Morphogenesis of a Highly Replicative EGFPVP22 Recombinant Marek's Disease Virus in Cell Culture

Abstract

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, ...

A Virulent Bioluminescent and Fluorescent Dual-Reporter Marek's Disease Virus Unveils an Alternative Spreading Pathway in Addition to Cell-to-Cell Contact

“…2). In contrast, the construct RnLuc-VP22, which lacks the 2A sequence between the luciferase and the VP22 sequences, led to a virus that was dramatically impaired in its spreading ability, consistent with previous work showing that tagging VP22 either at its C or N terminus is detrimental for MDV replication (12,16).…”

“…Indeed, with the most permissive cell culture system for MDV, total MDV production in vitro during 4 days of infection is, at best, 10 5 PFU/10 6 cells (6,12). This suggests that an adequately permissive and physiopathologically relevant MDV in vitro production system remains to be identified.…”

“…In previous studies, fluorescently tagged MDVs proved very useful in cell sorting of MDV-infected cells and in documenting differential tissue expression of tagged tegument protein (12,16). However, a bioluminescent MDV reporter virus expressing the highly sensitive luciferase enzyme has not yet been reported (19).…”

“…According to the current paradigm, MDV belongs to a unique class of viruses that rely exclusively on cell-to-cell transmission (12)(13)(14)(15). In contrast, it has been reported that infectious, cell-free MDVs are produced in feather follicle epithelial cells, and these may directly participate in viral transmission in chickens (16,17).…”

“…However, a bioluminescent MDV reporter virus expressing the highly sensitive luciferase enzyme has not yet been reported (19). In addition, tagging of viral proteins, either at the C or N terminus, is likely to be detrimental for virus replication (12,16). To overcome this issue, I used the cis-acting hydrolase element (CHYSEL) (20)(21)(22)(23).…”