Marek’s disease virus oncoprotein Meq physically interacts with the chicken infectious anemia virus-encoded apoptotic protein apoptin

Abstract

Marek's disease (MD) is a neoplastic disease of poultry caused by Marek's disease virus (MDV), a highly contagious alphaherpesvirus. Meq, the major MDV oncoprotein, induces neoplastic transformation of T-cells through several mechanisms, including inhibition of apoptosis. In contrast, the chicken anemia virus (CAV)-encoded protein apoptin (VP3) is a powerful inducer of apoptosis of tumor cells, a property that is exploited for anticancer therapeutics. Although the molecular mechanisms of selective induction of tumor cell apoptosis by apoptin are not fully understood, its tumor cell–restricted nuclear translocation is thought to be important. Co-infection with MDV and CAV is common in many countries, CAV antigens are readily detectable in MD lymphomas, and the MDV-transformed T-lymphoblastoid cell lines such as MSB-1 is widely used for propagating CAV for vaccine production. As MDV-transformed cell lines express high levels of Meq, we examined here whether CAV-encoded apoptin interacts with Meq in these cells. Using immunofluorescence microscopy, we found that apoptin and Meq co-localize to the nucleus, and biochemical analysis indicated that the two proteins do physically interact. Using a combination of Meq mutagenesis and co-immunoprecipitation, we demonstrate that apoptin interacts with Meq within a region between amino acids 130 and 140. Results from the IncuCyte assay suggested that Meq inhibits apoptin-induced apoptosis activity. In summary, our findings indicate that Meq interacts with and inhibits apoptin. Insights into this novel interaction between Meq and apoptin will relevance for pathogenesis of coinfections of the two viruses and in CAV vaccine production using MDV-transformed cell lines.

Novel Insights into the Roles of Bcl-2 Homolog Nr-13 (vNr-13) Encoded by Herpesvirus of Turkeys in the Virus Replication Cycle, Mitochondrial Networks, and Apoptosis Inhibition

“…Although HVT genome organization shows extensive similarity with the MDV genome, HVT is the only avian alphaherpesvirus, which encodes a Bcl-2 homolog, vNr-13, expressed from two identical copies from the gene HVT079 and HVT096 (9,34). Several MDV and ILTV proteins were shown to have anti-apoptotic properties but none of them have Bcl-2 homology (27,45,46). Multiple sequence alignments revealed that vNr-13 shared the highest sequence identity of 64.4% with chicken Nr-13, compared to only 30% with the frog homolog (10,11).…”

“…Briefly, total RNA extracted from HVTinfected CEF using RNeasy mini kit (QIAGEN, Crawley, UK) was reverse transcribed using Superscript III (Invitrogen) kit according to manufacturer's instructions. After denaturation of the reverse transcriptase at 70 °C for 15 min, 5 µl of the reaction was used in a 50 µl volume polymerase chain reaction (PCR) containing 10 mM dNTP, 10 µM of each primer (Table 1) (27). Plasmid pX330A-1x2 was used for cloning of guide RNAs (gRNAs) for CRISPR/Cas9-based gene editing (see below).…”

“…It is known that herpesviruses such as MDV-1, HSV and varicella zoster virus encode non-Bcl-2 oncoproteins with anti-apoptotic properties(74). We have recently shown that Meq, a non Bcl-2 oncoprotein of MDV inhibits apoptosis and interacts with chicken infectious anemia virus-encoded apoptotic protein apoptin(27,75). Finally, the apoptotic regulation of vNr-13 appears to be biphasic, first at early time points in low PFU infections, and then at later time points in high PFU infections, and it willon October 3, 2020 by guest http://jvi.asm.org/ Downloaded from be interesting to examine the roles of other viral/cellular factors involved in modulating apoptotic pathways particularly at later time points of HVT infection.…”

Out of Sight, but Not Out of Mind: Aspects of the Avian Oncogenic Herpesvirus, Marek’s Disease Virus

“…Recently Brown et al [61] reported for the first time that the meq protein can physically, and potentially functionally, interact with the chicken anemia virus (CAV) apoptin protein transactivating the CAV transcription [62]. CAV is an economically important immunosuppressive virus of chickens that causes anemia, lymphoid atrophy, morbidity, and mortality and synergizes with many co-infecting pathogens, including MDV, as demonstrated for avian respiratory viruses [22].…”

The bZIP Proteins of Oncogenic Viruses

“…MEQ also assists in MDV-associated oncogenesis by inhibiting the apoptosis of infected cells [ 134 , 149 , 150 ]. For example, MEQ binds and inhibits the apoptosis inducer apoptin in DF-1 cells [ 149 ].…”