The Marek’s disease virus (MDV) protein encoded by the UL17 ortholog is essential for virus growth

Marek’s disease virus type 1 (MDV-1) shows a strict dependency on the direct cell-to-cell spread for its propagation in cell culture. As MDV-1 shows an impaired nuclear egress in cell culture, we wished to address the characterization of capsid/tegument genes which may intervene in the maturation of intranuclear capsids. Orthologs of UL17 are present in all herpesviruses and, in all reported case, were shown to be essential for viral growth, playing a role in capsid maturation and DNA packaging. As only HSV-1 and PrV UL17 proteins have been characterized so far, we wished to examine the role of MDV-1 pUL17 in virus replication. To analyze MDV-1 UL17 gene function, we created deletion mutants or point mutated the open reading frame (ORF) to interrupt its coding phase. We established that a functional ORF UL17 is indispensable for MDV-1 growth. We chose to characterize the virally encoded protein by tagging the 729 amino-acid long protein with a repeat of the HA peptide that was fused to its C-terminus. Protein pUL17 was identified in infected cell extracts as an 82 kDa protein which localized to the nucleus, colocalizing with VP5, the major capsid protein, and VP13/14, a major tegument protein. By using green fluorescent protein fusion and HA tagged proteins expressed under the cytomegalovirus IE gene enhancer/promoter (PCMV IE), we showed that MDV-1 pUL17 nuclear distribution in infected cells is not an intrinsic property. Although our results strongly suggest that another viral protein retains (or relocate) pUL17 to the nucleus, we report that none of the tegument protein tested so far were able to mediate pUL17 relocation to the nucleus

ESCDL-1 cell-line derived from chicken ES cells is fully permissive to Mardiviruses

National audienc

ESCDL-1, a new cell line derived from chicken embryonic stem cells, supports efficient replication of Mardiviruses.

Marek's disease virus is the etiological agent of a major lymphoproliferative disorder in poultry and the prototype of the Mardivirus genus. Primary avian somatic cells are currently used for virus replication and vaccine production, but they are largely refractory to any genetic modification compatible with the preservation of intact viral susceptibility. We explored the concept of induction of viral replication permissiveness in an established pluripotent chicken embryonic stem cell-line (cES) in order to derive a new fully susceptible cell-line. Chicken ES cells were not permissive for Mardivirus infection, but as soon as differentiation was triggered, replication of Marek's disease virus was detected. From a panel of cyto-differentiating agents, hexamethylene bis (acetamide) (HMBA) was found to be the most efficient regarding the induction of permissiveness. These initial findings prompted us to analyse the effect of HMBA on gene expression, to derive a new mesenchymal cell line, the so-called ESCDL-1, and monitor its susceptibility for Mardivirus replication. All Mardiviruses tested so far replicated equally well on primary embryonic skin cells and on ESCDL-1, and the latter showed no variation related to its passage number in its permissiveness for virus infection. Viral morphogenesis studies confirmed efficient multiplication with, as in other in vitro models, no extra-cellular virus production. We could show that ESCDL-1 can be transfected to express a transgene and subsequently cloned without any loss in permissiveness. Consequently, ESCDL-1 was genetically modified to complement viral gene deletions thus yielding stable trans-complementing cell lines. We herein claim that derivation of stable differentiated cell-lines from cES cell lines might be an alternative solution to the cultivation of primary cells for virology studies

Cell cycle modulation by Marek's disease virus: the tegument protein VP22 triggers S-phase arrest and DNA damage in proliferating cells

International audienceMarek's disease is one of the most common viral diseases of poultry affecting chicken flocks worldwide. The disease is caused by an alphaherpesvirus, the Marek's disease virus (MDV), and is characterized by the rapid onset of multifocal aggressive T-cell lymphoma in the chicken host. Although several viral oncogenes have been identified, the detailed mechanisms underlying MDV-induced lymphomagenesis are still poorly understood. Many viruses modulate cell cycle progression to enhance their replication and persistence in the host cell, in the case of some oncogenic viruses ultimately leading to cellular transformation and oncogenesis. In the present study, we found that MDV, like other viruses, is able to subvert the cell cycle progression by triggering the proliferation of low proliferating chicken cells and a subsequent delay of the cell cycle progression into S-phase. We further identified the tegument protein VP22 (pUL49) as a major MDV-encoded cell cycle regulator, as its vector-driven overexpression in cells lead to a dramatic cell cycle arrest in S-phase. This striking functional feature of VP22 appears to depend on its ability to associate with histones in the nucleus. Finally, we established that VP22 expression triggers the induction of massive and severe DNA damages in cells, which might cause the observed intra S-phase arrest. Taken together, our results provide the first evidence for a hitherto unknown function of the VP22 tegument protein in herpesviral reprogramming of the cell cycle of the host cell and its potential implication in the generation of DNA damages

Number and percentage of capsids in the different cell compartments.

<p>Number and percentage of capsids in the different cell compartments.</p

Differentiation increases permissiveness of cES to GaHV-2.

<p>(A & B): cES cells were plated and infected 6 days post plating in reduced serum conditions with sorted CESC infected with vBAC20GFPVP22. Cells were fixed after an incubation of 6 days at 37°C. (A) cell monolayers were maintained in WE medium containing 1% FBS and 1.5% CS. (B) DMSO (64 μM) was added from day 4 after plating and until the end of the culture (scale bar represents 200 μm). (C) cES cells (passage 36) were exposed to HMBA and infected with sorted vBAC20GFPVP22-infected cells. Expression of VP22 was detected by the GFP signal and ICP4 by staining with monoclonal antibody E21 (red); cell nuclei were stained by Hoechst 33342. At late stages of infection, ICP4 is detected both in the cytoplasm and nucleus in VP22 expressing cells. At early stages of infection, when VP22 is barely detectable in the cells surrounding the highly infected cell, ICP4 staining is predominantly nuclear (arrow heads) indicating spread of virus from the originally infected cell to the neighbouring cells (scale bar represents 20 μm). (D) Induction of differentiation by HMBA increases susceptibility of cES cells to GaHV-2 infection. Comparison of the plaque counts at 4 days pi either on cES cells or on primary CESC exposed to differentiating drugs (2 independent experiments sampling 10 replicates for each condition with cES and 4 replicates with CESC). (E) Comparison of plaque sizes on either cES exposed to HMBA differentiation or CESC. For both cell types, HMBA was added in the maintenance medium after the infection with sorted vBAC20EGFPVP22-infected cells. Plaques appeared larger in cES differentiated cells. Plaque sizes from 80 plaques per experiment are shown as boxplots and whiskers (Tukey) (in B, P<0.001; Mann Whitney test).</p

Constitutive expression of pUL49 (VP22) in ESCDL-1 complements the deletion of UL49 in GaHV2 genome.

<p>(A & B) Constitutive expression of pUL49 (VP22) in uncloned cell populations (A) and in ESCDL-1-UL49/clone 2 (B). VP22 staining by anti pUL49 Mabs and an Alexa Fluor^® 488 goat anti-mouse detects filamentous material between 2 strongly positive nuclei that appear to be still bound after cell division (white arrowhead). (C to H) Complementation of replication for BAC20ΔUL49 on ESCDL-1-UL49/clone 7 (C, E, G) and absence of viral dissemination in non-complementing ESCDL-1 (D, F, H). Viral replication was detected using a chicken hyper immune serum revealed by an Alexa Fluor^® 488 goat anti-chicken conjugate together with an anti-ICP4 Mab (C, D), a mixture of anti-gI and -gE Mabs (E, F), or a mixture of anti-pUL49 (VP22) Mabs (G, H) all revealed by an Alexa Fluor^® 594 goat anti-mouse conjugate. The restoration of pUL49 (VP22) expression is associated with the viral replication (G). Early-late (ICP4) and late (gE-gI) antigens are detected in isolated ESCDL-1 cells (D & F) and in panel H the arrow points to an isolated cell in which vBAC20ΔUL49 undergoes an aborted replication cycle as revealed by the polyclonal anti-MDV serum without detection of VP22. Scale bar represents 50μm.</p

TEM analysis of vBAC20 morphogenesis in ESCDL-1.

<p>(A) Overview of an infected cell with intranuclear A (black triangle) and B (black arrowheads) capsids and intracytoplasmic C capsids (white ellipses). The white arrow points to an image of capsid tegumentation in the cytoplasm. (B) Intranuclear accumulation of small particles (SP– 30 to 35 nm in diameter) arranged in a pseudo-crystalline structure in the vicinity of A capsids (black triangle). (C) Accumulation of primary enveloped virions in distended cisternae of the nuclear membrane (black arrows point to enveloped C capsids). (D) C capsid undergoing secondary envelopment: electron dense material, possibly of tegument origin, surrounding the capsid is surrounded by a membrane in close vicinity to the Golgi (bar represents 0.2 μm). (E) Multiple intracytoplasmic C capsids (white ellipses) close to an enveloped cytoplasmic particle (white arrowhead).</p

Expression of pUL37 in ESCDL-1 complements the deletion of UL37 ORF in BACRB-1B.

<p>Upper panel (1–4): Transfection of BACRB-1BΔ37 yields viral plaques on complementing cells: viral plaques were detected in ESCDL-1-UL37 by staining with Mabs B17 (anti-VP22), K11 (anti-gB) and E21 (anti-ICP4) and an Alexa Fluor^® 488 GAM conjugate (1). In ESCDL-1, BACRB-1BΔ37 did not yield a virus that could disseminate and only isolated positive cells could be seen (2). As a control, BACRB-1BUL17mRFP was transfected in either complementing or non-complementing parental cells, producing viral plaques on both (3 & 4). Scale bar = 200 μm. Middle Panel (5 to 8): vBACRB-1BΔ37 can be serially passaged in complementing cells and virus multiplication induces pUL37 expression in the ESCDL-1-UL37: BACRB-1BΔ37 (5,6) or BAC RB-1B (7,8) were transfected either in ESCDL-1-UL37 complementing cells or in ESCDL-1 and passaged once in the same cells. The development of viral infection by passage 2 of the vBACRB-1BΔ37 virus is seen in complementing cells (green fluorescence in 5) and coincides with the expression of pUL37 in infected cells (red fluorescence in 5); in non-complementing cells the same virus passage does not replicate (6). The parental virus (vBACRB-1B) transfected and passaged in the same conditions replicated equally well on ESCDL-1 and on ESCDL-1-UL37 (7 & 8). Scale bar = 50μm. Lower Panel (9 to 12): vBACRB-1BΔ37 may be passaged at least 3 times in complementing cells and does not revert to a replicating virus when plated on non-complementing cells. The 3rd passage of vBACRB-1BΔ37 yielded typical viral plaques in complementing cells (9) whereas the same virus did not form plaques in ESCDL-1 (10). Again vRB-1B at the same passage developed equally well in both cells (staining as in the upper panel, except for HOECHST 33342). Scale bar = 200 μm.</p