Rhesus Macaque Rhadinovirus Encodes a Viral Interferon Regulatory Factor To Disrupt Promyelocytic Leukemia Nuclear Bodies and Antagonize Type I Interferon Signaling.

Interferon (IFN) production and the subsequent induction of IFN-stimulated genes (ISGs) are highly effective innate strategies utilized by cells to protect against invading pathogens, including viruses. Critical components involved in this innate process are promyelocytic leukemia nuclear bodies (PML-NBs), which are subnuclear structures required for the development of a robust IFN response. As such, PML-NBs serve as an important hurdle for viruses to overcome to successfully establish an infection. Both Kaposi's sarcoma-associated herpesvirus (KSHV) and the closely related rhesus macaque rhadinovirus (RRV) are unique for encoding viral homologs of IFN regulatory factors (termed vIRFs) that can manipulate the host immune response by multiple mechanisms. All four KSHV vIRFs inhibit the induction of IFN, while vIRF1 and vIRF2 can inhibit ISG induction downstream of the IFN receptor. Less is known about the RRV vIRFs. RRV vIRF R6 can inhibit the induction of IFN by IRF3; however, it is not known whether any RRV vIRFs inhibit ISG induction following IFN receptor signaling. In our present study, we demonstrate that the RRV vIRF R12 aids viral replication in the presence of the type I IFN response. This is achieved in part through the disruption of PML-NBs and the inhibition of robust ISG transcription.IMPORTANCE KSHV and RRV encode a unique set of homologs of cellular IFN regulatory factors, termed vIRFs, which are hypothesized to help these viruses evade the innate immune response and establish infections in their respective hosts. Our work elucidates the role of one RRV vIRF, R12, and demonstrates that RRV can dampen the type I IFN response downstream of IFN signaling, which would be important for establishing a successful infection in vivo.

Assembly of Epstein-Barr Virus Capsid in Promyelocytic Leukemia Nuclear Bodies.

The Epstein-Barr virus (EBV) capsid contains a major capsid protein, VCA; two minor capsid proteins, BDLF1 and BORF1; and a small capsid protein, BFRF3. During the lytic cycle, these capsid proteins are synthesized and imported into the host nucleus for capsid assembly. This study finds that EBV capsid proteins colocalize with promyelocytic leukemia (PML) nuclear bodies (NBs) in P3HR1 cells during the viral lytic cycle, appearing as nuclear speckles under a confocal laser scanning microscope. In a glutathione S-transferase pulldown study, we show that BORF1 interacts with PML-NBs in vitro. BORF1 also colocalizes with PML-NBs in EBV-negative Akata cells after transfection and is responsible for bringing VCA and the VCA-BFRF3 complex from the cytoplasm to PML-NBs in the nucleus. Furthermore, BDLF1 is dispersed throughout the cell when expressed alone but colocalizes with PML-NBs when BORF1 is also present in the cell. In addition, this study finds that knockdown of PML expression by short hairpin RNA does not influence the intracellular levels of capsid proteins but reduces the number of viral particles produced by P3HR1 cells. Together, these results demonstrate that BORF1 plays a critical role in bringing capsid proteins to PML-NBs, which may likely be the assembly sites of EBV capsids. The mechanisms elucidated in this study are critical to understanding the process of EBV capsid assembly. IMPORTANCE Capsid assembly is an important event during the Epstein-Barr virus (EBV) lytic cycle, as this process is required for the production of virions. In this study, confocal microscopy revealed that the EBV capsid protein BORF1 interacts with promyelocytic leukemia (PML) nuclear bodies (NBs) in the host nucleus and is responsible for transporting the other EBV capsid proteins, including VCA, BDLF1, and BFRF3, to these subnuclear locations prior to initiation of capsid assembly. This study also found that knockdown of PML expression by short hairpin RNA significantly reduces EBV capsid assembly capabilities. This enhanced understanding of capsid assembly offers potential for the development of novel antiviral strategies and therapies that can prevent the propagation and spread of EBV.

JC virus intranuclear inclusions associated with PML-NBs: analysis by electron microscopy and structured illumination microscopy.

Progressive multifocal leukoencephalopathy is a fatal demyelinating disorder caused by JC virus infection. JC virus was recently found to target promyelocytic leukemia nuclear bodies (PML-NBs), punctuate domains in the nuclei. Thus, the virus progenies cluster in dots as intranuclear inclusions (ie, as dot-shaped inclusions). In the present study, both the viral major and minor capsid proteins were expressed from polycistronic expression vectors with a powerful promoter, and formation into virus-like particles (VLPs) was examined by electron microscopy. When the upstream regulatory sequence including the agnogene (nt 275 to 490) was present, capsid protein expression was suppressed, but numerous VLPs were efficiently formed with restricted accumulation to PML-NBs. VLPs were uniform, and the cells were severely degraded. In contrast, when the 5' terminus of the agnogene (nt 275 to 409; 135 bp) was deleted, capsid protein expression was markedly enhanced, but VLPs were more randomly produced in the nucleus outside of PML-NBs. VLPs were pleomorphic, and cell degradation was minimal. JC virus association with PML-NBs was confirmed in human brain tissues by structured illumination microscopy. PML-NBs were shaped in spherical shells, with viral capsid proteins circumscribing the surface. These findings indicate that PML-NBs are intranuclear locations for pathogenic JC virus proliferation. Either the agnogene or its product likely supports efficient progeny production at PML-NBs, leading to subsequent degeneration of host glial cells.

Epstein - Barr virus Latent Membrane Protein 1 suppresses reporter activity through modulation of promyelocytic leukemia protein-nuclear bodies.

The Epstein-Barr virus (EBV) encoded Latent Membrane Protein 1 (LMP1) has been shown to increase the expression of promyelocytic leukemia protein (PML) and the immunofluorescent intensity of promyelocytic leukemia nuclear bodies (PML NBs). PML NBs have been implicated in the modulation of transcription and the association of reporter plasmids with PML NBs has been implicated in repression of reporter activity. Additionally, repression of various reporters in the presence of LMP1 has been noted. This study demonstrates that LMP1 suppresses expression of reporter activity in a dose responsive manner and corresponds with the LMP1 induced increase in PML NB intensity. Disruption of PML NBs with arsenic trioxide or a PML siRNA restores reporter activity. These data offer an explanation for previously conflicting data on LMP1 signaling and calls attention to the possibility of false-positives and false-negatives when using reporter assays as a research tool in cells expressing LMP1.

Influenza A (H1N1) in a pediatric patient with newly diagnosed acute promyelocytic leukemia and invasive pulmonary aspergillosis.

Influenza A (H1N1) pandemic reached its peak in Europe in autumn 2009. H1N1 infection can be a serious complication in patients with comorbidity or immunodepression. Here, we report of a boy with newly diagnosed acute promyelocytic leukemia with a very severe respiratory distress caused by influenza A (H1N1) infection in pulmonary aspergillosis, successfully treated with antifungal therapy, oseltamivir, and extracorporeal membrane oxygenation.

Crosstalk between viruses and PML nuclear bodies: a network-based approach.

Due to the recent advances in instrumental and scientific methods, cell biology data are generated with increasing speed and quantity. One of these fast developing fields is the crosstalk between promyelocytic leukemia protein nuclear bodies (PML-NBs) and viruses. PML-NBs are dynamic nuclear protein aggregates which are targeted by entire viral particles, viral proteins or viral nucleic acids. Their possible anti-viral properties motivated researchers to investigate the interaction between PML-NBs and viruses in depth. Based on extensive literature data mining, we created a comprehensive PML-NB/virus crosstalk Cytoscape network, which groups not only the most common relations but also less well described findings. The network is easy to navigate and provides a biologically relevant overview which can help finding interesting case studies.

Viral disruption of promyelocytic leukemia (PML) nuclear bodies by hijacking host PML regulators.

Epstein-Barr virus (EBV) latent infection promotes cell survival and proliferation, in some cases contributing to tumourigenesis. EBV-immortalized cells and EBV-induced tumours express the viral EBNA1 protein which, in addition to its roles in replicating and maintaining EBV genomes, can alter cellular processes, including the disruption of promyelocytic leukemia (PML) nuclear bodies (NBs) through the degradation of PML proteins. PML NBs are based on PML proteins and mediate several cellular processes including apoptosis, DNA repair and antiviral responses. Accordingly, EBNA1 expression decreases apoptosis and DNA repair which may contribute to malignant transformation. The ability of EBNA1 to disrupt PML NBs has recently been shown to require EBNA1 binding to two host proteins, the protein kinase CK2 and deubiquitylating protein USP7/HAUSP, both of which are known to be partially associated with PML NBs. EBNA1 increases the association of both CK2 and USP7 with PML NBs and, as a result, increases phosphorylation of PML proteins by CK2, a modification that is known to trigger PML polyubiquitylation and degradation. Recent data also implicates USP7 as a negative regulator of PML proteins and nuclear bodies by a mechanism independent of its intrinsic ubiquitin cleavage activity. The results suggest that EBNA1 usurps two host PML regulators in order to promote degradation of PML proteins and loss of PML NBs.

Acute promyelocytic leukemia in HIV-infected adults: a case report and review of therapeutic considerations.

The incidence of acute promyelocytic leukemia (APL) in patients with HIV is exceedingly rare, making the establishment of therapeutic approaches challenging and often individualized. We report the case of a 43-year-old female who presented with fatigue and malaise, and was concurrently diagnosed with APL and HIV. Induction and consolidation with all-trans-retinoic acid (ATRA), idarubicin, and mitoxantrone were initiated in conjunction with highly active anti-retroviral therapy (HAART) consisting of tenofovir/emtricitabine, fosamprenavir, and raltegravir. A complete morphologic, cytogenetic, and molecular response was achieved post-induction. Therapeutic strategies should consider overlapping effects of current agents in targeting both pathologies. ATRA has been found to induce apoptosis in HIV-infected leukemic cells, and protease inhibitor therapy has furthermore been reported to be synergistic with ATRA in inducing differentiation of APL cell lines. Pending further investigation, regimens with protease inhibitor backbones may represent a viable first-line strategy for patients elected to receive HAART in addition to ATRA and standard chemotherapy.

HPV-16 E7 reveals a link between DNA replication stress, fanconi anemia D2 protein, and alternative lengthening of telomere-associated promyelocytic leukemia bodies.

Expression of the high-risk human papillomavirus (HPV-16) E7 oncoprotein extends the life span of primary human keratinocytes and partially restores telomere length in the absence of telomerase. The molecular basis of this activity is incompletely understood. Here, we show that HPV-16 E7 induces an increased formation of alternative lengthening of telomeres (ALT)-associated promyelocytic leukemia bodies (APBs) in early passage primary human keratinocytes as well as HPV-negative tumor cells. This activity was found to require sequences of HPV-16 E7 involved in degradation of the retinoblastoma tumor suppressor protein as well as regions in the COOH terminus. HPV-16 E7-induced APBs contained ssDNA and several proteins that are involved in the response to DNA replication stress, most notably the Fanconi anemia D2 protein (FANCD2) as well as BRCA2 and MUS81. In line with these results, we found that FANCD2-containing APBs form in an ATR-dependent manner in HPV-16 E7-expressing cells. To directly show a role of FANCD2 in ALT, we provide evidence that knockdown of FANCD2 rapidly causes telomere dysfunction in cells that rely on ALT to maintain telomeres. Taken together, our results suggest a novel link between replication stress and recombination-based telomere maintenance that may play a role in HPV-16 E7-mediated extension of host cell life span and immortalization.

High prevalence of human T-lymphotropic virus type I carriers among patients with myelodysplastic syndrome refractory anemia with excess of blasts (RAEB), RAEB in transformation and acute promyelocytic leukemia.

We examined human T-lymphotropic virus type I (HTLV-I) infection among patients with myelodysplastic syndrome (MDS), refractory anemia with excess of blasts (RAEB)/RAEB in transformation (RAEBt) and acute myelogenous leukemia (AML). The study population consisted of 151 patients: 46 with MDS RAEB/RAEBt and 105 with AML (M1, n = 15; M2, n = 39; M3, n = 18; M4, n = 19; M5, n = 9; M6, n = 3; M7, n = 2). As a reference, we examined 92 patients with refractory anemia (RA) and 405 patients with cardiovascular diseases (CVD). Thirteen patients with RAEB/RAEBt (28.3%), 11 with AML (11.6%), 27 with RA (29.3%), and 45 with CVD (11.0%) were positive for HTLV-I. Seven AML patients with HTLV-I infection had M3 acute promyelocytic leukemia (APL). The prevalences of HTLV-I infection among patients with RAEB/RAEBt (P < 0.001), APL (P = 0.001), and RA (P < 0.001) were significantly higher than that in patients with CVD. The prevalences of HTLV-I infection were still significantly higher in patients with RAEB/RAEBt (P = 0.007), APL (P = 0.017) and RA (P < 0.001) than in those with CVD matched by sex and age. Platelet counts and survival times of RAEB/RAEBt patients with infection were significantly lower than those of patients without infection.

Acute promyelocytic leukemia and HIV-1 infection: case report and review of the literature.

We report a 27-year-old man with HIV-1 infection who developed acute promyelocytic leukemia (APL) with a novel complex three-way chromosomal translocation t(15;16;17). Induction of remission and consolidation with all-trans-retinoic acid (ATRA)- and anthracycline-based chemotherapy was followed by maintenance therapy consisting of ATRA, 6-mercaptopurine (6-MP), and methotrexate (MTX). Highly active antiretroviral therapy (HAART) was continued with brief interruptions. He remains in complete remission 40 months after diagnosis.

Herpes zoster during treatment with arsenic trioxide.

In vitro data suggest that arsenic compounds can suppress cell-mediated immunity by inducing apoptosis of T helper lymphocytes. We describe an occurrence of herpes zoster during treatment with arsenic trioxide (ATO) in two patients who were already in remission from acute promyelocytic leukemia and received ATO as consolidation treatment. During this complication, their leukocyte counts and differentials were within normal limits. Our report suggested the immunosuppressive effect of ATO in vivo. Both patients responded well to an oral antiviral. Clinicians should be aware of this complication during treatment with ATO since early antiviral treatment may help avoid complications including post-herpetic neuralgia.

Distinct roles of the Adenovirus E4 ORF3 protein in viral DNA replication and inhibition of genome concatenation.

Adenovirus early proteins E4 ORF3 and E4 ORF6 have complementary functions during viral infection. Both proteins facilitate efficient viral DNA replication, late protein expression, and prevention of concatenation of viral genomes. Additionally, E4 ORF6 is involved in the shutoff of the host cell protein synthesis through its interaction with the E1B 55K protein. This complex also leads to the degradation of p53. A unique function of E4 ORF3 is the reorganization of nuclear structures known as PML oncogenic domains (PODs). The function of these domains is unclear, but PODs have been implicated in a number of important cellular processes, including transcriptional regulation, apoptosis, transformation, and response to interferon. The goal of this study was to determine the functional significance of the reorganization of PODs by E4 ORF3. Point mutations were made in the E4 ORF3 gene. These mutants were recombined into a virus lacking E4 ORF6 and expressed under the control of the natural virus E4 promoter. The panel of mutant viruses was used to investigate the role of E4 ORF3 during the course of the viral infection program. One of the mutant viruses exhibited aberrant reorganization of PODs and had a severe defect in viral DNA replication, thus leading to a dramatic decrease in virus production. A number of mutants accumulated viral DNA and infectious virus particles to wild-type levels but showed significant viral genome concatenation. These data show that E4 ORF3 is a multifunctional protein and that a specific rearrangement of nuclear PML domains is coupled to efficient viral DNA replication. This function is distinct from the role of E4 ORF3 in the regulation of virus genome concatenation via inhibition of cellular double-strand break repair.

Acute promyelocytic leukemia in a HIV seropositive patient.

Acute myeloid leukemia (AML) is infrequent in patients with human immunodeficiency virus (HIV) infection. Among AML, acute promyelocytic leukemia (APL) has been rarely described in such patients, with only one case being published. We report a 30 years-old intravenous drug abuser HIV-infected male with APL who attained complete clinical, morphological, and molecular remission after differentiation therapy with all-trans-retinoic acid (ATRA) followed by intensive chemotherapy. The results of treatment in this patient and in other AML published cases suggest that therapy for AML should not be modified because of HIV infection if patients have an adequate performance status.

Agonist-stimulated free calcium in subcellular compartments. Delivery of recombinant aequorin to organelles using a replication deficient adenovirus vector.

Changes in the concentration of calcium ions ([Ca2+]) within cellular organelles play a central role in controlling cellular function. We have engineered the Ca2+ sensitive photoprotein aequorin to monitor selectively [Ca2+] within defined subcellular compartments, namely the cytosol, nucleus and endoplasmic reticulum. DNA encoding the engineered aequorins have been inserted into a replication deficient adenovirus (Ad) type 5 E1-vector, under control of the cytomegalovirus (CMV) major immediate early promoter. The Ad vector provides a simple and efficient method to express the photoproteins in a wide variety of mammalian cell types. Efficient targeting of the photoproteins to the appropriate cellular compartment was established immunocytochemically in COS7 cells, where it was expressed in up to 100% of the target population. Levels of expression could be controlled by virus dose and chemical agents which affect the activity of the CMV promoter. In HeLa cells expressing nuclear targeted aequorin or cytosolic aequorin, ATP or histamine induced immediate biphasic elevations of both nuclear and cytosolic [Ca2+]; subsequent challenge with agonist evoked similar responses. In addition to epithelial type adherent cell lines (COS7 and HeLa), aequorin expression was also readily detected in non-adherent cells of myeloid lineage (K562 and HL60) and non-adherent primary cells polymorphonuclear leucocytes (neutrophils). The Ad vectors can, therefore, be used to express targeted aequorin in a range of different cell types and represents a novel method to monitor changes in free [Ca2+] in cellular organelles.

Adenovirus replication is coupled with the dynamic properties of the PML nuclear structure.

Wild-type PML and at least four other novel proteins are localized within discrete nuclear structures known as PODs. We demonstrate here that during adenovirus infection, immediate early viral proteins from the E1 and E4 transcription units associate with the POD, which in turn undergoes a dramatic morphological change. During this process, the auto-antigen Sp-100 and NDP55 but not PML, relocate from the POD to the viral inclusion bodies, the sites of adenovirus DNA replication and late RNA transcription. The E4-ORF3 11-kD protein alone will induce this reorganization and reciprocally, viruses carrying mutations in the E4-domain fail to do so. These same viral mutants are defective in viral replication as well as the accumulation of late viral mRNAs and host cell transcription shutoff. We show that interferon (INF) treatment enhances the expression of PML, reduces or blocks PODs reorganization, and inhibits BrdU incorporation into viral inclusion bodies. In addition, cell lines engineered to overexpress PML prevent PODs from viral-induced reorganization and block or severely delay adenovirus replication. These results suggest that viral replication relies on components of the POD and that the structure is a target of early viral proteins.

[Acute myeloid leukemia (promyelocytic leukemia) with detection of virus particles in a boar. Histologic, histochemical and ultrastructural findings]. / Akute myeloische Leukose (Promyelozyten-Leukose) mit Nachweis von Viruspartikeln bei einem Eber. Histologische, histochemische und ultrastrukturelle Befunde.

Acute myeloid leukemia is a rare finding in swine. A case of acute promyelocytic leukemia is described by light microscopy, enzyme- and immunohistochemistry as well as electron microscopy. Tumour cells can be demonstrated in bone marrow, lymph nodes, liver, spleen, kidneys, small intestine and myocardium. The tumorous neutrophil precursor cells are positive for naphthol-AS-D-chloroacetate esterase and lysozyme. They show ultrastructurally numerous granules as typical for promyelocytes. Virus particles of the oncorna-c-type can be demonstrated within the tumour cells.