Mutations in the Meq oncoprotein of Marek's disease virus are selected by vaccination and mediate functional interactome changes
Date
2024
Authors
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Publisher
University of Delaware
Abstract
Marek’s disease (MD) of chickens, caused by the alphaherpesvirus Marek’s disease virus (MDV), is prevalent in the poultry industry with consequential economic losses. A hallmark of MDV infection is the rapid onset of T-lymphomas in its natural host. The MDV oncoprotein Meq (Marek’s EcoRI-Q-fragment-encoded protein) is essential for T-cell lymphoma formation. MD is currently controlled in poultry production through live-attenuated or apathogenic vaccines. Despite this control, field strains have continued to evolve in virulence since the 1970s. Nonsynonymous polymorphisms identified in the coding sequence of Meq have been correlated with this evolution of virulence, and our preliminary data suggest that these changes could be functioning at two stages of infection, namely, during the early replication of MDVs and in the T-cell transformation phase of infection. Therefore, this study focuses on the analysis of Meq isoforms at these stages of infection. Using a combination of recombinant MDVs, mass spectrometry (MS)-based-proteomic analyses, and follow-up biochemical and functional assays, we have identified key cellular components and pathways by which MDV field strains have evolved in virulence to overcome vaccine-mediated protection. ☐ MD vaccines provide immunity that protects against lymphomagenesis but does not prevent infection and transmission of field challenge strains. This non-sterilizing immunity allows MDV to mutate into emergent field strains of higher virulence that can escape vaccinal immunity. Meq is under positive selection, and the selection pressures are largely driven by vaccination. The present study, therefore, addresses the hypothesis that mutations in Meq have been selected by the innate immune response elicited by host vaccination during the early lytic infection stage. Mutations common to field strains overcoming protection conferred by vaccines commonly used in the US (herpesvirus of turkeys, HVT; and Mardivirus-2, SB-1) were identified in the C-terminal proline-rich repeat region (PRR) of Meq. These mutations map to second position sites of tetra-proline stretches, PPPP->P(Q/A)PP. For very virulent and hypervirulent strains arising in Europe, where an attenuated MDV-1 strain has been used as a vaccine since the early 1970s (CVI988/Rispens), mutations in the Meq of high virulence European strains map to the basic N-terminal and leucine zipper (bZIP) domains. ☐ In the Plateau State of Nigeria, regional field strains have arisen on farms that administered the CVI988 vaccine at hatch and subsequently re-vaccinated chickens with HVT at 21 days post-hatch at farms receiving these vaccinates. Using maximum likelihood phylogenetic analysis, we reconstruct the MDV phylogeny of North American, Eurasian, and African MDV lineages to address the phylogeographic relationship between international vaccination practices and discrete vaccine-mediated selection of Meq. Sequence analysis of the Meq genes from field strain virus identified mutations in both N-terminal and C-terminal domains (NTD and CTD). Our data, therefore, suggest a direct effect of vaccine strategy on the fitness selection of MDV field isolates. ☐ To designate virulence to mutations in Meq and their causal role in overcoming early vaccine responses, strain RB-1B-based recombinant-MDVs expressing Meq isoforms derived from the vaccine, virulent (v), very virulent (vv), and very virulent plus (vv+) pathotypes were constructed. Groups of unvaccinated and in ovo HVT-and HVT/SB1-vaccinated specific pathogen-free chickens were challenged at hatch with 2000 PFU of each recombinant virus. Pathogenicity was assessed by measuring viral replication kinetics, the induction of lymphoproliferative lesions, and mortality. The exchange of prototype Meq isoforms demonstrates pathotype-specific trends in MD-incidence and -mortality rates in the naïve host. However, in the MD-immunized chickens, there were no significant changes in MD incidence and mortality between the challenge viruses, indicating the complex roles of these isoforms in pathogenesis and providing insight into the discrete functional properties contributing to Meq pathobiology. ☐ Conceivably, these amino acid substitutions affect specificity at the binding interface of protein-protein interactions, which in turn may stabilize the disordered proline-rich composition of Meq. This work addresses the hypothesis that mutations in the Meq oncoprotein have been selected based on changes in the interactomes of Meq in latently infected, transformed T-cells. MDV-associated lymphoblastoid cell lines were subjected to anti-Meq immunoprecipitation and subsequent MS-based proteomic analysis to profile the endogenous Meq interactome among MDV pathotypes. Functional gene ontology analysis shows enrichment of DNA repair-associated proteins and chromatin remodeling complexes in Meq-host interactomes of high-virulence pathotypes. ☐ We speculate that Meq interacts with DNA repair-associated proteins during lytic infection, presumably to alter host genomic stability, leading to an accumulation of mutations. Somatic mutations in cancer driver genes are necessary to assist Meq in CD4+ T-cell transformation later in infection when the virus establishes latency. By proteomic analysis, we identified a candidate Meq interactor, Nedd4 Binding Protein 2 (N4BP2), involved in DNA repair and recombination through a small MutS-related domain. Domain mapping experiments show that the full-length Meq re-localizes N4BP2 from the cytoplasm to the nucleus and nucleolus in a CTD-dependent manner. ☐ The presence of the unspliced, full-length Meq throughout infection suggests the Meq CTD has a nonredundant role with Meq splice variants during early lytic infection when replicating virus elicits robust host responses of the innate immune system and DNA damage response pathways that are important selection pressures on the CTD. The functional constraints in this context may lend Meq pleiotropic properties to antagonize innate immune signaling pathways and DNA damage responses by interaction with DNA repair enzymes as a novel mechanism of vv+MDVs for increased efficiency of viral integration into the host genome during latency. ☐ The 33 amino acid CTD of Meq contains a highly conserved transactivation domain; however, distinct mutations within an adjacent disordered PPR are characteristic of v, vv, and vv+ MDV pathotypes. Embedded in the PRR are five to seven tetra-proline motifs; however, contrary to ancestral strains, fewer proline tetrads constitute the CTD of contemporary strains by proline to glutamine or alanine substitutions at position two (P[Q/A]PP) or partial PRR truncation. How these proline tetrads cooperate in the context of Meq biology remains uncharacterized. However, the prevailing dogma is that selection for the consecutive reduction of proline tetrads affects virulence and, therefore, plays a functional role in MDV pathogenesis. We hypothesized that these substitutions alter the specificity or affinity of protein-protein interactions, mediating a gain of function. ☐ In support of this hypothesis, we now report that the PRR of the vv+MDV Meq isoform mediates the nucleolar recruitment of a cellular ATP-dependent chromatin remodeler, BRG1. We show that this specific binding of BRG1 increases the efficiency in the transcriptional activity of the adjacent CTD in a manner that is dependent on amino acid residues at positions 153, 176, and 217, suggesting that these residues determine the unique BRG1 binding site. We propose a mechanism whereby Meq occupancy at repressed regulatory loci requires BRG1 cooperation to promote an open chromosome state by remodeling the chromatin topology. Thus, the complete transactivation potential of the conserved region in the C-terminal transactivation domain depends on accessibility to promoter and enhancer elements. This finding provides the first experimental evidence, with amino acid resolution of residues, that mutations within the PRR of the vv+MDV Meq isoform have been selected by changes in its interactome.
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Keywords
Chromatin remodeling complex, Evolution of virulence, Herpesvirus, Molecular virology, Oncoprotein, Pathogenesis