Purification and visualization of influenza a viral ribonucleoprotein complexes.

The influenza A viral genome consists of eight negative-sense, single stranded RNA molecules, individually packed with multiple copies of the influenza A nucleoprotein (NP) into viral ribonulceoprotein particles (vRNPs). The influenza vRNPs are enclosed within the viral envelope. During cell entry, however, these vRNP complexes are released into the cytoplasm, where they gain access to the host nuclear transport machinery. In order to study the nuclear import of influenza vRNPs and the replication of the influenza genome, it is useful to work with isolated vRNPs so that other components of the virus do not interfere with these processes. Here, we describe a procedure to purify these vRNPs from the influenza A virus. The procedure starts with the disruption of the influenza A virion with detergents in order to release the vRNP complexes from the enveloped virion. The vRNPs are then separated from the other components of the influenza A virion on a 33-70% discontinuous glycerol gradient by velocity sedimentation. The fractions obtained from the glycerol gradient are then analyzed on via SDS-PAGE after staining with Coomassie blue. The peak fractions containing NP are then pooled together and concentrated by centrifugation. After concentration, the integrity of the vRNPs is verified by visualization of the vRNPs by transmission electron microscopy after negative staining. The glycerol gradient purification is a modification of that from Kemler et al. (1994)(1), and the negative staining has been performed by Wu et al. (2007).(2).

Characterization and purification of the discoidin domain-containing protein retinoschisin and its interaction with galactose.

RS1, also known as retinoschisin, is an extracellular discoidin domain-containing protein that has been implicated in maintaining the cellular organization and synaptic structure of the vertebrate retina. Mutations in the gene encoding RS1 are responsible for X-linked retinoschisis, a retinal degenerative disease characterized by the splitting of the retinal cell layers and visual impairment. To better understand the role of RS1 in retinal cell biology and X-linked retinoschisis, we have studied the interaction of wild-type and mutant RS1 with various carbohydrates coupled to agarose supports. RS1 bound efficiently to galactose-agarose and to a lesser extent lactose-agarose, but not agarose, N-acetylgalactosamine-agarose, N-acetylglucosamine-agarose, mannose-agarose, or heparin-agarose. RS1 cysteine mutants (C59S/C223S and C59S/C223S/C40S) which prevent disulfide-linked octamer formation exhibited little if any binding to galactose-agarose. The disease-causing R141H mutant bound galactose-agarose at levels similar to that of wild-type RS1, whereas the R141S mutant resulted in a marked reduction in the level of galactose-agarose binding. RS1 bound to galactose-agarose could be effectively displaced by incubation with isopropyl beta- d-1-thiogalactopyranoside (IPTG). This property was used as a basis to develop an efficient purification procedure. Anion exchange and galactose affinity chromatography was used to purify RS1 from the culture media of stably transformed Sf21 insect cells that express and secrete RS1. This cell expression and protein purification method should prove useful in the isolation of RS1 for detailed structure-function studies.

Ultrastructural analysis of the nuclear localization sequences on influenza A ribonucleoprotein complexes.

The influenza A genome consists of eight single-stranded RNA molecules, each associated with an oligomeric core of the structural protein, nucleoprotein, to form a distinct viral ribonucleoprotein (vRNP) complex. vRNPs are the entities responsible for the transcription and replication of the influenza viral RNAs in the nuclei of host cells. Thus, nuclear targeting and localization of the vRNPs are a critical step in the infection and life cycle of influenza A. We have recently shown that the nuclear import of vRNPs derived from influenza A virions is independently mediated by two nuclear localization sequences (NLSs) on nucleoprotein: NLS1, spanning residues 1-13 at the N terminus, and NLS2, spanning residues 198-216 in the middle of the protein, with NLS1 being the principal mediator. To better understand the structural basis for the differences in the ability of NLS1 and that of NLS2 to mediate nuclear import of influenza vRNPs, we analyzed the levels of surface exposure of these NLSs on vRNPs by both dot blotting and immunogold labeling of vRNPs in their native state. We found that NLS1 is much more accessible to its corresponding antibody compared with NLS2. Electron microscopy of immunogold-labeled vRNPs further showed that 71% of vRNPs were labeled with one to six gold particles located throughout the vRNP for NLS1. In contrast, less than 10% of vRNPs were labeled with an antibody against NLS2, usually with a single gold particle located at one end of the vRNP. In addition, a regular periodicity of repeat was observed with gold particles labeling for NLS1, indicative of a highly regular helical conformation present in the vRNPs. These findings provide the underlying structural basis for the enhanced ability of NLS1 in mediating nuclear import of influenza vRNPs and add to our understanding of the ultrastructural features of vRNP complexes derived from influenza A virions.

Retinoschisin (RS1), the protein encoded by the X-linked retinoschisis gene, is anchored to the surface of retinal photoreceptor and bipolar cells through its interactions with a Na/K ATPase-SARM1 complex.

Retinoschisin or RS1 is a discoidin domain-containing protein encoded by the gene responsible for X-linked retinoschisis (XLRS), an early onset macular degeneration characterized by a splitting of the retina. Retinoschisin, expressed and secreted from photoreceptors and bipolar cells as a homo-octameric complex, associates with the surface of these cells where it serves to maintain the cellular organization of the retina and the photoreceptor-bipolar synaptic structure. To gain insight into the role of retinoschisin in retinal cell adhesion and the pathogenesis of XLRS, we have investigated membrane components in retinal extracts that interact with retinoschisin. Unlike the discoidin domain-containing blood coagulation proteins Factor V and Factor VIII, retinoschisin did not bind to phospholipids or retinal lipids reconstituted into unilamellar vesicles or immobilized on microtiter plates. Instead, co-immunoprecipitation studies together with mass spectrometric-based proteomics and Western blotting showed that retinoschisin is associated with a complex consisting of Na/K ATPase (alpha3, beta2 isoforms) and the sterile alpha and TIR motif-containing protein SARM1. Double labeling studies for immunofluorescence microscopy confirmed the co-localization of retinoschisin with Na/K ATPase and SARM1 in photoreceptors and bipolar cells of retina tissue. We conclude that retinoschisin binds to Na/K ATPase on photoreceptor and bipolar cells. This interaction may be part of a novel SARM1-mediated cell signaling pathway required for the maintenance of retinal cell organization and photoreceptor-bipolar synaptic structure.

Nuclear import of influenza A viral ribonucleoprotein complexes is mediated by two nuclear localization sequences on viral nucleoprotein.

BACKGROUND: The influenza A virus replicates in the nucleus of its host cell. Thus, entry of the influenza genome into the cell nucleus is necessary for establishing infection. The genome of the influenza A virus consists of eight single-stranded, negative-sense RNA molecules, individually packed with several copies of the viral nucleoprotein (NP) into ribonucleoprotein particles (vRNPs). These vRNPs are large, rod-shaped complexes containing a core of NP, around which the RNA is helically wrapped. The vRNPs are the entities that enter the nucleus, and their nuclear import must be mediated by nuclear localization sequences (NLSs) exposed on the vRNPs. NP contains at least two putative NLSs, one at the N-terminus (NLS1) and one in the middle (NLS2) of the protein. These NP NLSs have been shown to mediate the nuclear import of recombinant NP molecules. However, it remains to be determined which NLS mediates the nuclear import of influenza vRNP complexes. RESULTS: To directly track the nuclear import of the influenza A genome, we developed an experimental assay based on digitonin-permeabilized cells and fluorescently-labeled vRNPs isolated from the influenza A virus. We used this assay to determine the contribution of the two proposed NLSs on NP to the nuclear import of influenza vRNP complexes. Peptides that mimic each of the two NLSs on NP were used to compete with vRNPs for their nuclear import receptors. In addition, antibodies against the two NP NLSs were used to block the NLSs on the vRNP complexes, and thereby inhibit vRNP nuclear import. Both peptide competition and antibody inhibition of either sequence resulted in decreased nuclear accumulation of vRNPs. The two sequences act independently of each other, as inhibition of only one of the two NLSs still resulted in significant, though diminished, nuclear import of vRNPs. Furthermore, when both sequences were blocked, vRNP nuclear import was almost completely inhibited. Antibody inhibition studies further showed that NLS1 on NP is the main contributor to the nuclear import of vRNPs. CONCLUSION: Our results demonstrate that both NLS1 and NLS2 on NP can mediate the nuclear uptake of influenza A vRNPs.

RS1, a discoidin domain-containing retinal cell adhesion protein associated with X-linked retinoschisis, exists as a novel disulfide-linked octamer.

RS1, also known as retinoschisin, is an extracellular protein that plays a crucial role in the cellular organization of the retina. Mutations in RS1 are responsible for X-linked retinoschisis, a common, early-onset macular degeneration in males that results in a splitting of the inner layers of the retina and severe loss in vision. RS1 is assembled and secreted from photoreceptors and bipolar cells as a homo-oligomeric protein complex. Each subunit consists of a 157-amino acid discoidin domain flanked by two small segments of 39 and 5 amino acids. To begin to understand how the structure of RS1 relates to its role in retinal cell adhesion and X-linked retinoschisis, we have determined the subunit organization and disulfide bonding pattern of RS1 by SDS gel electrophoresis, velocity sedimentation, and mass spectrometry. Our results indicate that RS1 exists as a novel octamer in which the eight subunits are joined together by Cys(59)-Cys(223) intermolecular disulfide bonds. Subunits within the octamer are further organized into dimers mediated by Cys(40)-Cys(40) bonds. These cysteines lie just outside the discoidin domain indicating that these flanking segments primarily function in the octamerization of RS1. Within the discoidin domain, two cysteine pairs (Cys(63)-Cys(219) and Cys(110)-Cys(142)) form intramolecular disulfide bonds that are important in protein folding, and one cysteine (Cys(83)) exists in its reduced state. Because mutations that disrupt subunit assembly cause X-linked retinoschisis, the assembly of RS1 into a disulfide-linked homo-octamer appears to be critical for its function as a retinal cell adhesion protein.

Defective discoidin domain structure, subunit assembly, and endoplasmic reticulum processing of retinoschisin are primary mechanisms responsible for X-linked retinoschisis.

Retinoschisin is a 24-kDa discoidin domain-containing protein that is secreted from photoreceptor and bipolar cells as a large disulfide-linked multisubunit complex. It functions as a cell adhesion protein to maintain the cellular organization and synaptic structure of the retina. Over 125 different mutations in the RS1 gene are associated with X-linked juvenile retinoschisis, the most common form of early onset macular degeneration in males. To identify molecular determinants important for retinoschisin structure and function and elucidate molecular and cellular mechanisms responsible for X-linked juvenile retinoschisis, we have analyzed the expression, protein folding, disulfide-linked subunit assembly, intracellular localization, and secretion of wild-type retinoschisin, 15 Cys-to-Ser variants and 12 disease-linked mutants. Our studies, together with molecular modeling of the discoidin domain, identify Cys residues involved in intramolecular and intermolecular disulfide bonds essential for protein folding and subunit assembly. We show that misfolding of the discoidin domain, defective disulfide-linked subunit assembly, and inability of retinoschisin to insert into the endoplasmic reticulum membrane as part of the protein secretion process are three primary mechanisms responsible for the loss in the function of retinoschisin as a cell adhesion protein and the pathogenesis of X-linked juvenile retinoschisis.