The complete genome sequence of the largest known double-stranded DNA virus, mimivirus, reveals the presence of a gene (denoted R355) that potentially encodes a cysteine protease that is expressed late (after 6 h) in the infectious cycle of the virus. In order to verify a sequence-based functional prediction and understand its role during the infectious process, the R355 protein was produced to assay its proteolytic activity and solve its three-dimensional structure. Here, the preliminary crystallographic analysis of the recombinant viral protein is reported. The crystals belonged to the orthorhombic space group P2(1)2(1)2(1), with a monomer in the asymmetric unit. A MAD data set was used for preliminary phasing using the selenium signal from a selenomethionine-substituted protein crystal.
Abstract The mimivirus 1.2Mb genome was shown to be organized into a nucleocapsid-like genomic fiber encased in the nucleoid compartment inside the icosahedral capsid (1). The genomic fiber protein shell is composed of a mixture of two GMC-oxidoreductase paralogs, one of them being the main component of the glycosylated layer of fibrils at the surface of the virion (2). In this study, we determined the effect of the deletion of each of the corresponding genes on the genomic fiber and the layer of surface fibrils. First, we deleted the GMC-oxidoreductase the most abundant in the genomic fiber, and determined its structure and composition in the mutant. As expected, it was composed of the second GMC-oxidoreductase and contained 5- and 6-start helices similar to the wild-type fiber. This result led us to propose a model explaining their coexistence. Then, we deleted the GMC-oxidoreductase the most abundant in the layer of fibrils to analyze its protein composition in the mutant. Second, we showed that the fitness of single mutants and the double mutant were not decreased compared to the wild-type viruses in laboratory conditions. Third, we determined that deleting the GMC-oxidoreductase genes did not impact the glycosylation or the glycan composition of the layer of surface fibrils, despite modifying their protein composition. Since the glycosylation machinery and glycan composition of members of different clades are different (3, 4), we expanded the analysis of the protein composition of the layer of fibrils to members of the B and C clades and showed that it was different among the three clades and even among isolates within the same clade. Taken together, the results obtained on two distinct central processes (genome packaging and virion coating) illustrate an unexpected functional redundancy in members of the family Mimiviridae , suggesting this may be the major evolutionary force behind their giant genomes. One-Sentence Summary Functional redundancy preserves mimivirus genomic fiber and layer of fibrils formation.
Abstract Mimivirus is the prototype of the Mimiviridae family of giant dsDNA viruses, initially isolated in Acanthamoeba1. Little is known about the organization of the viral genome inside the membrane limited nucleoid2 and whether unpacking or other rearrangements are required prior to transcription and replication. Here we show that opening of its large icosahedral capsid in vitro leads to the release of electron dense, 30 nm diameter rod-shaped objects that appear to be expelled from the particles and unwinding. We developed a purification procedure and characterized the detailed structure at various stages of decompaction using cryo-electron microscopy single particle analyses and its composition by proteomics. This revealed that the viral genome is encased into a helical protein shell surprisingly made of the two GMC-type oxydoreductases that are also the major components of the glycosylated fibrils surrounding the capsid3. The 1.2 Mb genome is folded to follow a 5- or 6-start left-handed helix, depending on the nature of the GMC-oxydoreductase, with each helical strand lining the interior of the protein shell. Proteomic analyses of the purified genomic fibre revealed the presence of several RNA polymerase subunits as well as additional proteins involved in genome compaction that can fit into the central channel of the protein shield. Such an elegant supramolecular organization represents a remarkable evolutionary solution for packaging while protecting the viral genome, in a state ready for immediate transcription upon unwinding in the host cytoplasm. We expect that a dedicated energy-driven machinery is required for the assembly of this rod-shaped giant viral chromosome and its further compaction in the membrane limited electron dense nucleoid, characteristic of the mature Mimivirus particles2,4,5.The parsimonious implication of the same protein in two functionally unrelated substructures of the virion is also unexpected for a giant virus with a thousand genes at its disposal.
The analysis of the Acanthamoeba polyphaga mimivirus genome revealed the first virus-encoded nucleoside diphosphate kinase (NDK), an enzyme that is central to the synthesis of RNA and DNA, ubiquitous in cellular organisms, and well conserved among the three domains of life. In contrast with the broad specificity of cellular NDKs for all types of ribo- and deoxyribonucleotides, the mimivirus enzyme exhibits a strongly preferential affinity for deoxypyrimidines. In order to elucidate the molecular basis of this unique substrate specificity, we determined the three-dimensional (3D) structure of the Acanthamoeba polyphaga mimivirus NDK alone and in complex with various nucleotides. As predicted from a sequence comparison with cellular NDKs, the 3D structure of the mimivirus enzyme exhibits a shorter Kpn loop, previously recognized as a main feature of the NDK active site. The structure of the viral enzyme in complex with various nucleotides also pinpointed two residue changes, both located near the active site and specific to the viral NDK, which could explain its stronger affinity for deoxynucleotides and pyrimidine nucleotides. The role of these residues was explored by building a set of viral NDK variants, assaying their enzymatic activities, and determining their 3D structures in complex with various nucleotides. A total of 26 crystallographic structures were determined at resolutions ranging from 2.8 A to 1.5 A. Our results suggest that the mimivirus enzyme progressively evolved from an ancestral NDK under the constraints of optimizing its efficiency for the replication of an AT-rich (73%) viral genome in a thymidine-limited host environment.
Megavirus chilensis, a close relative of the Mimivirus giant virus, is able to replicate in Acanthamoeba castellanii. The first step of viral infection involves the internalization of the virions in host vacuoles. It has been experimentally demonstrated that Mimivirus particles contain many proteins capable of resisting oxidative stress, as encountered in the phagocytic process. These proteins are conserved in Megavirus, which has an additional gene (Mg277) encoding a putative superoxide dismutase. The Mg277 ORF product was overexpressed in Escherichia coli, purified and crystallized. A SAD data set was collected to 2.24 Å resolution at the selenium peak wavelength on the BM30 beamline at the ESRF from a single crystal of selenomethionine-substituted recombinant superoxide dismutase protein.
