Structure, function and unexplored properties of fuselloviruses-encoded transcription factors

Objective: Archaeal viruses are predicted to encode for a wide variety of transcriptional regulators. The majority of these gene products do not have detectable homologues in the databases other than in related hyperthermophilic viral genomes, thus leading to the necessity of performing structural and functional analyses to unravel their role. Archaeal transcription regulators resemble the bacterial ones at structural level but operate in an eukaryal-like transcriptional context (1). Therefore, it is expected that in this “hybrid” transcriptional apparatus, peculiar and not yet characterized mechanisms for the regulation of expression are employed and novel folds and/or functions might be discovered. Fuselloviridae is one of the best-characterized families of crenarchaeal viruses, whose members have been used as model systems to understand the mechanisms underpinning the transcription process at structural and functional level. Methods: Putative transcription factors encoded by pSSVx and SSV1 have been studied with the purpose of determining their structure and/or dissecting their regulative mechanisms. In this regard, a combination of approaches, ranging from DNA-protein interaction assays to structure determination techniques, has been used. Results: The discovery and characterization of the SSV1-encoded regulator F55 (2) helped to shed light on the molecular mechanisms governing the maintenance of the SSV1 lysogeny. This protein has been proved to bind, in vitro and in vivo, to the viral genome at the promoters of the UV-inducible (Tind), of the early transcripts (T5 and T6) as well as of its own transcript (Tlys). Indeed, band-shift and chromatin immunoprecipitation assays confirmed the interaction of F55 with these regulative regions and its dissociation soon after UV irradiation of SSV1 lysogenic cells. The structure of two proteins encoded by the hybrid plasmid/virus pSSVx has been solved by nuclear magnetic resonance spectroscopy and X-ray diffraction, revealing that ORFc-68 bears a looped-hinged-helix motif (3) whereas Stf76 assumes a winged helix-turn-helix fold (4). These DNA-binding proteins interact specifically with target sequences located within promoters of their gene, thus regulating their own expression. Despite the resemblance of the overall fold to that of bacterial transcription regulators, both ORFc68 and Stf76 display peculiar structural features. Interestingly, a 28aa-long stretch of the Stf76 amino acid sequence (named Pep37V-64R) that encompasses the DNA-binding domain was in silico predicted to be a potential cationic antimicrobial peptide (CAMP) and tested for antimicrobial and antifungal activities towards several strains. It was revealed that both Gram+ and Gram- strains were sensitive to the action of Pep37V-64R, possibly as a consequence of membrane disaggregation and/or of the retained ability of Pep37V-64R to interact with nucleic acids. Further biochemical and structural studies on this peptide are underway. Conclusion: Altogether these analyses expand the current knowledge about the structures adopted by archaeal transcription factors and their mechanisms of action. Moreover, it has been revealed that the domain of Achaea might represent a potential reservoir of bioactive peptides. References: 1. Contursi et al. (2013) Extremophiles, 17(6):881-895 2. Fusco et al., (2013) J Virol, 87(10):5926-5936 3. Contursi et al., (2011) Biochem J, 435(1):157-166 4. Contursi et al., (2014) Nucleic Acids Res, [Epub ahead of print] doi: 10.1093/nar/gku215