Three-Dimensional Structure of the Enveloped Bacteriophage Φ12: An Incomplete T = 13 Lattice Is Superposed on an Enclosed T = 1 Shell

BACKGROUND:Bacteriophage phi12 is a member of the Cystoviridae, a unique group of lipid containing membrane enveloped bacteriophages that infect the bacterial plant pathogen Pseudomonas syringae pv. phaseolicola. The genomes of the virus species contain three double-stranded (dsRNA) segments, and the virus capsid itself is organized in multiple protein shells. The segmented dsRNA genome, the multi-layered arrangement of the capsid and the overall viral replication scheme make the Cystoviridae similar to the Reoviridae. METHODOLOGY/PRINCIPAL FINDINGS:We present structural studies of cystovirus phi12 obtained using cryo-electron microscopy and image processing techniques. We have collected images of isolated phi12 virions and generated reconstructions of both the entire particles and the polymerase complex (PC). We find that in the nucleocapsid (NC), the phi12 P8 protein is organized on an incomplete T = 13 icosahedral lattice where the symmetry axes of the T = 13 layer and the enclosed T = 1 layer of the PC superpose. This is the same general protein-component organization found in phi6 NC's but the detailed structure of the entire phi12 P8 layer is distinct from that found in the best classified cystovirus species phi6. In the reconstruction of the NC, the P8 layer includes protein density surrounding the hexamers of P4 that sit at the 5-fold vertices of the icosahedral lattice. We believe these novel features correspond to dimers of protein P7. CONCLUSIONS/SIGNIFICANCE:In conclusion, we have determined that the phi12 NC surface is composed of an incomplete T = 13 P8 layer forming a net-like configuration. The significance of this finding in regard to cystovirus assembly is that vacancies in the lattice could have the potential to accommodate additional viral proteins that are required for RNA packaging and synthesis

Genome-wide RNAi screen for nuclear actin reveals a network of cofilin regulators

Nuclear actin plays an important role in many processes that regulate gene expression. Cytoplasmic actin dynamics are tightly controlled by numerous actin-binding proteins, but regulation of nuclear actin has remained unclear. Here, we performed a genome-wide RNA interference (RNAi) screen in Drosophila cells to identify proteins that influence either nuclear polymerization or import of actin.We validate 19 factors as specific hits, and show that Chinmo (known as Bach2 in mammals), SNF4Aγ (Prkag1 in mammals) and Rab18 play a role in nuclear localization of actin in both fly and mammalian cells. We identify several new regulators of cofilin activity, and characterize modulators of both cofilin kinases and phosphatase. For example, Chinmo/Bach2, which regulates nuclear actin levels also in vivo, maintains active cofilin by repressing the expression of the kinase Cdi (Tesk in mammals). Finally, we show that Nup98 and lamin are candidates for regulating nuclear actin polymerization. Our screen therefore reveals new aspects of actin regulation and links nuclear actin to many cellular processes

Characterization of recombinant amino-terminal NC4 domain of human collagen IX - Interaction with glycosaminoglycans and cartilage oligomeric matrix protein

The N-terminal NC4 domain of collagen IX is a globular structure projecting away from the surface of the cartilage collagen fibril. Several interactions have been suggested for this domain, reflecting its location and its characteristic high isoelectric point. In an attempt to characterize the NC4 domain in more detail, we set up a prokaryotic expression system to produce the domain. The purified 27.5-kDa product was analyzed for its glycosaminoglycan-binding potential by surface plasmon resonance and solid-state assays. The results show that the NC4 domain of collagen IX specifically binds heparin with a K-d of 0.6 muM, and the full-length recombinant collagen IX has an even stronger interaction with heparin, with an apparent K-d of 3.6 nM. The heparin-binding site of the NC4 domain was located in the extreme N terminus, containing a heparin-binding consensus sequence, whereas electron microscopy suggested the presence of at least three additional heparin-binding sites on full-length collagen IX. The NC4 domain was also shown to bind cartilage oligomeric matrix protein. This interaction and the association of cartilage oligomeric matrix protein with other regions of collagen IX were found to be heparin-competitive. Circular dichroism analyses of the NC4 domain indicated the presence of stabilizing disulfide bonds and a thermal denaturation point of about 80degreesC. The pattern of disulfide bond formation within the NC4 domain was identified by tryptic peptide mass mapping of the NC4 in native and reduced states. A similar pattern was demonstrated for the NC4 domain of full-length recombinant collagen IX

A Survey of Protein Structures from Archaeal Viruses

Viruses that infect the third domain of life, Archaea, are a newly emerging field of interest. To date, all characterized archaeal viruses infect archaea that thrive in extreme conditions, such as halophilic, hyperthermophilic, and methanogenic environments. Viruses in general, especially those replicating in extreme environments, contain highly mosaic genomes with open reading frames (ORFs) whose sequences are often dissimilar to all other known ORFs. It has been estimated that approximately 85% of virally encoded ORFs do not match known sequences in the nucleic acid databases, and this percentage is even higher for archaeal viruses (typically 90%–100%). This statistic suggests that either virus genomes represent a larger segment of sequence space and/or that viruses encode genes of novel fold and/or function. Because the overall three-dimensional fold of a protein evolves more slowly than its sequence, efforts have been geared toward structural characterization of proteins encoded by archaeal viruses in order to gain insight into their potential functions. In this short review, we provide multiple examples where structural characterization of archaeal viral proteins has indeed provided significant functional and evolutionary insight