FAP57/WDR65 targets assembly of a subset of inner arm dyneins and connects to regulatory hubs in cilia.

Ciliary motility depends on both the precise spatial organization of multiple dynein motors within the 96 nm axonemal repeat and the highly coordinated interactions between different dyneins and regulatory complexes located at the base of the radial spokes. Mutations in genes encoding cytoplasmic assembly factors, intraflagellar transport factors, docking proteins, dynein subunits, and associated regulatory proteins can all lead to defects in dynein assembly and ciliary motility. Significant progress has been made in the identification of dynein subunits and extrinsic factors required for preassembly of dynein complexes in the cytoplasm, but less is known about the docking factors that specify the unique binding sites for the different dynein isoforms on the surface of the doublet microtubules. We have used insertional mutagenesis to identify a new locus, IDA8/BOP2, required for targeting the assembly of a subset of inner dynein arms (IDAs) to a specific location in the 96 nm repeat. IDA8 encodes flagellar-associated polypeptide (FAP)57/WDR65, a highly conserved WD repeat, coiled coil domain protein. Using high resolution proteomic and structural approaches, we find that FAP57 forms a discrete complex. Cryo-electron tomography coupled with epitope tagging and gold labeling reveal that FAP57 forms an extended structure that interconnects multiple IDAs and regulatory complexes.

Induction of influenza-specific mucosal immunity by an attenuated recombinant Sendai virus.

BACKGROUND: Many pathogens initiate infection at the mucosal surfaces; therefore, induction of mucosal immune responses is a first level of defense against infection and is the most powerful means of protection. Although intramuscular injection is widely used for vaccination and is effective at inducing circulating antibodies, it is less effective at inducing mucosal antibodies. METHODOLOGY/PRINCIPAL FINDINGS: Here we report a novel recombinant, attenuated Sendai virus vector (GP42-H1) in which the hemagglutinin (HA) gene of influenza A virus was introduced into the Sendai virus genome as an additional gene. Infection of CV-1 cells by GP42-H1 resulted in cell surface expression of the HA protein. Intranasal immunization of mice with 1,000 plaque forming units (pfu) of GP42-H1 induced HA-specific IgG and IgA antibodies in the blood, bronchoalveolar lavage fluid, fecal pellet extracts and saliva. The HA-specific antibody titer induced by GP42-H1 closely resembles the titer induced by sublethal infection by live influenza virus; however, in contrast to infection by influenza virus, immunization with GP42-H1 did not result in disease symptoms or the loss of body weight. In mice that were immunized with GP42-H1 and then challenged with 5LD(50) (1250 pfu) of influenza virus, no significant weight loss was observed and other visual signs of morbidity were not detected. CONCLUSIONS: These results demonstrate that the GP42-H1 Sendai virus recombinant is able to confer full protection from lethal infection by influenza virus, supporting the conclusion that it is a safe and effective mucosal vaccine vector.

Inhibition of the catalytic function of activation-induced cytidine deaminase promotes apoptosis of germinal center B cells in BXD2 mice.

OBJECTIVE: To determine whether functional suppression of the catalytic domain of activation-induced cytidine deaminase (AID) can suppress the hyperreactive germinal center (GC) responses in BXD2 mice. METHODS: We generated transgenic BXD2 mice expressing a dominant-negative (DN) form of Aicda at the somatic hypermutation site (BXD2-Aicda-DN-transgenic mice). Real-time quantitative reverse transcriptase-polymerase chain reaction was used to determine the expression of Aicda and DNA damage/repair genes. Enzyme-linked immunosorbent assay was used to measure serum levels of autoantibodies and immune complexes (ICs). Development of GCs and antibody-containing ICs as well as numbers of proliferative and apoptotic cells were determined using flow cytometry and/or immunohistochemical analyses. Development of arthritis and kidney disease was evaluated histologically in 6-8-month-old mice. RESULTS: Suppression of the somatic hypermutation function of AID resulted in a significant decrease in autoantibody production without affecting the expression of DNA damage-related genes in GC B cells of BXD2-Aicda-DN-transgenic mice. There was decreased proliferation, increased apoptosis, increased expression of caspase 9 messenger RNA in GC B cells, and lower numbers of GCs in the spleens of BXD2-Aicda-DN-transgenic mice. Decreased GC response was associated with lower levels of IgG-containing ICs. Anti-IgM- and anti-CD40 plus anti-Ig-induced B cell proliferative responses were decreased in BXD2-Aicda-DN-transgenic mice. CONCLUSION: Inhibition of the AID somatic hypermutation function in BXD2 mice suppressed development of spontaneous GCs, generation of autoantibody-producing B cells, and autoimmunity in BXD2 mice. Suppression of AID catalytic function to limit selection-based survival of GC B cells could become a novel therapy for the treatment of autoimmune disease.

Intraclonal competition inhibits the formation of high-affinity antibody-secreting cells.

Protective immunity requires a diverse, polyclonal B cell repertoire. We demonstrate that affinity maturation of the humoral response to a hapten is impaired when preexisting clonally restricted cells recognizing the hapten are dominant in the B cell repertoire. B1-8i(+/-) mice, which feature a high frequency of B cells with nitrophenyl (NP)-binding specificity, respond to NP-haptenated proteins with the production of NP-specific Abs, but affinity maturation is impaired due to insufficient generation of high-affinity Ab-producing cells. We manipulated the frequency of NP-specific B cells by adoptive transfer of B1-8 B cells into naive, wild-type recipients. Remarkably, when 10(4) B1-8 B cells were transferred, these cells supported efficient affinity maturation and plasma cell differentiation. In contrast, when 10(6) B1-8 cells were transferred, affinity maturation did not occur. These data indicate that restricting the frequency of clonally related B cells is required to support affinity maturation.

Interleukin 17-producing T helper cells and interleukin 17 orchestrate autoreactive germinal center development in autoimmune BXD2 mice.

Interleukin 17 (IL-17) is a cytokine associated with inflammation, autoimmunity and defense against some bacteria. Here we show that IL-17 can promote autoimmune disease through a mechanism distinct from its proinflammatory effects. As compared with wild-type mice, autoimmune BXD2 mice express more IL-17 and show spontaneous development of germinal centers (GCs) before they increase production of pathogenic autoantibodies. We show that blocking IL-17 signaling disrupts CD4+ T cell and B cell interactions required for the formation of GCs and that mice lacking the IL-17 receptor have reduced GC B cell development and humoral responses. Production of IL-17 correlates with upregulated expression of the genes Rgs13 and Rgs16, which encode regulators of G-protein signaling, and results in suppression of the B cell chemotactic response to the chemokine CXCL12. These findings suggest a mechanism by which IL-17 drives autoimmune responses by promoting the formation of spontaneous GCs.

Nodavirus coat protein imposes dodecahedral RNA structure independent of nucleotide sequence and length.

The nodavirus Flock house virus (FHV) has a bipartite, positive-sense RNA genome that is packaged into an icosahedral particle displaying T=3 symmetry. The high-resolution X-ray structure of FHV has shown that 10 bp of well-ordered, double-stranded RNA are located at each of the 30 twofold axes of the virion, but it is not known which portions of the genome form these duplex regions. The regular distribution of double-stranded RNA in the interior of the virus particle indicates that large regions of the encapsidated genome are engaged in secondary structure interactions. Moreover, the RNA is restricted to a topology that is unlikely to exist during translation or replication. We used electron cryomicroscopy and image reconstruction to determine the structure of four types of FHV particles that differed in RNA and protein content. RNA-capsid interactions were primarily mediated via the N and C termini, which are essential for RNA recognition and particle assembly. A substantial fraction of the packaged nucleic acid, either viral or heterologous, was organized as a dodecahedral cage of duplex RNA. The similarity in tertiary structure suggests that RNA folding is independent of sequence and length. Computational modeling indicated that RNA duplex formation involves both short-range and long-range interactions. We propose that the capsid protein is able to exploit the plasticity of the RNA secondary structures, capturing those that are compatible with the geometry of the dodecahedral cage.

Packaging double-helical DNA into viral capsids.

DNA packaging in bacteriophage P4 has been examined using a molecular mechanics model with a reduced representation containing one pseudoatom per turn of the double helix. The model is a discretized version of an elastic continuum model. The DNA is inserted piecewise into the model capsid, with the structure being reoptimized after each piece is inserted. Various optimization protocols were investigated, and it was found that molecular dynamics at a very low temperature (0.3 K) produces the optimal packaged structure. This structure is a concentric spool, rather than the coaxial spool that has been commonly accepted for so many years. This geometry, which was originally suggested by Hall and Schellman in 1982 (Biopolymers Vol. 21, pp. 2011-2031), produces a lower overall elastic energy than coaxial spooling.