Marek's disease virus (MDV) is an alphaherpesvirus for which infection is strictly cell associated in permissive cell culture systems. In contrast to most other alphaherpesviruses, no comprehensive ultrastructural study has been published to date describing the different stages of MDV morphogenesis. To circumvent problems linked to nonsynchronized infection and low infectivity titers, we generated a recombinant MDV expressing an enhanced green fluorescent protein fused to VP22, a major tegument protein that is not implicated in virion morphogenesis. Growth of this recombinant virus in cell culture was decreased threefold compared to that of the parental Bac20 virus, but this mutant was still highly replicative. The recombinant virus allowed us to select infected cells by cell-sorting cytometry at late stages of infection for subsequent transmission electron microscopy analysis. Under these conditions, all of the stages of assembly and virion morphogenesis could be observed except extracellular enveloped virions, even at the cell surface. We observed 10-fold fewer naked cytoplasmic capsids than nuclear capsids, and intracellular enveloped virions were very rare. The partial envelopment of capsids in the cytoplasm supports the hypothesis of the acquisition of the final envelope in this cellular compartment. We demonstrate for the first time that, compared to other alphaherpesviruses, MDV seems deficient in three crucial steps of viral morphogenesis, i.e., release from the nucleus, secondary envelopment, and the exocytosis process. The discrepancy between the efficiency with which this MDV mutant spreads in cell culture and the relatively inefficient process of its envelopment and virion release raises the question of the MDV cell-to-cell spreading mechanism.Marek's disease virus (MDV), referred to as Gallid herpesvirus 2, is the etiological agent of Marek's disease in chickens, a multifaceted disease most widely recognized by the induction of a malignant T-cell lymphoma. This major pathogen of chickens is a herpesvirus classified in the Mardivirus genus (Marek's disease-like viruses) within the Alphaherpesvirinae subfamily. MDV can be efficiently propagated in cell culture but remains strictly cell associated without free viral particles being detectable in the supernatant (2, 38). Moreover, infectious MDV virion particles cannot be purified from infected cell lysates as has been described for varicella-zoster virus (VZV) or turkey herpesvirus. Therefore, homologous vaccines commonly used in poultry flocks are frozen viable MDV-infected cells, which require storage and transport in liquid nitrogen (4). This feature makes MDV a unique virus within the herpesvirus family and among animal viruses in general.From electron microscopy (EM) studies of cultured cells infected with various herpesviruses, including mutant viruses with deletions of different tegument proteins or glycoproteins genes, three different pathways for the assembly and morphogenesis of herpesviruses have been proposed (reviewed in references 7, 20, ...

In a previous study, we have demonstrated that the platelet adhesive glycoprotein thrombospondin-1 (TSP-1) interacts specifically with the cytoskeletal protein alpha-actinin in a solid-phase binding assay. Stored in the alpha-granules of platelets, TSP-1 is secreted during cell activation and binds to the plasma membrane promoting the platelet macroaggregate formation. However, the molecular mechanism by which TSP-1 reaches and binds to the platelet surface is to date unelucidated. alpha-Actinin is an actin-binding and actinin-cross-linking protein that is present in most cells and may act as a link between the bundles of F-actin and the plasma membrane. In this study, we have investigated a possible interaction of alpha-actinin with TSP-1 in platelets by examining their respective subcellular location during the platelet activation process. By indirect immunofluorescence. alpha-actinin was found to display a granular staining in resting platelets similar to that of TSP-1. Performing postembedding immunogold labeling for electron microscopy, we detected the presence of alpha-actinin throughout the cytoplasm, but the strongest gold staining was found in organelles identified as alpha-granules on the basis of their ultrastructure and TSP-1 content. With the use of double immunogold labeling on platelets at different stages of activation by thrombin, both alpha-actinin and TSP-1 were seen redistributing from the alpha-granules to the platelet surface via the open canalicular system (OCS). At the same time, the cytoplasmic alpha-actinin concentrated toward the plasma membrane, but no colocalization with the F-actin bundles was evidenced. Finally, preembedding immunogold labeling and immunoprecipitation of 125I-surface-labeled, thrombin-activated platelets further demonstrated that alpha-actinin was expressed on the plasma membrane in the absence of any detectable expression of actin and that it could from molecular complexes with TSP-1 on activated platelets. These results suggest that alpha-actinin found to be present on the platelet surface together with TSP-1 originates in the alpha-granules by fusion of the alpha-granules with the plasma membrane during platelet exocytosis.