Homomultimeric structure by assembly of sirv2 p98 proteins or p98 variants, conjugate and uses thereof

The present invention relates to a homomultimeric protein structure constituted by assembled monomers of the P98 protein of Sulfolobus islandicus rod-shaped virus 2 (SIRV2) or assembled monomer variants of said P98 protein. In a particular embodiment, this homomultimeric protein structure has a seven-fold rotational symmetry, and is found in an open conformation or closed conformation. A particular structure has the form of baseless 7-face pyramid. The invention also relates to a conjugate comprising or consisting of a homomultimeric protein structure of the invention to which one or more heterologous molecule(s) is attached. Furthermore, the invention also concerns a homomultimeric protein structure or a conjugate of the invention, inserted into or exposed at the surface of a lipid layer or bilayer, of a vesicle or of a cell, and their uses thereof

Homomultimeric structure by assembly of sirv2 p98 proteins or p98 variants, conjugate and uses thereof

The present invention relates to a homomultimeric protein structure constituted by assembled monomers of the P98 protein of Sulfolobus islandicus rod-shaped virus 2 (SIRV2) or assembled monomer variants of said P98 protein. In a particular embodiment, this homomultimeric protein structure has a seven-fold rotational symmetry, and is found in an open conformation or closed conformation. A particular structure has the form of baseless 7-face pyramid. The invention also relates to a conjugate comprising or consisting of a homomultimeric protein structure of the invention to which one or more heterologous molecule(s) is attached. Furthermore, the invention also concerns a homomultimeric protein structure or a conjugate of the invention, inserted into or exposed at the surface of a lipid layer or bilayer, of a vesicle or of a cell, and their uses thereof

AFV1, a novel virus infecting hyperthermophilic archaea of the genus acidianus

AbstractWe describe a novel virus, AFV1, of the hyperthermophilic archaeal genus Acidianus. Filamentous virions are covered with a lipid envelope and contain at least five different proteins with molecular masses in the range of 23–130 kDa and a 20.8-kb-long linear double-stranded DNA. The virus has been assigned to the family Lipothrixviridae on the basis of morphotypic characteristics. Host range is confined to several strains of Acidianus and the virus persists in its hosts in a stable carrier state. The latent period of virus infection is about 4 h. Viral DNA was sequenced and sequence similarities were found to the lipothrixvirus SIFV, the rudiviruses SIRV1 and SIRV2, as well as to conjugative plasmids and chromosomes of the genus Sulfolobus. Exceptionally for the linear genomes of archaeal viruses, many short direct repeats, with the sequence TTGTT or close variants thereof, are closely clustered over 300 bp at each end of the genome. They are reminiscent of the telomeric ends of linear eukaryal chromosomes

Dark matter in archaeal genomes: a rich source of novel mobile elements, defense systems and secretory complexes

International audienceMicrobial genomes encompass a sizable fraction of poorly characterized, narrowly spread fast-evolving genes. Using sensitive methods for sequences comparison and protein structure prediction, we performed a detailed comparative analysis of clusters of such genes, which we denote "dark matter islands", in archaeal genomes. The dark matter islands comprise up to 20% of archaeal genomes and show remarkable heterogeneity and diversity. Nevertheless, three classes of entities are common in these genomic loci: (a) integrated viral genomes and other mobile elements; (b) defense systems, and (c) secretory and other membrane-associated systems. The dark matter islands in the genome of thermophiles and mesophiles show similar general trends of gene content, but thermophiles are substantially enriched in predicted membrane proteins whereas mesophiles have a greater proportion of recognizable mobile elements. Based on this analysis, we predict the existence of several novel groups of viruses and mobile elements, previously unnoticed variants of CRISPR-Cas immune systems, and new secretory systems that might be involved in stress response, intermicrobial conflicts and biogenesis of novel, uncharacterized membrane structures

Solution structure of an archaeal DNA binding protein with an eukaryotic zinc finger fold

International audienceWhile the basal transcription machinery in archaea is eukaryal-like, transcription factors in archaea and their viruses are usually related to bacterial transcription factors. Nevertheless, some of these organisms show predicted classical zinc fingers motifs of the C2H2 type, which are almost exclusively found in proteins of eukaryotes and most often associated with transcription regulators. In this work, we focused on the protein AFV1p06 from the hyperthermophilic archaeal virus AFV1. The sequence of the protein consists of the classical eukaryotic C2H2 motif with the fourth histidine coordinating zinc missing, as well as of N- and C-terminal extensions. We showed that the protein AFV1p06 binds zinc and solved its solution structure by NMR. AFV1p06 displays a zinc finger fold with a novel structure extension and disordered N- and C-termini. Structure calculations show that a glutamic acid residue that coordinates zinc replaces the fourth histidine of the C2H2 motif. Electromobility gel shift assays indicate that the protein binds to DNA with different affinities depending on the DNA sequence. AFV1p06 is the first experimentally characterised archaeal zinc finger protein with a DNA binding activity. The AFV1p06 protein family has homologues in diverse viruses of hyperthermophilic archaea. A phylogenetic analysis points out a common origin of archaeal and eukaryotic C2H2 zinc fingers

A novel archaeal regulatory protein, Sta1, activates transcription from viral promoters

While studying gene expression of the rudivirus SIRV1 in cells of its host, the hyperthermophilic crenarchaeon Sulfolobus, a novel archaeal transcriptional regulator was isolated. The 14 kDa protein, termed Sulfolobus transcription activator 1, Sta1, is encoded on the host chromosome. Its activating effect on transcription initiation from viral promoters was demonstrated in in vitro transcription experiments using a reconstituted host system containing the RNA polymerase, TATA-binding protein (TBP) and transcription factor B (TFB). Most pronounced activation was observed at low concentrations of either of the two transcription factors, TBP or TFB. Sta1 was able to bind viral promoters independently of any component of the host pre-initiation complex. Two binding sites were revealed by footprinting, one located in the core promoter region and the second ∼30 bp upstream of it. Comparative modeling, NMR and circular dichroism of Sta1 indicated that the protein contained a winged helix–turn–helix motif, most probably involved in DNA binding. This strategy of the archaeal virus to co-opt a host cell regulator to promote transcription of its genes resembles eukaryal virus–host relationships

Characterization of Sulfolobus islandicus rod-shaped virus 2 gp19, a single-strand specific endonuclease

The hyperthermophilic Sulfolobus islandicus rod-shaped virus 2 (SIRV2) encodes a 25-kDa protein (SIRV2gp19) annotated as a hypothetical protein with sequence homology to the RecB nuclease superfamily. Even though SIRV2gp19 homologs are conserved throughout the rudivirus family and presumably play a role in the viral life cycle, SIRV2gp19 has not been functionally characterized. To define the minimal requirements for activity, SIRV2gp19 was purified and tested under varying conditions. SIRV2gp19 is a single-strand specific endonuclease that requires Mg2+ for activity and is inactive on double-stranded DNA. A conserved aspartic acid in RecB nuclease superfamily Motif II (D89) is also essential for SIRV2gp19 activity and mutation to alanine (D89A) abolishes activity. Therefore, the SIRV2gp19 cleavage mechanism is similar to previously described RecB nucleases. Finally, SIRV2gp19 single-stranded DNA endonuclease activity could play a role in host chromosome degradation during SIRV2 lytic infection