Marek's disease virus (MDV) causes a general malaise in chickens that is mostly characterized by the development of lymphoblastoid tumors in multiple organs. The use of bacterial artificial chromosomes (BACs) for cloning and manipulation of the MDV genome has facilitated characterization of specific genes and genomic regions. The development of most MDV BACs, including pRB-1B-5, derived from a very virulent MDV strain, involved replacement of the U S 2 gene with mini-F vector sequences. However, when reconstituted viruses based on pRB-1B were used in pathogenicity studies, it was discovered that contact chickens housed together with experimentally infected chickens did not contract Marek's disease (MD), indicating a lack of horizontal transmission. Staining of feather follicle epithelial cells in the skins of infected chickens showed that virus was present but was unable to be released and/or infect susceptible chickens. Restoration of U S 2 and removal of mini-F sequences within viral RB-1B did not alter this characteristic, although in vivo viremia levels were increased significantly. Sequence analyses of pRB-1B revealed that the U L 13, U L 44, and U S 6 genes encoding the U L 13 serine/threonine protein kinase, glycoprotein C (gC), and gD, respectively, harbored frameshift mutations. These mutations were repaired individually, or in combination, using two-step Red mutagenesis. Reconstituted viruses were tested for replication, MD incidence, and their abilities to horizontally spread to contact chickens. The experiments clearly showed that U S 2, U L 13, and gC in combination are essential for horizontal transmission of MDV and that none of the genes alone is able to restore this phenotype.Marek's disease virus (MDV), also known as Gallid herpesvirus 2, causes Marek's disease (MD), a lymphoproliferative disease in chickens characterized by the development of tumors in the viscera and other organs. The pathogenesis of infection with MDV can be divided into four phases (4). In the first phase, between 3 and 6 days postinfection (p.i.), the primary target cells of infection are bursa-derived (B) lymphocytes. Infection of activated CD4 ϩ thymus-derived (T) lymphocytes follows in the second phase. During the second phase, viral replication typically decreases and a latent infection is established between 5 and 10 days p.i. in activated CD4 ϩ T lymphocytes. The third phase is characterized by reactivation of MDV replication between 14 and 21 days p.i. and infection of feather follicle epithelium (FFE) cells. After the third phase, virus is shed from the chicken in dried FFE cells, and clinical MD symptoms and lymphomas may develop depending on the genetic susceptibility of the chickens and the virulence of the virus strain.The MDV genome consists of the unique short (U S ) and long (U L ) regions, flanked by the inverted repeat long (IR L ) and short (IR S ) regions, and the terminal repeat long (TR L ) and short (TR S ) regions. Through manipulation of the MDV genome using multiple approaches, specific genes or re...

Herpesvirus telomeric repeats facilitate virus integration into host telomeres, a process which is required for the establishment of virus latency.

Marek's disease (MD) in chickens is caused by the alphaherpesvirus MD virus (MDV) and is characterizedby the development of lymphoblastoid tumors in multiple organs. The recent identification and cloning of RLORF4 and the finding that four of six attenuated strains of MDV contained deletions within RLORF4 suggested that it is involved in the attenuation process of MDV. To assess the role of RLORF4 in MD pathogenesis, its coding sequence was deleted in the pRB-1B bacterial artificial chromosome clone. Additionally, RLORF5a was deleted separately to examine its importance for oncogenesis. The sizes of plaques produced by MDV reconstituted from pRB-1B⌬RLORF5a (rRB-1B⌬RLORF5a) were similar to those produced by the parental pRB-1B virus (rRB-1B). In contrast, virus reconstituted from pRB-1B⌬RLORF4 (rRB-1B⌬RLORF4) produced significantly larger plaques. Replication of the latter virus in cultured cells was higher than that of rRB-1B or rRB-1B⌬RLORF5a using quantitative PCR (qPCR) assays. In vivo, both deletion mutants and rRB-1B replicated at comparable levels at 4, 7, and 10 days postinoculation (p.i.), as determined by virus isolation and qPCR assays. At 14 days p.i., the number of PFU of virus isolated from chickens infected with rRB-1B⌬RLORF4 was comparable to that from chickens infected with highly attenuated RB-1B and significantly lower than that from rRB-1B-infected birds. The number of tumors and kinetics of tumor production in chickens infected with rRB-1B⌬RLORF5a were similar to those of P2a chickens infected with rRB-1B. In stark contrast, none of the chickens inoculated with rRB-1B⌬RLORF4 died up to 13 weeks p.i.; however, two chickens had tumors at the termination of the experiment. The data indicate that RLORF4 is involved in attenuation of MDV, although the function of RLORF4 is still unknown.Marek's disease (MD) is a lymphoproliferative disease in chickens caused by a member of the Alphaherpesvirinae subfamily, MD virus (MDV). The pathogenesis of infection with MDV can be divided into four phases (3). Bursa-derived (B) lymphocytes are infected between 3 and 6 days postinfection (p.i.) and are the primary target cells for the first phase of lytic infection, which is followed by infection of activated CD4 ϩ thymus-derived (T) lymphocytes in the second phase. During the second phase, viral replication typically decreases and a latent infection is established between 5 and 10 days p.i. in activated CD4ϩ T lymphocytes. The third phase is characterized by reactivation of MDV replication between 14 and 21 days p.i. After the third phase, lymphomas may develop depending on the genetic susceptibility of the chickens and the virulence of the virus strain.The MDV genome consists of two unique regions, the unique short region (U S ) and the unique long region (U L ), flanked by inverted repeats known as the inverted repeat long (IR L ) and short (IR S ), and the terminal repeat long (TR L ) and short (TR S ). The MDV Eco Q (Meq or RLORF7) and viral interleukin-8 (IL-8) homologue genes and the open reading frames ...

The herpesvirus ubiquitin-specific protease (USP) family, whose founding member was discovered as a protease domain embedded in the large tegument protein of herpes simplex virus 1 (HSV-1), is conserved across all members of the Herpesviridae. Whether this conservation is indicative of an essential function of the enzyme in vivo has not yet been established. As reported here, USP activity is conserved in Marek's disease virus (MDV), a tumorigenic alphaherpesvirus. A single amino acid substitution that abolishes the USP activity of the MDV large tegument protein diminishes MDV replication in vivo, and severely limits the oncogenic potential of the virus. Expression of the USP transcripts in MDV-transformed cell lines further substantiates this hypothesis. The herpesvirus USP thus appears to be required not only to maintain a foothold in the immunocompetent host, but also to contribute to malignant outgrowths.chicken ͉ deubiquitinating enzyme ͉ herpes ͉ Marek's disease virus T he ubiquitin-proteasome system controls cytosolic proteolysis, certain aspects of transcription, antigen presentation via major histocompatibility complex (MHC) class I products, and the trafficking of surface-exposed receptors (1-5). As for many other posttranslational modifications, both ubiquitin conjugation and its reversal by ubiquitin-specific proteases (USPs) determine the biological outcome of the reaction. The enzyme families that catalyze ubiquitin conjugation and removal are quite diverse (6-9). Consequently, bioinformatic analysis is not always adequate to identify novel USPs. To target such enzymes biochemically, we developed activity-based probes for USPs and enzymes that act on ubiquitin-like modifiers (10). These probes are equipped with an affinity handle to allow retrieval and identification of the enzymes targeted by the electrophilic warhead installed at the probe's carboxyl terminus.Through the use of one of these probes, HA-ubiquitin vinylmethylester (HA-UbVME), we identified the large tegument protein of herpes simplex virus 1 (HSV-1), viral protein (VP) 1/2, encoded by the unique-long (U L ) 36 gene, as the source of an active USP. Its sequence showed no obvious similarity to known eukaryotic USPs and failed to yield an obvious signature of residues diagnostic of known cysteine protease families. Nonetheless, sequence comparisons across the Herpesviridae show the presence of a few absolutely conserved residues (Cys, Asp, His, Glu), all of which are consistent with involvement in a potential thiol protease active site. We have, meanwhile, confirmed both the mechanism of action of such ubiquitin-based probes (11)(12)(13)(14)(15) and the identity of the viral cysteine protease domain as an authentic USP by crystallographic analysis of the homologous segment of the murine cytomegalovirus (MCMV) M48 protein (16). Notwithstanding the conservation of the identified USP activity in all herpesviruses, we do not know whether this activity makes a contribution to the replicative success and pathogenicity of herpesviruses in vivo...

Marek's disease (MD) is caused by a ubiquitous, lymphotropic alphaherpesvirus, MD virus (MDV). MD has been a major concern in the poultry industry owing to the emergence of increasingly virulent strains over the last few decades that were isolated in the face of comprehensive vaccination. The disease is characterized by a variety of clinical signs; among them are neurological symptoms, chronic wasting and, most notably, the development of multiple lymphomas that manifest as solid tumors in the viscera and musculature. Much work has been devoted to study MD-induced oncogenesis and the genes involved in this process. Among the many genes encoded by MDV, a number have been shown recently to affect the development of tumors in chickens, one protein directly causing transformation of cells (Meq) and another being involved in maintaining transformed cells (vTR). Other MDV gene products modulate and are involved in early lytic in vivo replication, thereby increasing the chance of transformation occurring. In this review, we will summarize specific genes encoded by MDV that are involved in the initiation and/or maintenance of transformation and will focus mostly on current vaccination and control strategies against MD, particularly how modern molecular biological methods may be used to improve strategies to combat the disease in the future.

Marek's disease virus (MDV), a lymphotropic alphaherpesvirus, causes Marek's disease (MD) in chickens. MD is characterized by neurological signs, chronic wasting, and T cell lymphomas that predominate in the visceral organs. MDV replicates in a highly cell-associated manner in vitro and in vivo, with infectious virus particles being released only from feather follicle epithelial (FFE) cells in the skin. Virus produced and shed from FFE cells allows transmission of MDV from infected to naïve chickens, but the mechanisms or roles of differential virus gene expression have remained elusive. Here, we generated recombinant MDV in which we fused enhanced green fluorescent protein (EGFP) to the C terminus of the tegument protein pUL47 (vUL47-EGFP) or pUL49 (vUL49-EGFP). While vUL49-EGFP was highly attenuated in vitro and in vivo, vUL47-EGFP showed unaltered pathogenic potential and stable production of pUL47-EGFP, which facilitated direct analysis of pUL47 expression in cells and tissues. Our studies revealed that pUL47-EGFP is expressed at low levels and localizes to the nucleus during lytic replication in vitro and in lymphocytes in the spleen in vivo, while it is undetectable in tumors. In contrast, pUL47-EGFP is highly abundant and localizes predominantly in the cytoplasm in FFE cells in the skin, where MDV is shed into the environment. We concluded that differential expression and localization of MDV pUL47-EGFP tegument protein is potentially important for the unique cell-associated nature of MDV in vitro and in lymphocytes in vivo, as well as production of free virus in FFE cells. Marek's disease (MD) in chickens is caused by the alphaherpesvirus Gallid herpesvirus 2 (GaHV-2), better known as MD virus (MDV). The most prominent sign of MD is the development of solid lymphomas in the viscera and other organs (5, 24). According to our current understanding, infection begins through the respiratory route by inhalation of cell-free virus or infected cells in chickens. The virus is presumably taken up by macrophages or dendritic cells and transported to lymphoid organs, where primary cytolytic infection occurs in B lymphocytes. MDV then infects T lymphocytes, where it can establish latency, predominantly in activated CD4 ϩ T cells, which can also undergo oncogenic transformation, ultimately resulting in lymphoma formation. Irrespective of the transformation event, migrating lymphocytes transport MDV to feather follicle epithelial (FFE) cells in the skin. MDV is highly cell associated in tissue culture cells and during in vivo replication in B and T lymphocytes (2, 9, 41), while the only known cell type in which fully infectious virus is produced is FFE cells in the skin (6).Many aspects of MDV replication and pathogenesis have remained unknown, partly due to the inability to track the virus in specific organs. In many other viruses, including herpesvirus, expression of fluorescent proteins in a soluble form or fused to a viral protein has allowed tracking of virus-infected cells and the localization of specific viral p...

Previous studies in this laboratory have shown that the SH‐2 domain‐containing protein tyrosine phosphatase SHP‐1 is expressed in CNS glia and functions to modulate cytokine activities in these cells. The present study demonstrates that SHP‐1 is expressed within multiple regions of the CNS in vivo, especially in white matter. Interestingly, we show that mice genetically lacking in SHP‐1 (motheaten mice) in the CNS displayed dysmyelination. We therefore examined the expression of SHP‐1 in the myelin‐forming oligodendrocytes. Oligodendrocytes present in either mixed glial cultures or pure cultures expressed high levels of SHP‐1 in the cytoplasm of cell bodies and processes. Oligodendrocytes isolated from motheaten mice did not express SHP‐1. To test possible functions for SHP‐1 in oligodendrocytes in controlling cytokine signaling, we compared the responsiveness of oligodendrocytes isolated from either motheaten or normal littermate mice with IL‐6. IL‐6 induced higher levels of STAT3 phosphorylation and STAT3‐responsive c‐fos gene expression in pure oligodendrocyte cultures of motheaten compared with normal littermate mice. These studies demonstrate that oligodendrocytes express SHP‐1 and that SHP‐1 functions to control IL‐6 signaling. SHP‐1 may therefore be a critical regulator of oligodendrocyte differentiation in response to IL‐6 family cytokines. Further, these findings may relate to dysmyelination in mice lacking SHP‐1. GLIA 29:376–385, 2000. © 2000 Wiley‐Liss, Inc.

Varicella-zoster virus (VZV) is an extremely cell-associated herpesvirus with limited egress of viral particles. The induction of autophagy in VZV-infected monolayers is easily detectable; inhibition of autophagy leads to decreased VZV glycoprotein biosynthesis and diminished viral titers. To explain how autophagic flux could exert a proviral effect on the VZV infectious cycle, we postulated that the VZV exocytosis pathway following secondary envelopment may converge with the autophagy pathway. This hypothesis depended on known similarities between VZV gE and autophagy-related (Atg) Atg9/Atg16L1 trafficking pathways. Investigations were carried out with highly purified fractions of VZV virions. When the virion fraction was tested for the presence of autophagy and endosomal proteins, microtubule-associated protein 1 light chain (MAP1LC3B) and Ras-like GTPase 11 (Rab11) were detected. By two-dimensional (2D) and 3D imaging after immunolabeling, both proteins also colocalized with VZV gE in a proportion of cytoplasmic vesicles. When purified VZV virions were enumerated after immunoelectron microscopy, gold beads were detected on viruses following incubation with antibodies to VZV gE (∼100%), Rab11 (50%), and LC3B (30%). Examination of numerous electron micrographs demonstrated that enveloped virions were housed in single-membraned vesicles; viral particles were not observed in autophagosomes. Taken together, our data suggested that some viral particles after secondary envelopment accumulated in a heterogeneous population of single-membraned vesicular compartments, which were decorated with components from both the endocytic pathway (Rab11) and the autophagy pathway (LC3B). The latter cytoplasmic viral vesicles resembled an amphisome.IMPORTANCE VZV infection leads to increased autophagic flux, while inhibition of autophagy leads to a marked reduction in virus spread. In this investigation of the proviral role of autophagy, we found evidence for an intersection of viral exocytosis and autophagy pathways. Specifically, both LC3-II and Rab11 proteins copurified with some infectious VZV particles. The results suggested that a subpopulation of VZV particles were carried to the cell surface in single-walled vesicles with attributes of an amphisome, an organelle formed from the fusion of an endosome and an autophagosome. Our results also addressed the interpretation of autophagy/xenophagy results with mutated herpes simplex virus lacking its ICP34.5 neurovirulence gene (HSVΔ34.5). The VZV genome lacks an ICP34.5 ortholog, yet we found no evidence of VZV particles housed in a double-membraned autophagosome. In other words, xenophagy, a degradative process documented after infection with HSVΔ34.5, was not observed in VZV-infected cells.