calves, levels of infectious virus were 2 to 3 logs lower in ocular or nasal swabs with 51g than with 51gR. Calves latently infected with the 51g mutant did not reactivate from latency because virus shedding did not occur in ocular or nasal cavities. As expected, calves latently infected with 51gR reactivated from latency following dexamethasone treatment. These studies demonstrate that mutation of a single well-conserved cysteine residue in the C3HC4 zinc RING finger of bICP0 has dramatic effects on the growth properties of BHV-1

Toll-Like Receptor 2 Activation by Chlamydia trachomatis Is Plasmid Dependent, and Plasmid-Responsive Chromosomal Loci Are Coordinately Regulated in Response to Glucose Limitation by C. trachomatis but Not by C. muridarum▿

We previously demonstrated that plasmid-deficient Chlamydia muridarum retains the ability to infect the murine genital tract but does not elicit oviduct pathology because it fails to activate Toll-like receptor 2 (TLR2). We derived a plasmid-cured derivative of the human genital isolate Chlamydia trachomatis D/UW-3/Cx, strain CTD153, which also fails to activate TLR2, indicating this virulence phenotype is associated with plasmid loss in both C. trachomatis and C. muridarum. As observed with plasmid-deficient C. muridarum, CTD153 displayed impaired accumulation of glycogen within inclusions. Transcriptional profiling of the plasmid-deficient strains by using custom microarrays identified a conserved group of chromosomal loci, the expression of which was similarly controlled in plasmid-deficient C. muridarum strains CM972 and CM3.1 and plasmid-deficient C. trachomatis CTD153. However, although expression of glycogen synthase, encoded by glgA, was greatly reduced in CTD153, it was unaltered in plasmid-deficient C. muridarum strains. Thus, additional plasmid-associated factors are required for glycogen accumulation by this chlamydial species. Furthermore, in C. trachomatis, glgA and other plasmid-responsive chromosomal loci (PRCLs) were transcriptionally responsive to glucose limitation, indicating that additional regulatory elements may be involved in the coordinated expression of these candidate virulence effectors. Glucose-limited C. trachomatis displayed reduced TLR2 stimulation in an in vitro assay. During human chlamydial infection, glucose limitation may decrease chlamydial virulence through its effects on plasmid-responsive chromosomal genes

Sec Biosynthesis in Eukaryotes

<p>The pathway of Sec biosynthesis is shown (see text for details and abbreviations are defined in the text with the exception of SerS [seryl-tRNA synthetase] and PSTK [<i>O</i>-phosphoseryl-tRNA^[Ser]Sec kinase]).</p