In vitro characterization of the Meq proteins of Marek's disease virus vaccine strain CVI988

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Abstract

Gallid herpesvirus 2 (GaHV-2), commonly known as Marek's disease virus serotype-1 (MDV-1), causes T cell lymphomas in chickens. Vaccines prepared from the attenuated CVI988/Rispens MDV-1 strain currently offer the best protection. Although attenuated CVI988/Rispens is non-oncogenic, it codes for at least two forms of the MDV oncoprotein Meq, and these proteins (CVI-Meq and CVI-LMeq) have not been fully characterized. Here, we report that both CVI-Meq proteins, like the Meq protein of Md5 (a very virulent oncogenic strain), were capable of transforming Rat-2 and NIH3T3 cells. Both CVI-Meq and CVI-LMeq proteins activated the meq promoter only in the presence of chicken c-Jun (CK-Jun) whereas Md5-Meq activated the same promoter irrespective of CK-Jun co-expression. However, Meq proteins of both Md5 and CVI988 bound the meq promoter in a ChIP assay regardless of whether CK-Jun was co-expressed. To understand the role of Meq DNA binding and transactivation/repression domains in transcription, we constructed three chimeric Meq proteins, namely, Md5-CVI-Meq, CVI-Md5-Meq, and Md5-CVI-L by exchanging domains between Md5 meq and CVI meq genes. Although these chimeric Meq proteins, unlike CVI-Meq proteins, transactivated the meq promoter, the activation was significantly less than Md5-Meq. To determine the role of individual amino acids, point mutations were introduced corresponding to the amino acid changes of CVI-Meq into Md5-Meq. Amino acid residues at positions 71 and 320 of the Md5-Meq protein were found to be important for transactivation of the meq promoter. All three Meq proteins activated the MDV gB, MMP-3 and Bcl-2 promoters and suppressed transcription from the MDV pp38/pp14 bidirectional promoter. Although no significant differences were observed, decreased transactivation activity was observed with CVI-Meq proteins when compared to Md5-Meq. Collectively, the data presented here indicate that CVI-Meq proteins are generally weak transactivators, which might contribute to the non-oncogenic phenotype of CVI988 virus in chickens.

Introduction

Gallid herpesvirus 2 (GaHV-2), commonly known as Marek's disease virus serotype 1 (MDV-1), causes Marek's disease (MD) in chickens. MDV-1 is classified in the genus Mardivirus of the subfamily Alphaherpesvirinae along with two other non-oncogenic poultry viruses, Gallid herpesvirus 3 (GaHV-3 or MDV serotype 2) and Meleagrid herpesvirus 1 (MeHV-1, MDV serotype 3 or Turkey herpesvirus). MD is characterized by T cell tumors, partial or complete paralysis of legs and wings, immunosuppression, skin leukosis, depression, and death (Calnek, 2001, Marek, 1907, Pappenheimer et al., 1929). MDV is prevalent in commercial poultry due to its high infectivity and long-term survivability outside its host and is responsible for significant economic losses worldwide. Based on the severity of the disease in vaccinated chickens, MDV-1 strains in North America are classified as mild (m), virulent (v), very virulent (vv) and very virulent plus (vv+) (Witter, 1997). MDV replicates in B and T lymphocytes during early cytolytic infection and subsequently establishes latency, a common feature of herpesvirus infections, in T lymphocytes. The virus transforms activated T lymphocytes that infiltrate several visceral organs, peripheral nerves, and skin as early as 3 weeks post-infection (Calnek, 2001).

The MDV genome consists of a unique long (UL), and a unique short (US) region flanked by inverted repeat regions, commonly referred to as terminal and internal repeats (TRL, IRL, IRS and TRS) and the sizes vary depending upon the strain; HVT strain FC-126 (159,160 bp) (Afonso et al., 2001), MDV-2 strain HPRS24 (164,270 bp) (Izumiya et al., 2001), MDV-1 strain GA (174,000 bp) (Lee et al., 2000), and MDV-1 strain Md5 (177,874 bp) (Tulman et al., 2000). The organization of the MDV genome resembles that of human herpesvirus 1 (HSV-1) with a co-linear organization of genes in both the unique long and unique short regions. MDV-1 specific genes, such as vIL-8 (Cui et al., 2004, Parcells et al., 2001), viral telomerase RNA (vTR) (Fragnet et al., 2003), viral lipase (Kamil et al., 2005), meq (Brown et al., 2006, Lupiani et al., 2004), and pp38 (Reddy et al., 2002), have been implicated in pathogenesis. Only MDV-1 strains are oncogenic and code for a unique basic leucine zipper (bZIP) protein, Meq (MDV EcoRI Q fragment). Two identical copies of the meq oncogene are present in the repeat long regions (TRL and IRL) and is commonly expressed in MDV lymphoma cells (Jones et al., 1992). Meq has been shown to transform fibroblast cell lines (Rat-2 and DF-1 cells), protect fibroblasts against serum starvation and apoptosis inducers, as well as promote proliferation of cells, all characteristics of oncoproteins (Levy et al., 2003a, Levy et al., 2005, Liu et al., 1998). More importantly, a recent work in our laboratory with a MDV mutant virus lacking both copies of meq has conclusively shown that Meq is required for transformation of T lymphocytes but not necessary for early viral replication (Lupiani et al., 2004).

Meq is 339 amino acids long and contains a basic amino acid rich DNA binding domain at the amino terminus (1–120) and a proline rich transactivation domain at the carboxy terminus (121–339). Meq also contains both nucleus and nucleolus localization signals (Liu et al., 1997) and co-localizes with Cdk2 in coiled bodies (Liu et al., 1999b). Like other bZIP proteins, Meq posses transactivation activity and is capable of forming homodimers with itself as well as heterodimers with other bZIP proteins such as c-Jun. Meq homodimers have been shown to repress MDV early promoters such as pp38 and pp14 while Meq/c-Jun heterodimers have been reported to activate the meq promoter (Levy et al., 2003b). Meq, like v-Jun, has also been shown to increase transcription of genes involved in growth and anti-apoptosis, suggesting the Jun pathway is involved in MDV transformation (Levy et al., 2005). In addition, Meq has been shown to interact with a cellular co-repressor, C-terminal-binding protein (CtBP), and this interaction is essential for MDV oncogenesis (Brown et al., 2006).

MD vaccines are effective in the prevention of tumor development but not infection. Studies have shown that field isolates continuously evolve towards greater virulence (Witter, 1997) and vaccination has been suggested as one of the driving forces in this evolution (Schat and Baranowski, 2007, Witter, 1998). Consequently, current vaccines may not be effective against MD in the near future. Understanding the molecular mechanisms of T cell transformation is critical for designing the next generation of vaccines against these evolving MD viruses. Several researchers have explored the use of cell culture attenuated MDV-1 strains as vaccines with limited success (Churchill et al., 1969, Nazerian, 1970, Rispens et al., 1972, Vielitz and Landgraf, 1971, Witter, 1982). Currently, attenuated CVI988/Rispens MDV-1 strain is used as a vaccine in the USA, and in Europe (Baigent et al., 2006) and confers the best protection. Interestingly, although attenuated vaccine strains of CVI988/Rispens encode and express Meq, they are non-oncogenic in chickens (Witter et al., 1995). Additionally, CVI988/Rispens encode and expresses a longer form of Meq (LMeq, 398 amino acids), which contains 59 amino acids in the form of a proline rich repeat in the transactivation domain (Chang et al., 2002a). Comparison of the predicted amino acid sequences of Meq from the very virulent strain Md5 and the vaccine strain CVI988/Rispens revealed two and four amino acid differences in the DNA binding and transactivation domains, respectively (Shamblin et al., 2004) (Fig. 1); however, the significance of these differences is currently unknown.

The objective of the current study was to determine how these amino acid differences affect the in vitro transformation and transactivation properties of Meq proteins expressed by CVI988/Rispens. Our findings indicate Meq proteins from attenuated CVI988/Rispens vaccine strain differ from Md5 Meq protein in transactivation activity but not in in vitro transformation properties. Based on these results, the lack of oncogenicity of CVI988/Rispens in chickens may be more related to transactivation activity than fibroblast-transforming ability of Meq proteins.

Section snippets

Cell culture

DF-1, a chicken embryo fibroblast continuous cell line, was maintained in Leibowitz–McCoy media supplemented with 4% fetal bovine serum (FBS) and penicillin–streptomycin at 37 °C. Cell lines 293T (human embryonic kidney cells containing SV40 T antigen), NIH 3T3 (mouse embryonic fibroblast cell line) and Rat-2 (rat embryonic fibroblast cell line) were cultured in Dulbecco's minimum essential medium (DMEM) supplemented with 10% FBS and penicillin–streptomycin at 37 °C.

Cloning of meq and chicken c-jun (CK-jun) genes

The Meq open reading frames

Md5-Meq, CVI-Meq and CVI-LMeq proteins transform fibroblasts

Previous work in our laboratory has demonstrated that deletion of the meq gene from Md5 inhibited T cell tumor development in chickens, confirming a direct role in MDV oncogenesis (Lupiani et al., 2004). Comparison of the predicted amino acid sequences of CVI-Meq and CVI-LMeq from the vaccine strain CVI988/Rispens with the Meq protein of the very virulent Md5 strain revealed two amino acid differences in the DNA binding domain (positions 71 and 77) and four amino acid differences in the

Discussion

MDV vaccines prepared from attenuated GaHV-2, GaHV-3, and MeHV-1 (MDV serotypes 1, 2, and 3) are largely used to control MD in chickens (Witter, 1998). Only MDV-1 induces T cell tumors while MDV-2 and MDV-3 do not cause MD in chickens. MDV-1 includes oncogenic strains and can be attenuated into non-oncogenic strains by serial passage in cell culture (Churchill et al., 1969, Nazerian, 1970, Rispens et al., 1972, Vielitz and Landgraf, 1971, Witter, 1982) or recombinant DNA techniques (Brown et

Acknowledgements

We thank Drs. David Everly (for pBabe plasmids) and David Ernest (for NIH3T3 cells). We also thank Vinayak Brahmakshatriya for technical help and Pam Ferro for critically reviewing of the manuscript. This work was supported by the National Research Initiative of the USDA Cooperative State Research, Education and Extension Service, grant 2004-35204-14840 (to S.M.R and B.L.) and Formula Animal Health grant TEX09098 (to B.L.). P.S. was supported by an NIH pre-doctoral training grant T32-AI052072.

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