Marek’s disease virus (MDV) homologues of herpes simplex virus type 1 UL49 (VP22) and UL48 (VP16) genes: high-level expression and characterization of MDV-1 VP22 and VP16

Abstract

Genes UL49 and UL48 of Marek's disease virus 1 (MDV-1) strain RB1B, encoding the respective homologues of herpes simplex virus type 1 (HSV-1) genes VP22 and VP16, were cloned into a baculovirus vector. Seven anti-VP22 MAbs and one anti-VP16 MAb were generated and used to identify the tegument proteins in cells infected lytically with MDV-1. The two genes are known to be transcribed as a single bicistronic transcript, and the detection of only one of the two proteins (VP22) in MSB-1 lymphoma and in chicken embryo skin cells infected with MDV-1 prompted the study of the transcription/translation of the UL49-48 sequence in an in vivo and in vitro expression system. VP16 was expressed in vitro at detectable levels, whereas it could only be detected at a lower level in a more controlled environment. It was demonstrated that VP22 is phosphorylated in insect cells and possesses the remarkable property of being imported into all cells in a monolayer. VP22 localized rapidly and efficiently to nuclei, like its HSV-1 counterpart. The DNA-binding property of VP22 is also reported and a part of the region responsible for this activity was identified between aa 16 and 37 in the N-terminal region of the protein.

A full UL13 open reading frame in Marek's disease virus (MDV) is dispensable for tumor formation and feather follicle tropism and cannot restore horizontal virus transmission of rRB-1B in vivo

“…K11 Mab (IgG1) against MDV-1 gB was obtained by immunizing a Balb/c mouse with baculovirus-expressed gB, and following a procedure described earlier [7]. The K11 Mab was found to react with both MDV and the serologically related herpesvirus of turkeys (HVT).…”

“…Chicken embryonic skin cells (CESC) were prepared and cultivated as previously described [7] from 12-day chicken embryos (LD1 Brown Leghorn chicken strain) [25]. The pRB-1B bacmid, herein referred to as 'pRB-1B', consists of the genome of the highly virulent RB-1B strain cloned into a bacterial artificial chromosome (BAC).…”

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

“…CESC were obtained from specific-pathogen-free embryos by following standard protocols as previously described (10). Recombinant viruses were recovered by transfecting the corresponding bacterial artificial chromosomal DNA into CESC cultures by using the Amaxa nucleofector kit and protocol.…”

“…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.…”

“…MDV is highly transmissible in vivo, whereas the in vitro production of MDV is in the range of 3 orders of magnitude lower than for other alphaherpesvirus, and this represents a barrier for MDV research (5)(6)(7)(8)(9)(10)(11). 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).…”