title: The Viral Gene ORF79 Encodes a Repressor Regulating Induction of the Lytic Life Cycle in the Haloalkaliphilic Virus ϕCh1.
abstract: In this study, we describe the construction of the first genetically modified mutant of a halovirus infecting haloalkaliphilicArchaeaBy random choice, we targeted ORF79, a currently uncharacterized viral gene of the haloalkaliphilic virus ϕCh1. We used a polyethylene glycol (PEG)-mediated transformation method to deliver a disruption cassette into a lysogenic strain of the haloalkaliphilic archaeonNatrialba magadiibearing ϕCh1 as a provirus. This approach yielded mutant virus particles carrying a disrupted version of ORF79. Disruption of ORF79 did not influence morphology of the mature virions. The mutant virus was able to infect cured strains ofN. magadii, resulting in a lysogenic, ORF79-disrupted strain. Analysis of this strain carrying the mutant virus revealed a repressor function of ORF79. In the absence of gp79, onset of lysis and expression of viral proteins occurred prematurely compared to their timing in the wild-type strain. Constitutive expression of ORF79 in a cured strain ofN. magadiireduced the plating efficiency of ϕCh1 by seven orders of magnitude. Overexpression of ORF79 in a lysogenic strain ofN. magadiiresulted in an inhibition of lysis and total absence of viral proteins as well as viral progeny. In further experiments, gp79 directly regulated the expression of the tail fiber protein ORF34 but did not influence the methyltransferase gene ORF94. Further, we describe the establishment of an inducible promoter forin vivostudies inN. magadiiIMPORTANCEGenetic analyses of haloalkaliphilicArchaeaor haloviruses are only rarely reported. Therefore, only little insight into thein vivoroles of proteins and their functions has been gained so far. We used a reverse genetics approach to identify the function of a yet undescribed gene of ϕCh1. We provide evidence that gp79, a currently unknown protein of ϕCh1, acts as a repressor protein of the viral life cycle, affecting the transition from the lysogenic to the lytic state of the virus. Thus, repressor genes in other haloviruses could be identified by sequence homologies to gp79 in the future. Moreover, we describe the use of an inducible promoter ofN. magadiiOur work provides valuable tools for the identification of other unknown viral genes by our approach as well as for functional studies of proteins by inducible expression.
authors.affiliation: Department of Microbiology, Immunobiology and Genetics, MFPL Laboratories, University of Vienna, Vienna, Austria.
authors.forename: Regina
authors.initials: R
authors.lastname: Selb
authors.affiliation: Department of Microbiology, Immunobiology and Genetics, MFPL Laboratories, University of Vienna, Vienna, Austria.
authors.forename: Christian
authors.initials: C
authors.lastname: Derntl
authors.affiliation: IMC FH Krems, University of Applied Sciences, Krems, Austria.
authors.forename: Reinhard
authors.initials: R
authors.lastname: Klein
authors.affiliation: Department of Microbiology, Immunobiology and Genetics, MFPL Laboratories, University of Vienna, Vienna, Austria.
authors.forename: Beatrix
authors.initials: B
authors.lastname: Alte
authors.affiliation: Department of Microbiology, Immunobiology and Genetics, MFPL Laboratories, University of Vienna, Vienna, Austria.
authors.forename: Christoph
authors.initials: C
authors.lastname: Hofbauer
authors.affiliation: Department of Microbiology, Immunobiology and Genetics, MFPL Laboratories, University of Vienna, Vienna, Austria.
authors.forename: Martin
authors.initials: M
authors.lastname: Kaufmann
authors.affiliation: Department of Microbiology, Immunobiology and Genetics, MFPL Laboratories, University of Vienna, Vienna, Austria.
authors.forename: Judith
authors.initials: J
authors.lastname: Beraha
authors.affiliation: Department of Microbiology, Immunobiology and Genetics, MFPL Laboratories, University of Vienna, Vienna, Austria.
authors.forename: Léa
authors.initials: L
authors.lastname: Schöner
authors.affiliation: Department of Microbiology, Immunobiology and Genetics, MFPL Laboratories, University of Vienna, Vienna, Austria angela.witte@univie.ac.at.
authors.forename: Angela
authors.initials: A
authors.lastname: Witte
mesh_terms: D004279:DNA, Viral / Q000235:genetics; D005814:Genes, Viral / Q000235:genetics; D006216:Halobacteriaceae / Q000821:virology*; D008242:Lysogeny / Q000235:genetics*; D017900:Myoviridae / Q000235:genetics*; D016366:Open Reading Frames / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics; D012097:Repressor Proteins / Q000235:genetics*; D014764:Viral Proteins / Q000235:genetics; D018406:Virus Physiological Phenomena / Q000235:genetics
chemical_list: D004279:DNA, Viral; D012097:Repressor Proteins; D014764:Viral Proteins
keywords: Archaea; Natrialba magadii; halophiles; virus; ϕCh1