Yoko Ushijima

Herpes Simplex Virus Type 2 UL56 Interacts with the Ubiquitin Ligase Nedd4 and Increases Its Ubiquitination

ABSTRACT The herpes simplex virus UL56 gene is conserved among most members of the Alphaherpesvirinae family and plays a critical role in viral pathogenicity in vivo. The HSV-2 UL56 protein (UL56) is a C-terminally anchored type II membrane protein that is predicted to be inserted into the virion envelope, leaving its N-terminal domain in the tegument. UL56 interacts with KIF1A and UL11. Here we report that UL56 also interacts with the ubiquitin ligase Nedd4 and increases its ubiquitination. Nedd4 was identified as a UL56-interacting protein by a yeast two-hybrid screen. UL56 bound to Nedd4 via its PY motifs. Nedd4 was phosphorylated and degraded in wild-type HSV-2-infected cells but not in cells infected with a UL56-deficient mutant. Ubiquitination assays revealed that UL56 increased ubiquitinated Nedd4, which was actively degraded in infected cells. UL56 also caused a decrease in Nedd4 protein levels and the increased ubiquitination in cotransfected cells. However, UL56 itself was not ubiquitinated, despite its interaction with Nedd4. Based on these findings, we propose that UL56 regulates Nedd4 in HSV-2-infected cells, although deletion of UL56 had no apparent effect on viral growth in vitro.

Bortezomib induces apoptosis in T lymphoma cells and natural killer lymphoma cells independent of Epstein-Barr virus infection

Epstein‐Barr virus (EBV), which infects not only B cells, but also T cells and natural killer (NK) cells, is associated with multiple lymphoid malignancies. Recently, the proteasome inhibitor bortezomib was reported to induce apoptosis of EBV‐transformed B cells. We evaluated the killing effect of this proteasome inhibitor on EBV‐associated T lymphoma cells and NK lymphoma cells. First, we found that bortezomib treatment decreased the viability of multiple T and NK cell lines. No significant difference was observed between EBV‐positive and EBV‐negative cell lines. The decreased viability in response to bortezomib treatment was abrogated by a pan‐caspase inhibitor. The induction of apoptosis was confirmed by flow cytometric assessment of annexin V staining. Additionally, cleavage of caspases and polyadenosine diphosphate‐ribose polymerase, increased expression of phosphorylated IκB, and decreased expression of inhibitor of apoptotic proteins were detected by immunoblotting in bortezomib‐treated cell lines. We found that bortezomib induced lytic infection in EBV‐positive T cell lines, although the existence of EBV did not modulate the killing effect of bortezomib. Finally, we administered bortezomib to peripheral blood mononuclear cells from five patients with EBV‐associated lymphoproliferative diseases. Bortezomib had a greater killing effect on EBV‐infected cells. These results indicate that bortezomib killed T or NK lymphoma cells by inducing apoptosis, regardless of the presence or absence of EBV.

Herpes simplex virus type 2 tegument protein UL56 relocalizes ubiquitin ligase Nedd4 and has a role in transport and/or release of virions.

BackgroundThe ubiquitin system functions in a variety of cellular processes including protein turnover, protein sorting and trafficking. Many viruses exploit the cellular ubiquitin system to facilitate viral replication. In fact, herpes simplex virus (HSV) encodes a ubiquitin ligase (E3) and a de-ubiquitinating enzyme to modify the hosts ubiquitin system. We have previously reported HSV type 2 (HSV-2) tegument protein UL56 as a putative adaptor protein of neuronal precursor cell-expressed developmentally down-regulated 4 (Nedd4) E3 ligase, which has been shown to be involved in protein sorting and trafficking.ResultsIn this study, we visualized and characterized the dynamic intracellular localization of UL56 and Nedd4 using live-cell imaging and immunofluorescence analysis. UL56 was distributed to cytoplasmic vesicles, primarily to the trans-Golgi network (TGN), and trafficked actively throughout the cytoplasm. Moreover, UL56 relocalized Nedd4 to the vesicles in cells transiently expressing UL56 and in cells infected with HSV-2. We also investigated whether UL56 influenced the efficiency of viral replication, and found that extracellular infectious viruses were reduced in the absence of UL56.ConclusionThese data suggest that UL56 regulates Nedd4 and functions to facilitate the cytoplasmic transport of virions from TGN to the plasma membrane and/or release of virions from the cell surface.

Microarray analysis of transcriptional responses to infection by herpes simplex virus types 1 and 2 and their US3-deficient mutants.

Herpes simplex virus types 1 (HSV-1) and 2 (HSV-2) induce similar responses in infected cells and animals but differ in several significant respects. Previous studies have shown that defects in the US3-encoded protein kinase greatly affect both viruses in their interactions with cells and hosts. To investigate the impact of infection with HSV-1, HSV-2 and their US3-deficient mutants (DeltaUS3) on cellular transcriptional responses, we performed a global microarray analysis on human epithelial HEp-2 cells that were mock-infected, or infected with wild-type (WT) HSV-1, HSV-2 and their DeltaUS3 mutants. Among 54,765 probe sets examined, only 1156 (approximately 2.1%) and 2006 (approximately 3.7%) genes increased by at least fourfold at 9h postinfection in WT HSV-1 and HSV-2-infected cells, respectively. Unexpectedly, HSV-2 infection increases mRNA levels for a larger number of cellular genes than HSV-1 infection. Additionally, DeltaUS3 infection upregulated the expression of a larger number of cellular genes than WT infection. The genes affected by HSV infection were assigned to various groups of functional classes and cellular pathways. We have thus identified cellular genes whose expression was similarly or differently changed by infection with each virus.

Herpes simplex virus UL56 interacts with and regulates the Nedd4-family ubiquitin ligase Itch

BackgroundHerpes simplex virus type 2 (HSV-2) is one of many viruses that exploits and modifies the cellular ubiquitin system. HSV-2 expresses the tegument protein UL56 that has been implicated in cytoplasmic transport and/or release of virions, and is a putative regulatory protein of Nedd4 ubiquitin ligase. In order to elucidate the biological function of UL56, this study examined the interaction of UL56 with the Nedd4-family ubiquitin ligase Itch and its role in the regulation of Itch. Additionally, we assessed the similarity between UL56 and regulatory proteins of Itch and Nedd4, Nedd4-family-interactins proteins (Ndfip).ResultsUL56 interacted with Itch, independent of additional viral proteins, and mediated more striking degradation of Itch, compared to Nedd4. Moreover, it was suggested that the lysosome pathway as well as the proteasome pathway was involved in the degradation of Itch. Other HSV-2 proteins with PY motifs, such as VP5 and VP16, did not mediate the degradation of endogenous Itch. Ndfip1 and Ndfip2 were similar in subcellular distribution patterns to UL56 and colocalized with UL56 in co-transfected cells.ConclusionsWe believe that this is the first report demonstrating the interaction of a HSV-specific protein and Itch. Thus, UL56 could function as a regulatory protein of Itch. The mechanism, function and significance of regulating Itch in HSV-2 infection remain unclear and warrant further investigation.

Oligonucleotide Microarray Analysis of Gene Expression Profiles followed by Real-Time Reverse-Transcriptase Polymerase Chain Reaction Assay in Chronic Active Epstein-Barr Virus Infection

Chronic active Epstein-Barr virus infection (CAEBV) is characterized by recurrent infectious mononucleosis-like symptoms and has high mortality and morbidity. To clarify the mechanisms of CAEBV, the gene-expression profiles of peripheral blood obtained from patients with CAEBV were investigated. Twenty genes were differentially expressed in 4 patients with CAEBV. This microarray result was verified using a real-time reverse-transcriptase polymerase chain reaction assay in a larger group of patients with CAEBV. Eventually, 3 genes were found to be significantly upregulated: guanylate binding protein 1, tumor necrosis factor-induced protein 6, and guanylate binding protein 5. These genes may be associated with the inflammatory reaction or with cell proliferation.

Replication-competent, oncolytic herpes simplex virus type 1 mutants induce a bystander effect following ganciclovir treatment

Cells expressing herpes simplex virus (HSV) thymidine kinase (tk) are killed by ganciclovir (GCV). Adjacent cells without HSV‐tk also die, a phenomenon known as the ‘bystander effect’. However, there is no evidence that replication‐competent HSV induces a bystander effect in the presence of GCV. Therefore, we investigated the bystander effect in HEp‐2 cells infected with replication‐competent, oncolytic HSV‐1 mutants, hrR3 and HF10. In cells infected at a multiplicity of infection (MOI) of 3, GCV did not induce apoptosis. At low MOIs of 0.3 and 0.03, however, a number of adjacent, uninfected cells apoptosed following GCV treatment. Irrespective of GCV treatment, HEp‐2 cells expressed minimal levels of connexin 43 (Cx43). However, Cx43 expression was enhanced by GCV in response to infection with HF10 at an MOI of 0.3, but not at an MOI of 3. Expression of other proteins involved in gap junctions, including Cx26 and Cx40, was not augmented under these conditions. The PKA and PI3K signal transduction pathways are likely involved in enhanced Cx43 expression as inhibitors of these pathways prevented Cx43 upregulation. These results suggest that infection with replication‐competent HSV‐1 induces the bystander effect in cells treated with GCV because of efficient intercellular transport of active GCV through abundant gap junctions. Copyright

Generation and characterization of UL21-null herpes simplex virus type 1

UL21 of herpes simplex virus type 1 (HSV-1) is an accessory gene that encodes a component of the tegument. Homologs of this protein have been identified in the alpha, beta, and gamma herpesvirus subfamilies, although their functions are unclear. To clarify the functions of UL21, we generated a UL21-null HSV-1 mutant. Growth analysis showed that the synthesis of infectious UL21-null HSV-1 in glial cells was delayed and that the overall yield was low. The plaque sizes of the UL21-null mutant were smaller than those of wild-type HSV-1. We identified several candidate UL21-interacting proteins, including intermediate filaments, by yeast two-hybrid screening. The distribution of glial fibrillary acidic protein (GFAP), which is the main component of intermediate filaments, was altered in UL21-null mutant-infected glial cells compared to wild-type virus-infected cells. These results will help clarify the function of UL21 and broaden our understanding of the life cycle of HSV.

Phase II study of intrabone single unit cord blood transplantation for hematological malignancies

The outcomes of cord blood transplantation with non‐irradiated reduced‐intensity conditioning for hematological malignancies need to be improved because of graft failure and delayed engraftment. Intrabone infusion of cord blood cells has the potential to resolve the problems. In this phase II study, 21 adult patients with hematological malignancy received intrabone transplantation of serological HLA‐A, B, and DR ≥4/6 matched single cord blood with a median number of cryopreserved total nucleated cells of 2.7 × 107/kg (range, 2.0–4.9 × 107/kg) following non‐irradiated fludarabine‐based reduced‐intensity conditioning. Short‐term methotrexate and tacrolimus were given as graft‐versus‐host disease prophylaxis, and granulocyte colony‐stimulating factor was given after transplantation. No severe adverse events related to intrabone injection were observed. The cumulative incidences of neutrophils ≥0.5 × 109/L, reticulocytes ≥1%, and platelets ≥20 × 109/L recoveries were 76.2%, 71.4%, and 76.2%, respectively, with median time to recoveries of 17, 28, and 32 days after transplantation, respectively. The probability of survival with neutrophil engraftment on day 60 was 71.4%, and overall survival at 1 year after transplantation was 52.4%. The incidences of grade II–IV and III–IV acute graft‐versus‐host disease were 44% and 19%, respectively, with no cases of chronic graft‐versus‐host disease. The present study showed the safety of direct intrabone infusion of cord blood. Further analysis is required to confirm the efficacy of intrabone single cord blood transplantation with non‐irradiated reduced‐intensity conditioning for adult patients with hematological malignancy. This study was registered with UMIN‐CTR, number 000000865.

Identification of the novel deletion-type PML-RARA mutation associated with the retinoic acid resistance in acute promyelocytic leukemia

All-trans retinoic acid (ATRA) and arsenic trioxide (ATO) are essential for acute promyelocytic leukemia (APL) treatment. It has been reported that mutations in PML-RARA confer resistance to ATRA and ATO, and are associated with poor prognosis. Although most PML-RARA mutations were point mutations, we identified a novel seven amino acid deletion mutation (p.K227_T233del) in the RARA region of PML-RARA in a refractory APL patient. Here, we analyzed the evolution of the mutated clone and demonstrated the resistance of the mutated clone to retinoic acid (RA). Mutation analysis of PML-RARA was performed using samples from a chemotherapy- and ATRA-resistant APL patient, and the frequencies of mutated PML-RARA transcript were analyzed by targeted deep sequencing. To clarify the biological significance of the identified PML-RARA mutations, we analyzed the ATRA-induced differentiation and PML nuclear body formation in mutant PML-RARA-transduced HL-60 cells. At molecular relapse, the p.K227_T233del deletion and the p.R217S point-mutation in the RARA region of PML-RARA were identified, and their frequencies increased after re-induction therapy with another type of retinoiec acid (RA), tamibarotene. In deletion PML-RARA-transduced cells, the CD11b expression levels and NBT reducing ability were significantly decreased compared with control cells and the formation of PML nuclear bodies was rarely observed after RA treatment. These results indicate that this deletion mutation was closely associated with the disease progression during RA treatment.