*Article* **Effect of Insertion and Deletion in the Meq Protein Encoded by Highly Oncogenic Marek's Disease Virus on Transactivation Activity and Virulence**

**Jumpei Sato <sup>1</sup> , Shiro Murata 1,2,\* , Zhiyuan Yang 1,3, Benedikt B. Kaufer <sup>4</sup> , Sotaro Fujisawa <sup>1</sup> , Hikari Seo <sup>1</sup> , Naoya Maekawa <sup>2</sup> , Tomohiro Okagawa <sup>2</sup> , Satoru Konnai 1,2, Nikolaus Osterrieder 4,5, Mark S. Parcells <sup>6</sup> and Kazuhiko Ohashi 1,2**


**Abstract:** Marek's disease virus (MDV) causes malignant lymphoma in chickens (Marek's disease, MD). Although MD is currently controlled by vaccination, MDV strains have continuously increased in virulence over the recent decades. Polymorphisms in Meq, an MDV-encoded oncoprotein that serves as a transcription factor, have been associated with the enhanced virulence of the virus. In addition, insertions and deletions in Meq have been observed in MDV strains of higher virulence, but their contribution to said virulence remains elusive. In this study, we investigated the contribution of an insertion (L-Meq) and a deletion in the Meq gene (S-Meq) to its functions and MDV pathogenicity. Reporter assays revealed that both insertion and deletion enhanced the transactivation potential of Meq. Additionally, we generated RB-1B-based recombinant MDVs (rMDVs) encoding each Meq isoform and analyzed their pathogenic potential. rMDV encoding L-Meq indueced the highest mortality and tumor incidence in infected animals, whereas the rMDV encoding S-Meq exhibited the lowest pathogenicity. Thus, insertion enhanced the transactivation activity of Meq and MDV pathogenicity, whereas deletion reduced pathogenicity despite having increased transactivation activity. These data suggest that other functions of Meq affect MDV virulence. These data improve our understanding of the mechanisms underlying the evolution of MDV virulence.

**Keywords:** Marek's disease virus; Marek's disease; Meq; CVI988; tumorigenesis; pathogenicity; transactivation activity

## **1. Introduction**

Marek's disease virus (MDV) is an alphaherpesvirus that belongs to the Herpesviridae family (subfamily: *Alphaherpesvirinae*, genus: *Mardivirus*, species: *Gallid alphaherpesvirus 2*). MDV causes Marek's disease (MD), which is characterized by neurological disorders, malignant lymphomas, and immunosuppression in infected chickens. Although MD has previously caused severe economic losses in the poultry industry, the disease is currently controlled by administering attenuated and/or non-pathogenic strains as vaccines [1]. *Meleagrid alphaherpesvirus 1* (turkey herpesvirus, HVT) was initially introduced to the poultry industry,

**Citation:** Sato, J.; Murata, S.; Yang, Z.; Kaufer, B.B.; Fujisawa, S.; Seo, H.; Maekawa, N.; Okagawa, T.; Konnai, S.; Osterrieder, N.; et al. Effect of Insertion and Deletion in the Meq Protein Encoded by Highly Oncogenic Marek's Disease Virus on Transactivation Activity and Virulence. *Viruses* **2022**, *14*, 382. https://doi.org/10.3390/v14020382

Academic Editor: Chi-Young Wang

Received: 26 January 2022 Accepted: 11 February 2022 Published: 14 February 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

followed by bivalent vaccines consisting of HVT and a strain of non-oncogenic *Gallid alphaherpesvirus 3*, and an attenuated *Gallid alphaherpesvirus 2* strain, Rispens CVI988, was introduced and is widely used as a gold standard vaccine against MD [2]. Introduction of the vaccines minimized the economic losses caused by virulent MDVs; however, the virulence of MDV field strains has continuously increased. Sporadic occurrences of MD are still reported even in vaccinated flocks in some countries [3–8], and the costs to the poultry industry are estimated at approximately USD 2 billion annually [9]. The emergence of MDV strains with increased virulence is considered to be a result of the selection of field strains adapted to vaccine-induced immune pressure [10–12]. Therefore, MDV strains with higher virulence may cause future outbreaks [13].

The *meq* oncogene encodes a 339-amino-acid protein that is expressed during the lytic and latent phases of infection and is essential for transformation of chicken T-cells [14]. Meq consists of a basic region (BR) and a leucine zipper (ZIP), similar to those of the oncoproteins Jun and Fos, at the N-terminus, and a transactivation domain characterized by proline-rich repeats (PRRs) at the C-terminal [14]. Meq can dimerize with itself, c-Jun, JunB, and Fos through its ZIP domain [15]. Microarray analysis identified that the same genes induced by v-Jun transformation, including *JTAP1*, *JAC*, and *HB-EGF*, were also upregulated by the expression of Meq in transformed DF-1 cells [16]. Additionally, Meq upregulated the expression of Bcl-2 and Ski and downregulated the expression of DAP5 and Fas, consistent with its anti-apoptotic properties [16,17]. Furthermore, *meq*-deleted recombinant MDV (rMDV) failed to induce lymphomas in infected chickens, indicating that Meq plays an essential role in the transformation induced by virulent MDV [18]. The distinct diversity in Meq proteins of MDV strains with varying virulence has been highlighted as a factor involved in the increased virulence of MDV [19]. Among them, the polymorphisms in the BR and PRRs in the transactivation domain have been associated with virulence-level, and several amino acid substitutions in these regions affect the transactivation activities of Meq proteins [20–22]. Moreover, in vivo studies using rMDVs generated based on the very virulent strain RB-1B strain revealed that rMDVs encoding Meq from highly virulent MDV strains showed higher virulence than those from low virulent strains [23]. Thus, polymorphisms in the *meq* gene appear to be directly involved in the increased virulence of MDV field strains.

In addition to the polymorphisms in Meq proteins, insertion or deletion in the transactivation domain of Meq have been detected in several MDV strains [8,20,24–28]. A long isoform of Meq containing an insertion (L-Meq) is characterized by a 59/60-amino acid insertion in the transactivation domain [24,27,29], and its insertion causes an increase in the number of PRRs. L-Meq was first identified in CVI988 [29] and has subsequently been detected in low-virulence strains isolated in the USA [19]. Therefore, it was initially believed that the L-Meq isoform contributes to decreased virulence of MDVs encoding this form. However, it was clarified that virulent MDV strains circulating in Australia encode an L-Meq, which also contains polymorphisms in PRRs [24]. Furthermore, recent in vivo experiments using RB-1B-based rMDVs harboring L-Meq or Meq encoded by CVI988, rMDV encoding CVI988-L-Meq exhibited markedly higher pathogenicity and tumorigenicity than did CVI988 [30]. Surprisingly, the rMDV encoding CVI988-L-Meq showed similar or even greater virulence than the rMDV encoding RB-1B-Meq, although RB-1B is an MDV strain with high virulence. Thus, amino acid insertions into the transactivation domain appear to contribute to enhanced virulence.

Since the 2010s, MDV strains encoding the short isoform of Meq (S-Meq) have been detected in some countries [20,25,28,31]. S-Meq contains a 41-amino acid deletion in the transactivation domain, resulting in a decrease in the number of PRRs. In addition, an in vitro study revealed that S-Meq had higher transactivation activity than Meq, suggesting that deletion in the transactivation domain enhances the protein functions of Meq [20]. However, MDV strains encoding S-Meq were observed in chickens without clinical signs in Italy [25] and in diseased but unvaccinated chickens in Japan [20]. Therefore, the contribution of S-Meq to MDV pathogenicity remains unclear. However, because deletion

in the transactivation domain was observed in recent field strains and could enhance the transactivation activity of Meq, this deletion may be involved in the evolution towards a greater virulence of these strains [20].

In this study, we systematically investigated the contribution of a common insertion and deletion in the transactivation domain of Meq and evaluated these for their effects on transactivation and MDV pathogenicity. We analyzed the transactivation activities of Meq, L-Meq, and S-Meq, the background sequences of which were identical to those of RB-1B-Meq. In addition, we generated RB-1B-based rMDVs encoding each Meq isoform by replacing the *meq* gene of the RB-1B genome cloned as a bacterial artificial chromosome (BAC) with the L-*meq*/S-*meq* gene that was introduced, and animal experiments were conducted to examine whether insertion/deletion affected MDV pathogenicity.

## **2. Materials and Methods**

#### *2.1. Ethics Statement*

All animal experiments were approved by the Institutional Animal Care and Use Committee of Hokkaido University (approval number: 19-0081). All experiments were performed in accordance with the relevant guidelines and regulations of the Faculty of Veterinary Medicine of Hokkaido University, which is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International.

#### *2.2. Cells and Virus*

Chicken embryo fibroblasts (CEFs) were prepared from 10-day-old fertilized eggs (Iwamura Hatchery Co., Ltd., Shibata, Japan) as described previously [32]. CEFs were cultured in Eagle's minimal essential medium (Nissui Pharmaceutical Co., Ltd., Tokyo, Japan) supplemented with 10% bovine calf serum (Sigma-Aldrich, St. Louis, MO, USA), 10% tryptose phosphate broth (Difco Laboratories, Detroit, MI, USA), 0.03% L-glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin, and 0.1% NaHCO3. DF-1 cells, a chicken fibroblast cell line, were cultured with 0.5 mL of Dulbecco's modified Eagle's medium (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), containing 10% fetal bovine serum, and incubated at 39 ◦C under 5% CO2. Viruses were reconstituted by transfecting BAC DNA into CEFs as described previously [33]. Viruses were propagated on CEFs for seven passages, and virus stocks were frozen in Cell Banker 1 (Nippon Zenyaku Kogyo Co., Ltd., Fukushima, Japan) and titrated on CEFs using plaque assays as described previously [34,35].

#### *2.3. Plasmids*

The expression plasmids for S-Meq and L-Meq were constructed, and some mutations were introduced by site-directed mutagenesis, according to a previous report [22]. The open reading frames (ORFs) of the S-*meq* and L-*meq* genes derived from the MDV strains Kgw-c2, an MDV strain isolated from unvaccinated chickens with MD symptoms in Japan in the 2010s (accession number: LC385874), and CVI988, an attenuated vaccine strain (accession number: AF493555) were amplified and cloned into the pCI-neo vector (Promega, Madison, WI, USA). The pathotype of Kgw-c2 has not been determined. To match the amino acid sequences with those of Meq from the very virulent RB-1B strain, aside from deletion or insertion in the transactivation domain, we introduced some mutations in the S-*meq* and L-*meq* genes, as shown in Table 1, and their S-Meq and L-Meq were designated as S-Meq (RB-1B) and L-Meq (RB-1B), respectively. In addition, we constructed an expression plasmid for Meq from MDV strain RB-1B (accession number: HM488349.1). For the assay to measure the transactivation activity, a c-Jun expression plasmid was constructed [22], and a reporter plasmid was prepared by inserting the Meq promoter region upstream of the firefly luciferase-coding region in the pGL3-Basic vector (Promega) [22]; a pRL-TK Renilla luciferase plasmid (Promega) was used as the control plasmid.


**Table 1.** Introduction of the mutation into the S-*meq* and/or L-*meq* genes.

#### *2.4. Dual-Luciferase Reporter Assay*

First, DF-1 cells were seeded in 24-well plates at 2.0 <sup>×</sup> <sup>10</sup><sup>5</sup> cells per well and incubated for 24 h. The cells in each well were transfected with 300 ng of expression plasmids, Meq, S-Meq (wild-type), S-Meq (RB-1B), L-Meq (wild-type), or L-Meq (RB-1B); 200 ng of the c-Jun expression plasmid; 500 ng of the reporter plasmid; and 5 ng of control pRL-TK using Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's instructions. The transfected cells were lysed 24 h after transfection using 1 × Passive Lysis Buffer (Promega). Luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega) and a Luminescencer-JNR AB-2100 (Atto Corp., Tokyo, Japan). The luminescence intensity of firefly luciferase was normalized to that of *Renilla* luciferase, and the results are indicated relative to the value of the luciferase activity in cells transfected with the pCI-neo vector.

#### *2.5. Generation of Recombinant Viruses*

To generate recombinant viruses encoding S-Meq (RB-1B), Meq, or L-Meq (RB-1B), we used a BAC clone of the very virulent RB-1B strain that lacks most of the internal repeat long (IRL) region as described previously [35]. The deleted IRL region is rapidly restored during virus reconstitution in the cell culture, resulting in a recombinant virus that harbors the respective *meq* gene in both repeat regions [35]. Therefore, we replaced the *meq* gene in the terminal repeat long (TRL) with the RB-1B-*meq*, S-*meq* (RB-1B), or L-*meq* (RB-1B) gene by two-step Red-mediated mutagenesis as previously described [36,37]. To screen for clones in which each *meq*-isoform was accurately inserted, the obtained plasmids encoding each rMDV genome were digested with BamHI-HF (New England Biolabs Japan Inc., Tokyo, Japan) overnight and subjected to electrophoresis on a 0.8% agarose gel. The insertion of each *meq*-isoform was further confirmed by polymerase chain reaction (PCR) and DNA sequencing, as previously reported [35]. The BAC-based infectious clones were transfected into CEFs using a CalPhos Mammalian Transfection Kit (Takara Bio Inc., Kyoto, Japan) according to the manufacturer's instructions. The pCAGGS-Cre plasmid (Gene Bridges GmbH, Heidelberg, Germany) was co-transfected to remove the BAC sequence from the virus genome. The reconstituted recombinant viruses were referred to as vRB-1B\_Meq, vRB-1B\_S-Meq, and vRB-1B\_L-Meq, respectively, and all viruses were passaged on CEFs. As the IRL region is rapidly restored during viral reconstitution, the restoration of the IRL, in addition to the deletion of the BAC sequence, in each virus was confirmed by PCR [35]. Each virus was titrated using a plaque assay and stored as described previously [34,35].

#### *2.6. In Vitro Replication of the Recombinant Viruses*

CEFs were seeded in six-well plates and infected with 50 plaque-forming units (PFU) of recombinant viruses after reaching confluence. The infected cells were collected daily for 5 days. The viral loads in the infected cells were analyzed by quantitative PCR (qPCR) to assess the replication and spread of recombinant viruses in vitro.

#### *2.7. Confirmation of Meq Expression Levels by RT-PCR*

A reverse transcription (RT)-PCR assay was used to investigate the mRNA expression of each *meq* isoform. In brief, we first seeded CEFs in 24-well plates at 2.0 <sup>×</sup> <sup>10</sup><sup>5</sup> cells per well, and after 24 h incubation, the CEFs were infected with 50 PFU of recombinant viruses. At 6 days post-infection (dpi), the infected CEFs were lysed with TRI reagent (Molecular Research Center, Inc., Cincinnati, OH, USA) and the total RNA was extracted according to the manufacturer's instructions. After the total RNA was treated with DNase I (Promega), cDNA was synthesized using PrimeScript Reverse Transcriptase (Takara Bio Inc.). The expression of each *meq* isoform in the cDNA samples was detected by PCR using TaKaRa Ex Taq (Takara Bio Inc.). The primer sets used are listed in Table 2.

**Table 2.** Primers used for reverse transcription-polymerase chain reaction and quantitative polymerase chain reaction analyses.


Abbreviations: qPCR, quantitative polymerase chain reaction; RT-PCR, reverse transcription polymerase chain reaction.

#### *2.8. In Vivo Characterization of Recombinant Viruses*

#### 2.8.1. Experimental Chickens

Fertilized eggs (Iwamura Hatchery Co., Ltd.) from commercial white leghorn chickens were hatched in the authors' laboratory and the chicks were raised in isolators. To analyze the recombinant viruses, we performed animal experiments as follows:

#### 2.8.2. 1st Animal Experiment

A total of 98 1-day-old chicks (Iwamura Hatchery Co., Ltd.) were randomly divided into four groups and housed separately in different isolators. The chickens were inoculated via the intraabdominal route with 5000 PFU of vRB-1B\_Meq (*n* = 28), vRB-1B\_S-Meq (*n* = 27), vRB-1B\_L-Meq (*n* = 27), or PBS (*n* = 16) as a negative control.
