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 haloalkaliphilic Archaea. By random choice, we targeted ORF79, a currently uncharacterized viral gene of the haloalkaliphilic virus Ch1. We used a polyethylene glycol (PEG)-mediated transformation method to deliver a disruption cassette into a lysogenic strain of the haloalkaliphilic archaeon Natrialba magadii bearing Ch1 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 of N. 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 of N. magadii reduced the plating efficiency of Ch1 by seven orders of magnitude. Overexpression of ORF79 in a lysogenic strain of N. magadii resulted 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 for in vivo studies in N. magadii. IMPORTANCE Genetic analyses of haloalkaliphilic Archaea or haloviruses are only rarely reported. Therefore, only little insight into the in vivo roles 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 Ch1. We provide evidence that gp79, a currently unknown protein of Ch1, 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 of N. magadii. Our 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.KEYWORDS Archaea, Natrialba magadii, Ch1, halophiles, virus H alovirus Ch1 is a model for haloalkaliphilic viruses and infects the haloalkaliphilic archaeon Natrialba magadii (1). As typical for members of the Myoviridae family, Ch1 consists of an icosahedral head and a contractible tail and contains a doublestranded DNA (dsDNA) genome (2). The halovirus Ch1 is adapted to the extreme growth conditions of its host N. magadii, depending on high salt concentrations (4 to

Comparative Genomics of Two New HF1-like Haloviruses

“…Most HF1-group members carry a N-6 methylase gene. In the myohalovirus phiCh1, the function of its N-6 methylase gene has been well studied [43,44]. The phiCh1 gene is expressed only late in infection, and modifies a proportion of sites on genomes that have not yet been packaged, the proportion varying between 5-50% depending upon growth conditions.…”

Viruses of archaea: Structural, functional, environmental and evolutionary genomics

“…Archaeal viruses frequently encode transcription factors with diverse DNA-binding motifs, including ribbon-helix-helix (RHH), winged helix-turn-helix (wHTH) and zinc-fingers (Peeters et al, 2013; Prangishvili et al, 2006), which due to their generally small size are amenable to crystallization and NMR. Structures of 10 such putative transcription factors have been determined thus far and some of them have been experimentally characterized revealing intricate patterns of transcriptional control during infection (Fusco et al, 2015b; Guilliere et al, 2009; Peixeiro et al, 2013; Selb et al, 2017). These studies have validated the conclusions initially reached by protein sequence analysis (Prangishvili et al, 2006), namely, that whereas the basal transcription machinery of archaea, in terms of the structure of promoters and subunit composition of the RNA polymerase, closely resembles the eukaryotic counterparts (Peeters et al, 2013; Werner and Grohmann, 2011), many of the transcription factors encoded by archaea and their viruses are bacterial-like.…”

“…Studies on bacterial and eukaryotic viruses have benefited from the availability of well-established genetic tools developed for the respective hosts and, more generally, from the broad knowledge base on the host biology. This, unfortunately, has not been the case for most of the archaeal virus-host systems, although new genetic tools are being developed for an increasing number of archaea and their viruses (Iverson and Stedman, 2012; Iverson et al, 2017; Jaubert et al, 2013; Selb et al, 2017; Wang et al, 2016; Wirth et al, 2011). However, during the past few years, high-throughput functional genomics approaches have been adapted to study archaeal viruses, yielding valuable information on their biology.…”

Archaeal Viruses from High-Temperature Environments

“…A final example: combined genetics and biochemical assays to suggest the function of viral gene, ORF79 , from the halophilic virus ϕCh1. Bioinformatically, ORF79 shows low homology to another halophilic viral protein, gp5 from Haloarcula hispanica tailed virus 2 (HHTV-2), to the adenovirus E1A protein, and to chromatin remodeling proteins [ 84 ]. Transformation of a ORF79 disruption cassette into a strain of the host that carried a proviral ϕCh1 yielded viruses that carried a disrupted version of ORF79.…”