Florencia Meyer

Kaposi Sarcoma-associated Herpesvirus Degrades Cellular Toll-Interleukin-1 Receptor Domain-containing Adaptor-inducing β-Interferon (TRIF)

Kaposi sarcoma-associated herpesvirus (KSHV) is a human γ-herpesvirus associated with several human malignancies. The replication and transcription activator (RTA) is necessary and sufficient for the switch from KSHV latency to lytic replication. Toll-interleukin-1 receptor (TIR) domain-containing adaptor-inducing β-interferon (TRIF, also called TIR-domain-containing adaptor molecule-1 (TICAM-1)) is a signaling adaptor molecule that is critically involved in the Toll-like receptor 3 (TLR-3) and TLR-4 signaling pathways for type I interferon (IFN) production, a key component of innate immunity against microbial infection. In this report, we find a new mechanism by which RTA blocks innate immunity by targeting cellular TRIF. RTA specifically degrades TRIF by shortening the half-life of TRIF protein. This RTA-mediated degradation is at least partially mediated through the ubiquitin-proteasome pathway because proteasome inhibitors as well as knockdown of cellular ubiquitin expression alleviate the degradation. RTA may not directly interact with TRIF and may activate TRIF degradation indirectly through an unknown mediator(s). RTA targets multiple regions of TRIF and may use its ubiquitin ligase domain for the degradation. In addition, physiological levels of TRIF protein are down-regulated during KSHV lytic replication when RTA is expressed. Finally, RTA down-regulates double-stranded RNA-initiated activation of TLR-3 pathway, in the absence of degradation of IFN regulatory factor 7 (IRF-7). Taken together, these data suggest that KSHV employs a novel mechanism to block the innate immunity by degrading TRIF protein. This work may contribute to our understandings on how KSHV evades host immunity for its survival in vivo.

A Protein Encoded by the Bovine Herpesvirus 1 Latency-Related Gene Interacts with Specific Cellular Regulatory Proteins, Including CCAAT Enhancer Binding Protein Alpha

ABSTRACT Following acute infection, bovine herpesvirus 1 establishes latency in sensory neurons of trigeminal ganglia (TG). Reactivation from latency occurs periodically, resulting in the shedding of infectious virus. The latency-related (LR) RNA is abundantly expressed in TG of latently infected calves, and the expression of LR proteins is necessary for dexamethasone-induced reactivation from latency. Previously published studies also identified an alternatively spliced LR transcript which is abundantly expressed in TG at 7 days after infection and has the potential to encode a novel LR fusion protein. Seven days after infection is when extensive viral gene expression is extinguished in TG and latency is established, suggesting that LR gene products influence the establishment of latency. In this study, we used a bacterial two-hybrid assay to identify cellular proteins that interact with the novel LR fusion protein. The LR fusion protein interacts with two proteins that can induce apoptosis (Bid and Cdc42) and with CCAAT enhancer binding protein alpha (C/EBP-α). Additional studies confirmed that the LR fusion protein interacts with human or insect C/EBP-α. C/EBP-α protein expression is induced in TG neurons of infected calves and after dexamethasone-induced reactivation from latency. Wild-type C/EBP-α, but not a DNA binding mutant of C/EBP-α, enhances plaque formation in bovine cells. We hypothesize that interactions between the LR fusion protein and C/EBP-α promote the establishment of latency.

Interferon regulatory factor 4 (IRF-4) targets IRF-5 to regulate Epstein-Barr virus transformation

The cellular interferon regulatory factor-4 (IRF-4), which is a member of IRF family, is involved in the development of multiple myeloma and Epstein-Barr virus (EBV)-mediated transformation of B lymphocytes. However, the molecular mechanism of IRF-4 in cellular transformation is unknown. We have found that knockdown of IRF-4 leads to high expression of IRF-5, a pro-apoptotic member in the IRF family. Overexpression of IRF-4 represses IRF-5 expression. Reduction of IRF-4 leads to growth inhibition, and the restoration of IRF-4 by exogenous plasmids correlates with the growth recovery and reduces IRF-5 expression. In addition, IRF-4 negatively regulates IRF-5 promoter reporter activities and binds to IRF-5 promoters in vivo and in vitro. Knockdown of IRF-5 rescues IRF-4 knockdown-mediated growth inhibition, and IRF-5 overexpression alone is sufficient to induce cellular growth inhibition of EBV-transformed cells. Therefore, IRF-5 is one of the targets of IRF-4, and IRF-4 regulates the growth of EBV-transformed cells partially through IRF-5. This work provides insight on how IRFs interact with one another to participate in viral pathogenesis and transformation.

Identification of a novel protein encoded by the latency-related gene of bovine herpesvirus 1

The latency-related (LR) RNA encoded by bovine herpesvirus 1 (BHV-1) is abundantly expressed and alternatively spliced in trigeminal ganglia. A mutant BHV-1 strain that contains three stop codons at the beginning of LR open reading frame (ORF)-2 (LR mutant virus) does not express ORF-2 or an adjacent reading frame that lacks an initiating ATG (RF-C). Calves latently infected with wild-type (wt) BHV-1, but not with the LR mutant virus, reactivate from latency, indicating that proteins encoded by the LR gene regulate the latency-reactivation cycle. The LR gene also contains another large ORF (ORF-1) that is approximately 200 bp downstream of stop codons inserted at the N-terminus of ORF-2. To test whether the LR mutant virus can expresses ORF-1, the authors developed antiserum directed against ORF-1. The ORF-1 antiserum recognizes specific proteins in bovine cells productively infected with wt BHV-1. ORF-1 protein expression is reduced, but not blocked, when bovine cells are infected with the LR mutant virus. Confocal microscopy demonstrated ORF-1 is present in the cytoplasm and nucleus of productively infected cells, whereas RF-C or a fusion protein containing RF-C localizes to the cytoplasm. Trigeminal ganglia from calves latently infected with wt BHV-1 contain neurons specifically stained with the ORF-1 antiserum. These studies suggest ORF-1 expression may be important for the BHV-1 latency-reactivation cycle.

Bovine herpesvirus 1 productive infection and immediate early transcription unit 1 promoter are stimulated by the synthetic corticosteroid dexamethasone

The primary site for life-long latency of bovine herpesvirus 1 (BHV-1) is sensory neurons. The synthetic corticosteroid dexamethasone consistently induces reactivation from latency; however the mechanism by which corticosteroids mediate reactivation is unclear. In this study, we demonstrate for the first time that dexamethasone stimulates productive infection, in part, because the BHV-1 genome contains more than 100 potential glucocorticoid receptor (GR) response elements (GREs). Immediate early transcription unit 1 (IEtu1) promoter activity, but not IEtu2 or VP16 promoter activity, was stimulated by dexamethasone. Two near perfect consensus GREs located within the IEtu1 promoter were necessary for dexamethasone-mediated stimulation. Electrophoretic mobility shift assays and chromatin immunoprecipitation studies demonstrated that the GR interacts with IEtu1 promoter sequences containing the GREs. Although we hypothesize that DEX-mediated stimulation of IEtu1 promoter activity is important during productive infection and perhaps reactivation from latency, stress likely has pleiotropic effects on virus-infected cells.

TLR-TRIF pathway enhances the expression of KSHV replication and transcription activator

Background: Host innate immunity is against virus infection and replication. Results: Toll-like receptor 3 activation leads to enhanced expression of a key Kaposis sarcoma-associated herpesvirus (KSHV) protein. Conclusion: KSHV uses host Toll-like receptor pathway to augment its critical gene expression. Significance: A virus may usurp host innate immunity for its own benefits. Kaposis sarcoma-associated herpesvirus (KSHV) is a human γ-herpesvirus. KSHV replication and transcription activator (RTA) is necessary and sufficient for KSHV reactivation from latency. Toll-like receptors (TLRs) recognize pathogen-associated molecular patterns, act through adaptors, and initiate innate and adaptive immune responses against pathogens. Toll/interleukin-1-receptor domain containing adaptor protein inducing interferon-β (TRIF) is an adaptor associated with TLR3 and TLR4 signaling, and is closely related to antiviral signaling to activate type I interferon (IFN) production. We previously found that KSHV RTA degrades TRIF indirectly and blocks TLR3 pathways. In this report, we find that TRIF, as well as TLR3 activation, enhances KSHV RTA protein expression. The C-terminal region of the RTA is involved in the responding TRIF-mediated enhancement. The degradation of TRIF and the enhancement of RTA expression are using two different pathways. The enhancement by TLR-TRIF is at least partially via promoting translational efficiency of RTA mRNA. Finally, the receptor-interacting protein 1 (RIP1) may be involved in TRIF-mediated enhancement of RTA expression, but not in the RTA-mediated degradation of TRIF. Therefore, the activation of TLR-TRIF pathway enhances KSHV RTA protein expression, and KSHV RTA in turn degrades TRIF to block innate immunity. The putative KSHV-TLR-adaptor-interacting loop may be a critical element to evade and usurp host innate immunity in KSHV life-cycle.

A protein encoded by the bovine herpesvirus 1 open reading frame E gene induces neurite-like morphological changes in mouse neuroblastoma cells and is expressed in trigeminal ganglionic neurons

Bovine herpes virus 1 (BHV-1), like other α-herpesvirinae subfamily members, establishes latency in sensory neurons. Periodically BHV-1 reactivates from latency, resulting in virus shedding and spread to uninfected cattle. Although reactivation from latency does not usually lead to recurrent disease, the latency-reactivation cycle is crucial for virus transmission. The latency-related (LR) RNA is abundantly expressed during latency, and expression of a LR encoded protein is necessary for dexamethasone-induced reactivation from latency in cattle. Within LR promoter sequences, a small open reading frame (ORF) was identified (ORF-E) that is antisense to the LR-RNA, and downstream of the bICP0 gene. ORF-E transcription is consistently detected in trigeminal ganglia (TG) of latently infected calves, suggesting ORF-E expression plays a role in the latency-reactivation cycle. Polyclonal antiserum directed against an ORF-E peptide or the entire ORF-E protein specifically recognizes the nucleus of sensory neurons in TG of latently infected calves. The ORF-E peptide—specific antiserum also recognizes a protein when mouse neuroblastoma cells (neuro-2A) are transfected with an ORF-E expression construct. In contrast to the growth inhibiting properties of the LR gene, stably transfected ORF-E—expressing cells were obtained. Neuro-2A cells stably transfected with a plasmid expressing ORF-E induced morphological changes that resembled neurite-like projections. In contrast, neurite-like projections were not observed following transfection of neuro-2A cells with an empty vector. These studies suggest that a protein encoded by ORF-E has the potential to alter the physiology or metabolism of neuronal cell types, which may be important for long-term latency.

The cellular transcription factor, CCAAT enhancer–binding protein alpha (C/EBP-α), has the potential to activate the bovine herpesvirus 1 immediate-early transcription unit 1 promoter

Following acute infection, bovine herpesvirus-1 (BHV-1) establishes a lifelong latent infection in sensory neurons of trigeminal ganglia. BHV-1 periodically reactivates from latency and is shed as infectious virus. The latency-related (LR) gene is abundantly expressed in trigeminal ganglia of infected calves, and proteins encoded by the LR gene are necessary for reactivation from latency. We previously demonstrated that a novel LR protein interacts with a host transcription factor, CCAAT enhancer-binding protein alpha (C/EBPα). C/EBPα increases plaque-forming efficiency when cotransfected with BHV-1 DNA and its expression is induced in neurons during reactivation from latency (Meyer et al, 2007, J Virol 81: 59–67). The ability of C/EBPα to bind DNA is necessary for stimulating productive infection, suggesting C/EBPα stimulates viral transcription. We tested whether C/EBPα could trans-activate the BHV-1 immediate early transcription unit 1 (IEtu1) promoter because the IEtu1 promoter activates expression of two viral genes (bICP0 and bICP4) that stimulate producitve infection. In the current study, We demonstrate that C/ EBPα and the BHV-1 trans-inducing factor (bTIF) synergistically trans-activate IEtu1 promoter activity. However, bICP0 and C/EBPα did not synergistically trans-activate IEtu1 promoter activity. Deletion of IEtu1 promoter sequences demonstrated that C/EBPα by itself could trans-activate a truncated IEtu1 promoter, suggesting sequences in the distal region of the IEtu1 promoter negatively regulate C/EBPα activtiy. These studies suggest that C/EBPα stimulates productive infection and reactivation from latency, in part, by cooperating with bTIF to activate IEtu1 promoter activity.

Proteogenomic Identification of a Novel Protein-Encoding Gene in Bovine Herpesvirus 1 That Is Expressed during Productive Infection

Bovine herpesvirus 1 (BoHV-1) is one of several microbes that contributes to the development of the bovine respiratory disease (BRD) and can also induce abortions in cattle. As other alpha-herpesvirinae subfamily members, BoHV-1 efficiently replicates in many cell types and subsequently establishes a life-long latent infection in sensory neurons. BoHV-1 encodes more than 70 proteins that are expressed in a well-defined manner during productive infection. However, in silico open reading frame (ORF) prediction of the BoHV-1 genome suggests that the virus may encode more than one hundred proteins. In this study we used mass spectrometry followed by proteogenomic mapping to reveal the existence of 92 peptides that map to previously un-annotated regions of the viral genome. Twenty-one of the newly termed “intergenic peptides” were predicted to have a viable ORF around them. Twelve of these produced an mRNA transcript as demonstrated by strand-specific RT-PCR. We further characterized the 5′ and 3′ termini of one mRNA transcript, ORF-A, and detected a 55 kDa protein produced during active infection using a custom-synthesized antibody. We conclude that the coding potential of BoHV-1 is underestimated.