ABSTRACT
The structural and functional analysis of the protein AvtR encoded by Acidianus filamentous virus 6 (AFV6), which infects the archaeal genus Acidianus, revealed its unusual structure and involvement in transcriptional regulation of several viral genes. The crystal structure of AvtR (100 amino acids) at 2.6-Å resolution shows that it is constituted of a repeated ribbon-helix-helix (RHH) motif, which is found in a large family of bacterial transcriptional regulators. The known RHH proteins form dimers that interact with DNA using their ribbon to create a central ß-sheet. The repeated RHH motifs of AvtR superpose well on such dimers, but its central sheet contains an extra strand, suggesting either conformational changes or a different mode of DNA binding. Systematic evolution of ligands by exponential enrichment (SELEX) experiments combined with systematic mutational and computational analysis of the predicted site revealed 8 potential AvtR targets in the AFV6 genome. Two of these targets were studied in detail, and the complex role of AvtR in the transcriptional regulation of viral genes was established. Repressing transcription from its own gene, gp29, AvtR can also act as an activator of another gene, gp30. Its binding sites are distant from both genes' TATA boxes, and the mechanism of AvtR-dependent regulation appears to include protein oligomerization starting from the protein's initial binding sites. Many RHH transcriptional regulators of archaeal viruses could share this regulatory mechanism.
Subject(s)
Acidianus/virology , DNA-Binding Proteins/chemistry , Lipothrixviridae/chemistry , Viral Proteins/chemistry , Acidianus/genetics , Amino Acid Sequence , Crystallography, X-Ray , DNA Mutational Analysis , DNA, Viral/metabolism , DNA-Binding Proteins/genetics , Electrophoretic Mobility Shift Assay , Lipothrixviridae/genetics , Models, Molecular , Molecular Sequence Data , Mutant Proteins/chemistry , Mutant Proteins/genetics , Protein Binding , Protein Conformation , Protein Multimerization , Viral Proteins/geneticsABSTRACT
Acidianus filamentous virus 1 (AFV1) (Lipothrixviridae) is an enveloped filamentous virus that was characterized from a crenarchaeal host. It infects Acidianus species that thrive in the acidic hot springs (>85 degrees C and pH <3) of Yellowstone National Park, WY. The AFV1 20.8-kb, linear, double-stranded DNA genome encodes 40 putative open reading frames whose sequences generally show little similarity to other genes in the sequence databases. Because three-dimensional structures are more conserved than sequences and hence are more effective at revealing function, we set out to determine protein structures from putative AFV1 open reading frames (ORF). The crystal structure of ORF157 reveals an alpha+beta protein with a novel fold that remotely resembles the nucleotidyltransferase topology. In vitro, AFV1-157 displays a nuclease activity on linear double-stranded DNA. Alanine substitution mutations demonstrated that E86 is essential to catalysis. AFV1-157 represents a novel class of nuclease, but its exact role in vivo remains to be determined.
Subject(s)
Acidianus/virology , Deoxyribonucleases/chemistry , Deoxyribonucleases/metabolism , Lipothrixviridae/chemistry , Lipothrixviridae/enzymology , Viral Proteins/chemistry , Viral Proteins/metabolism , Amino Acid Sequence , Amino Acid Substitution , Crystallography, X-Ray , DNA/metabolism , DNA Mutational Analysis , Deoxyribonucleases/genetics , Hot Springs/microbiology , Lipothrixviridae/genetics , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Open Reading Frames , Protein Structure, Tertiary , Viral Proteins/geneticsABSTRACT
Acidianus filamentous virus 1 (AFV1), a member of the Lipothrixviridae family, infects the hyperthermophilic, acidophilic crenarchaeaon Acidianus hospitalis. The virion, covered with a lipidic outer shell, is 9,100-A long and contains a 20.8-kb linear dsDNA genome. We have identified the two major coat proteins of the virion (MCPs; 132 and 140 amino acids). They bind DNA and form filaments when incubated with linear dsDNA. A C-terminal domain is identified in their crystal structure with a four-helix-bundle fold. In the topological model of the virion filament core, the genomic dsDNA superhelix wraps around the AFV1-132 basic protein, and the AFV1-140 basic N terminus binds genomic DNA, while its lipophilic C-terminal domain is imbedded in the lipidic outer shell. The four-helix bundle fold of the MCPs from AFV1 is identical to that of the coat protein (CP) of Sulfolobus islandicus rod-shaped virus (SIRV), a member of the Rudiviridae family. Despite low sequence identity between these proteins, their high degree of structural similarity suggests that they could have derived from a common ancestor and could thus define an yet undescribed viral lineage.
Subject(s)
Capsid Proteins/chemistry , Lipothrixviridae/chemistry , Protein Folding , Acidianus/virology , Crystallography, X-Ray , DNA-Binding Proteins/chemistry , Genome, Viral , Lipothrixviridae/genetics , Molecular Sequence Data , Phylogeny , Protein Structure, Secondary , Structural Homology, Protein , Sulfolobus/chemistryABSTRACT
Viruses infecting hyperthermophilic archaea have intriguing morphologies and genomic properties. The vast majority of their genes do not have homologs other than in other hyperthermophilic viruses, and the biology of these viruses is poorly understood. As part of a structural genomics project on the proteins of these viruses, we present here the structure of a 102 amino acid protein from acidianus filamentous virus 1 (AFV1-102). The structure shows that it is made of two identical motifs that have poor sequence similarity. Although no function can be proposed from structural analysis, tight binding of the gateway tag peptide in a groove between the two motifs suggests AFV1-102 is involved in protein protein interactions.
Subject(s)
Acidianus/virology , Crystallography, X-Ray , Lipothrixviridae/chemistry , Viral Proteins/chemistry , Lipothrixviridae/metabolism , Peptides/chemistry , Peptides/metabolism , Protein Binding , Protein Conformation , Viral Proteins/metabolismABSTRACT
A large-scale programme has been embarked upon aiming towards the structural determination of conserved proteins from viruses infecting hyperthermophilic archaea. Here, the crystallization of protein 14 from the archaeal virus SIFV is reported. This protein, which contains 111 residues (MW 13 465 Da), was cloned and expressed in Escherichia coli with an N-terminal His(6) tag and purified to homogeneity. The tag was subsequently cleaved and the protein was crystallized using PEG 1000 or PEG 4000 as a precipitant. Large crystals were obtained of the native and the selenomethionine-labelled protein using sitting drops of 100-300 nl. Crystals belong to space group P6(2)22 or P6(4)22, with unit-cell parameters a = b = 68.1, c = 132.4 A. Diffraction data were collected to a maximum acceptable resolution of 2.95 and 3.20 A for the SeMet-labelled and native protein, respectively.
Subject(s)
Lipothrixviridae/chemistry , Sulfolobus/virology , Viral Proteins/chemistry , Cloning, Molecular , Crystallization , Sulfolobus/genetics , Viral Proteins/genetics , X-Ray Diffraction/methodsABSTRACT
Bacteriophages are classified into one order and 13 families. Over 5100 phages have been examined in the electron microscope since 1959. At least 4950 phages (96%) are tailed. They constitute the order Caudovirales and three families. Siphoviridae or phages with long, noncontractile tails predominate (61% of tailed phages). Polyhedral, filamentous, and pleomorphic phages comprise less than 4% of bacterial viruses. Bacteriophages occur in over 140 bacterial or archaeal genera. Their distribution reflects their origin and bacterial phylogeny. Bacteriophages are polyphyletic, arose repeatedly in different hosts, and constitute 11 lines of descent. Tailed phages appear as monophyletic and as the oldest known virus group.
