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Characterizing the molecular basis of attenuation of Marek's disease virus via in vitro serial passage identifies de novo mutations in the helicase-primase subunit gene UL5 and other candidates associated with reduced virulence.

ABSTRACT: Marek's disease (MD) is a lymphoproliferative disease of chickens caused by the oncogenic Gallid herpesvirus 2, commonly known as Marek's disease virus (MDV). MD vaccines, the primary control method, are often generated by repeated in vitro serial passage of this highly cell-associated virus to attenuate virulent MDV strains. To understand the genetic basis of attenuation, we used experimental evolution by serially passing three virulent MDV replicates generated from an infectious bacterial artificial chromosome (BAC) clone. All replicates became completely or highly attenuated, indicating that de novo mutation, and not selection among quasispecies existing in a strain, is the primary driving force for the reduction in virulence. Sequence analysis of the attenuated replicates revealed 41 to 95 single-nucleotide variants (SNVs) at 2% or higher frequency in each population and several candidate genes containing high-frequency, nonsynonymous mutations. Five candidate mutations were incorporated into recombinant viruses to determine their in vivo effect. SNVs within UL42 (DNA polymerase auxiliary subunit) and UL46 (tegument) had no measurable influence, while two independent mutations in LORF2 (a gene of unknown function) improved survival time of birds but did not alter disease incidence. A fifth SNV located within UL5 (helicase-primase subunit) greatly reduced in vivo viral replication, increased survival time of birds, and resulted in only 0 to 11% disease incidence. This study shows that multiple genes, often within pathways involving DNA replication and transcriptional regulation, are involved in de novo attenuation of MDV and provides targets for the rational design of future MD vaccines.Marek's disease virus (MDV) is a very important pathogen in chickens that costs the worldwide poultry industry $1 billion to $2 billion annually. Marek's disease (MD) vaccines, the primary control method, are often produced by passing virulent strains in cell culture until attenuated. To understand this process, we identified all the changes in the viral genome that occurred during repeated cell passage. We find that a single mutation in the UL5 gene, which encodes a viral protein necessary for DNA replication, reduces disease incidence by 90% or more. In addition, other candidate genes were identified. This information should lead to the development of more effective and rationally designed MD vaccines leading to improved animal health and welfare and lower costs to consumers.

SUBMITTER: Hildebrandt E

PROVIDER: S-EPMC4093847 | biostudies-literature | 2014 Jun

REPOSITORIES: biostudies-literature

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Characterizing the molecular basis of attenuation of Marek's disease virus via in vitro serial passage identifies de novo mutations in the helicase-primase subunit gene UL5 and other candidates associated with reduced virulence.

Hildebrandt Evin E Dunn John R JR Perumbakkam Sudeep S Niikura Masahiro M Cheng Hans H HH

Journal of virology 20140319 11

<h4>Unlabelled</h4>Marek's disease (MD) is a lymphoproliferative disease of chickens caused by the oncogenic Gallid herpesvirus 2, commonly known as Marek's disease virus (MDV). MD vaccines, the primary control method, are often generated by repeated in vitro serial passage of this highly cell-associated virus to attenuate virulent MDV strains. To understand the genetic basis of attenuation, we used experimental evolution by serially passing three virulent MDV replicates generated from an infect ...[more]

PMID: 24648463

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Project description:UnlabelledPoxviridae are viruses with a large linear double-stranded DNA genome coding for up to 250 open reading frames and a fully cytoplasmic replication. The double-stranded DNA genome is covalently circularized at both ends. Similar structures of covalently linked extremities of the linear DNA genome are found in the African swine fever virus (asfarvirus) and in the Phycodnaviridae We are studying the machinery which replicates this peculiar genome structure. From our work with vaccinia virus, we give first insights into the overall structure and function of the essential poxvirus virus helicase-primase D5 and show that the active helicase domain of D5 builds a hexameric ring structure. This hexamer has ATPase and, more generally, nucleoside triphosphatase activities that are indistinguishable from the activities of full-length D5 and that are independent of the nature of the base. In addition, hexameric helicase domains bind tightly to single- and double-stranded DNA. Still, the monomeric D5 helicase construct truncated within the D5N domain leads to a well-defined structure, but it does not have ATPase or DNA-binding activity. This shows that the full D5N domain has to be present for hexamerization. This allowed us to assign a function to the D5N domain which is present not only in D5 but also in other viruses of the nucleocytoplasmic large DNA virus (NCLDV) clade. The primase domain and the helicase domain were structurally analyzed via a combination of small-angle X-ray scattering and, when appropriate, electron microscopy, leading to consistent low-resolution models of the different proteins.ImportanceSince the beginning of the 1980s, research on the vaccinia virus replication mechanism has basically stalled due to the absence of structural information. As a result, this important class of pathogens is less well understood than most other viruses. This lack of information concerns in general viruses of the NCLDV clade, which use a superfamily 3 helicase for replication, as do poxviruses. Here we provide for the first time information about the domain structure and DNA-binding activity of D5, the poxvirus helicase-primase. This result not only refines the current model of the poxvirus replication fork but also will lead in the long run to a structural basis for antiviral drug design.

Dataset Information

Characterizing the molecular basis of attenuation of Marek's disease virus via in vitro serial passage identifies de novo mutations in the helicase-primase subunit gene UL5 and other candidates associated with reduced virulence.

Publications

Characterizing the molecular basis of attenuation of Marek's disease virus via in vitro serial passage identifies de novo mutations in the helicase-primase subunit gene UL5 and other candidates associated with reduced virulence.

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