Cytoplasmic polyhedrosis virus (CPV) is unique within the Reoviridae family in having a turreted single-layer capsid contained within polyhedrin inclusion bodies, yet being fully capable of cell entry and endogenous RNA transcription. Biochemical data have shown that the amino-terminal 79 residues of the CPV turret protein (TP) is sufficient to bring CPV or engineered proteins into the polyhedrin matrix for micro-encapsulation. Here we report the three-dimensional structure of CPV at 3.88 A resolution using single-particle cryo-electron microscopy. Our map clearly shows the turns and deep grooves of alpha-helices, the strand separation in beta-sheets, and densities for loops and many bulky side chains; thus permitting atomic model-building effort from cryo-electron microscopy maps. We observed a helix-to-beta-hairpin conformational change between the two conformational states of the capsid shell protein in the region directly interacting with genomic RNA. We have also discovered a messenger RNA release hole coupled with the mRNA capping machinery unique to CPV. Furthermore, we have identified the polyhedrin-binding domain, a structure that has potential in nanobiotechnology applications.

Murine gammaherpesvirus 68 (MHV68 [also known as ) is distinguished by its ability to replicate to high titers in cultured cells, making it an excellent candidate for studying gammaherpesvirus virion composition. Extracellular MHV68 virions were isolated, and abundant virion-associated proteins were identified by mass spectrometry. Five nucleocapsid protein homologues, the tegument protein homologue encoded by open reading frame (ORF) 75c, and envelope glycoproteins B and H were detected. In addition, gene products from MHV68 ORF20, ORF24, ORF28, ORF45, ORF48, and ORF52 were identified in association with virions, suggesting that these gammaherpesvirus genes are involved in the early phase of infection or virion assembly and egress.The herpesvirus virion is composed of an icosahedral nucleocapsid surrounded by a proteinacious layer of tegument, which in turn is enclosed by a glycoprotein-containing lipid envelope (50). The structure and protein composition of the nucleocapsid have been shown to be conserved among the three subfamilies (␣Ϫ, ␤Ϫ, and ␥Ϫ) of herpesviruses (11, 14, 62-64, 72, 74). The icosahedral nucleocapsid contains at least four integral structural proteins (the major capsid protein, triplex-1 protein, triplex-2 protein, and small capsid protein) surrounding a core of viral DNA (11,14,27,42,56,62,72,76). The other components of the virion, the envelope and the tegument in particular, are less well understood (38). The envelope contains viral glycoproteins critical for virion binding, entry, and signaling upon infection of a host cell (4,15,26,34,55,67). The tegument is the electron-dense component of the virion surrounding the capsid and interacting with the envelope (14,38,75). While the tegument component of alphaherpesviruses and betaherpesviruses is known to contain a number of gene products involved in assembly and egress of infectious virus (38) or modulation of the host cell environment upon initial infection (10,13,21,25,30,40), little is known about the protein composition of the gammaherpesvirus tegument nor about the functions of gammaherpesvirus tegument proteins immediately after infection of the cell.Study of the functions of tegument proteins in the two human gammaherpesviruses, Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV), is hampered by the lack of cell culture systems capable of supporting productive replication of these viruses. However, murine gammaherpesvirus 68 (MHV68, or ␥HV-68) is not constrained in this manner, replicating to high titers in conventional tissue culture systems. MHV68 is a model for studying de novo gammaherpesvirus infection and pathogenesis (16,20,36,66,73). The virus is found in wild murid rodents and is capable of infecting laboratory strains of mice (8,39,48). MHV68 establishes productive infection in lung epithelia and a latent infection in splenocytes, macrophages, dendritic cells, and lung epithelial cells (23,48,57,61,69).The MHV68 virion exhibits morphological similarity to the virion organization of other gammaherpe...

Cystic fibrosis transmembrane conductance regulator (CFTR), a member of the ABC (ATP binding cassette) transporter family, is a chloride channel whose activity is controlled by protein kinase–dependent phosphorylation. Opening and closing (gating) of the phosphorylated CFTR is coupled to ATP binding and hydrolysis at CFTR's two nucleotide binding domains (NBD1 and NBD2). Recent studies present evidence that the open channel conformation reflects a head-to-tail dimerization of CFTR's two NBDs as seen in the NBDs of other ABC transporters (Vergani et al., 2005). Whether these two ATP binding sites play an equivalent role in the dynamics of NBD dimerization, and thus in gating CFTR channels, remains unsettled. Based on the crystal structures of NBDs, sequence alignment, and homology modeling, we have identified two critical aromatic amino acids (W401 in NBD1 and Y1219 in NBD2) that coordinate the adenine ring of the bound ATP. Conversion of the W401 residue to glycine (W401G) has little effect on the sensitivity of the opening rate to [ATP], but the same mutation at the Y1219 residue dramatically lowers the apparent affinity for ATP by >50-fold, suggesting distinct roles of these two ATP binding sites in channel opening. The W401G mutation, however, shortens the open time constant. Energetic analysis of our data suggests that the free energy of ATP binding at NBD1, but not at NBD2, contributes significantly to the energetics of the open state. This kinetic and energetic asymmetry of CFTR's two NBDs suggests an asymmetric motion of the NBDs during channel gating. Opening of the channel is initiated by ATP binding at the NBD2 site, whereas separation of the NBD dimer at the NBD1 site constitutes the rate-limiting step in channel closing.

Bluetongue virus (BTV) is transmitted by blood-feeding insects (Culicoides sp.) and causes hemorrhagic diseases in livestock. BTV is a nonenveloped, double-stranded RNA (dsRNA) virus with two capsids: a well-studied, stable core enclosing the dsRNA genome and a highly unstable, poorly studied coat responsible for host cell attachment and entry. Here, based on cryo-electron microscopy (cryoEM), we report a 7-Å resolution structure of the infectious BTV virion, including the coat proteins. We show that unlike other dsRNA viruses, the VP2 attachment trimer has a triskelion shape composed of three tip domains branching from a central hub domain. We identify three putative sialic acid-binding pockets in the hub and present supporting biochemical data indicating sugar moiety binding is important for BTV infection. Despite being a nonenveloped virus, the putative VP5 membrane penetration trimer, located slightly inward of the VP2 attachment trimer, has a central coiled-coil α-helical bundle, similar to the fusion proteins of many enveloped viruses (e.g., HIV, herpesviruses, vesicular stomatitis virus, and influenza virus). Moreover, mapping of the amino acid sequence of VP5 to the secondary structural elements identified by cryoEM locates 15 amphipathic α-helical regions on the external surface of each VP5 trimer. The cryoEM density map also reveals few, weak interactions between the VP5 trimer and both the outer-coat VP2 trimer and the underlying core VP7 trimer, suggesting that the surface of VP5 could unfurl like an umbrella during penetration and shedding of the coat to release the transcriptionally active core particle.cryo-electron microscopy | dsRNA virus structure | membrane penetration protein | sialic acid-binding protein B luetongue virus (BTV) is a segmented double-stranded RNA (dsRNA) virus in the Orbivirus genus of the Reoviridae family. It infects both ruminants and blood-feeding insects of the Culicoides genus that vector the virus between ruminant hosts. BTV has recently emerged in European countries with severe economic consequences (1, 2), possibly due to climate change and the increased distribution of insect vectors (3).The virus has four major structural proteins, two (VP2 and VP5) in the coat and two (VP3 and VP7) in the core. The virus also contains an RNA polymerase (4), a helicase (5), an mRNA capping enzyme (6), and the genome composed of 10 linear dsRNA molecules (7). In contrast to other members of the Reoviridae family, the coat of BTV is highly fragile. Upon entry into the cytoplasm, the unstable BTV coat is shed to release a stable core particle. High-resolution structures (3.5 Å) (8) are therefore available for the two core proteins, but only low-resolution structures (24 Å) (9) exist for the proteins that make up the unstable coat and that mediate attachment (10) and entry (11). Although the low-resolution structure places coat proteins VP2 and VP5 in sites consistent with their functions-VP2 (attachment) protruding outward and VP5 (membrane penetration) in a slightly more inward locat...

The three-dimensional structure of the intact human cytomegalovirus (HCMV) was determined to 18-A resolution by electron cryomicroscopy and computer reconstruction. Its capsid shell is composed of pentons, hexons, and triplexes arranged on a T = 16 icosahedral lattice and is identical to that of the B-capsid isolated from host cell nuclei. An icosahedrally ordered tegument layer formed by 960 copies of filamentous density is also visualized, which interacts with the pentons, hexons, and triplexes of the underlying capsid. The observed structural similarities and differences of HCMV with those of herpes simplex virus offer insights into the significance of the different tegument components for their infection processes while maintaining similar capsids.

SummaryGrass carp reovirus (GCRV) is a member of the Aquareovirus genus of the family Reoviridae, a large family of dsRNA viruses infecting plants, insects, fishes and mammals. We report the first subnanometer-resolution three-dimensional (3D) structures of both GCRV core and virion by cryoelectron microscopy (cryoEM). These structures have allowed the delineation of interactions among the over 1000 molecules in this enormous macromolecular machine, and a detail comparison with other dsRNA viruses at the secondary structure level. The GCRV core structure shows that the inner proteins have strong structural similarities even at the level of secondary structure elements with those of orthoreoviruses, indicating that the structures involved in viral dsRNA interaction and transcription are highly conserved. In contrast, the level of similarity in structures decreases in the proteins situated in the outer layers of the virion. The proteins involved in host recognition and attachment exhibit the least similarities to other members of Reoviridae. Furthermore, in GCRV, the RNA-translocating turrets are in an open state and lack a counterpart for the σ1 protein situated on top of the close turrets observed in mammalian orthoreovirus (MRV). Interestingly, the distribution and organization of GCRV core proteins resembles those of the cytoplasmic polyhedrosis virus (CPV), a cypovirus and the structurally simplest member of the Reoviridae family. Our results suggest that GCRV occupies a unique structure niche between the simpler cypoviruses and the considerably more complex MRV, thus providing an important model for understanding the structural and functional conservation and diversity of this enormous family of dsRNA viruses.

The cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel regulated by cAMP-dependent phosphorylation, which is expressed in epithelial cells of a wide variety of tissues including the reproductive tracts. Mutations in the gene encoding CFTR cause cystic fibrosis, a common genetic disease in Caucasian populations with a multitude of clinical manifestations including infertility/subfertility in both sexes. However, the physiological role of CFTR in reproduction and its involvement in the pathogenesis of reproductive diseases remain largely unknown. This review discusses the role of CFTR in regulating fluid volume and bicarbonate secretion in the reproductive tracts and their importance in various reproductive events. We also discuss the contribution of CFTR dysfunction to a number of pathological conditions. The evidence presented is consistent with an important role of CFTR in reproductive health and disease, suggesting that CFTR might be a potential target for the diagnosis and treatment of reproductive diseases including infertility.

Copper formulations have been used for decades for antimicrobial and antifouling applications. With the development of nano-formulations of copper that are more effective than their ionic and micron-sized analogs, a key regulatory question is whether these materials should be treated as new or existing materials. To address this issue, here we compare the magnitude and mechanisms of toxicity of a series of Cu species (at concentration ranging from 2–250 µg/mL), including nano Cu, nano CuO, nano Cu(OH)2 (CuPro and Kocide), micro Cu, micro CuO, ionic Cu2+ (CuCl2 and CuSO4) in two species of bacteria (Escherichia coli and Lactobacillus brevis). The primary size of the particles studied ranged from 10 nm to 10 µm. Our results reveal that Cu and CuO nanoparticles (NPs) are more toxic than their micron-sized counterparts at the same Cu concentration, with toxicities approaching that of the ionic Cu species. Strikingly, these NPs showed distinct differences in their mode of toxicity when compared to the ionic and micron-sized Cu, highlighting the unique toxicity properties of materials at the nanoscale. In vitro DNA damage assays reveal that both nano Cu and micron-sized Cu are capable of causing complete degradation of plasmid DNA but electron tomography results show that only nanoformulations of Cu are internalized as intact intracellular particles. These studies suggest that nano Cu at the concentration of 50 µg/mL may have unique genotoxicity in bacteria compared to ionic and micron-sized Cu.