Preliminary Evaluation of a Bunyavirus Vector for Cancer Immunotherapy

Replicon particles of Rift Valley fever virus, referred to as nonspreading Rift Valley fever virus (NSR), are intrinsically safe and highly immunogenic. Here, we demonstrate that NSR-infected human dendritic cells can activate CD8⁺ T cells in vitro and that prophylactic and therapeutic vaccinations of mice with NSR encoding a tumor-associated CD8 peptide can control the outgrowth of lymphoma cells in vivo. These results suggest that the NSR system holds promise for cancer immunotherapy.     

Identification of the potential active site of the septal peptidoglycan polymerase FtsW

SEDS (Shape, Elongation, Division and Sporulation) proteins are widely conserved peptidoglycan (PG) glycosyltransferases that form complexes with class B penicillin-binding proteins (bPBPs, with transpeptidase activity) to synthesize PG during bacterial cell growth and division. Because of their crucial roles in bacterial morphogenesis, SEDS proteins are one of the most promising targets for the development of new antibiotics. However, how SEDS proteins recognize their substrate lipid II, the building block of the PG layer, and polymerize it into glycan strands is still not clear. In this study, we isolated and characterized dominant-negative alleles of FtsW, a SEDS protein critical for septal PG synthesis during bacterial cytokinesis. Interestingly, most of the dominant-negative FtsW mutations reside in extracellular loops that are highly conserved in the SEDS family. Moreover, these mutations are scattered around a central cavity in a modeled FtsW structure, which has been proposed to be the active site of SEDS proteins. Consistent with this, we found that these mutations blocked septal PG synthesis but did not affect FtsW localization to the division site, interaction with its partners nor its substrate lipid II. Taken together, these results suggest that the residues corresponding to the dominant-negative mutations likely constitute the active site of FtsW, which may aid in the design of FtsW inhibitors.     

The actin-based motor protein myosin II regulates MHC class II trafficking and BCR-driven antigen presentation

Antigen (Ag) capture and presentation onto major histocompatibility complex (MHC) class II molecules by B lymphocytes is mediated by their surface Ag receptor (B cell receptor [BCR]). Therefore, the transport of vesicles that carry MHC class II and BCR-Ag complexes must be coordinated for them to converge for processing. In this study, we identify the actin-associated motor protein myosin II as being essential for this process. Myosin II is activated upon BCR engagement and associates with MHC class II-invariant chain complexes. Myosin II inhibition or depletion compromises the convergence and concentration of MHC class II and BCR-Ag complexes into lysosomes devoted to Ag processing. Accordingly, the formation of MHC class II-peptides and subsequent CD4 T cell activation are impaired in cells lacking myosin II activity. Therefore, myosin II emerges as a key motor protein in BCR-driven Ag processing and presentation.     

Production of IGF-binding proteins by vascular endothelial cells

Conditioned serum-free media from cultured human, bovine and rodent endothelial cells contained binding proteins with high affinity for the insulin-like growth factors (IGFs). After partial purifications on heparin or Multiplication Stimulating Activity (MSA)-affinity columns, 2 species of binding protein were identified, a major protein having Mr approximately 35,000 and a minor 22-28,000 protein. The binding proteins had greater affinity for IGF-I than IGF-II with no affinity for insulin or proinsulin. Substantial amounts of the binding proteins remained cell-associated, loosely bound to the outer cell surface of the endothelial cell. Binding protein(s) from human endothelial cells cross-reacted with antibodies to the 53,000 dalton acid-stable human serum binding protein. Production of endothelial binding proteins was not stimulated by growth hormone or insulin. We conclude that endothelial cells in culture produce large quantities of specific IGF binding proteins. Such binding proteins should be relevant in understanding the complex metabolism and function of the IGFs in the intact host.     

The bacterial cell division protein fragment EFtsN binds to and activates the major peptidoglycan synthase PBP1b

Peptidoglycan (PG) is an essential constituent of the bacterial cell wall. During cell division, the machinery responsible for PG synthesis localizes mid-cell, at the septum, under the control of a multiprotein complex called the divisome. In Escherichia coli, septal PG synthesis and cell constriction rely on the accumulation of FtsN at the division site. Interestingly, a short sequence of FtsN (Leu⁷⁵–Gln⁹³, known as ^EFtsN) was shown to be essential and sufficient for its functioning in vivo, but what exactly this sequence is doing remained unknown. Here, we show that ^EFtsN binds specifically to the major PG synthase PBP1b and is sufficient to stimulate its biosynthetic glycosyltransferase (GTase) activity. We also report the crystal structure of PBP1b in complex with ^EFtsN, which demonstrates that ^EFtsN binds at the junction between the GTase and UB2H domains of PBP1b. Interestingly, mutations to two residues (R141A/R397A) within the ^EFtsN-binding pocket reduced the activation of PBP1b by FtsN but not by the lipoprotein LpoB. This mutant was unable to rescue the ΔponB-ponA^ts strain, which lacks PBP1b and has a thermosensitive PBP1a, at nonpermissive temperature and induced a mild cell-chaining phenotype and cell lysis. Altogether, the results show that ^EFtsN interacts with PBP1b and that this interaction plays a role in the activation of its GTase activity by FtsN, which may contribute to the overall septal PG synthesis and regulation during cell division.     

Membrane specializations and endosome maturation in dendritic cells and B cells

Interest in the cell biology of antigen presentation is centered on dendritic cells (DCs) as initiators of the immune response. The ability to examine primary antigen-presenting cells, as opposed to cell lines, has opened a new window for study of antigen processing and peptide acquisition by Class II major histocompatibility complex (MHC) products, especially where intracellular trafficking of peptide-Class-II complexes is concerned. Here, we review the dynamics of Class II MHC-positive intracellular structures in dendritic cells as well as B cells. We focus on the generation of multivesicular bodies, where Class II MHC products acquire antigenic peptide, on the endosomal transport of peptide-loaded Class II MHC to the cell surface and on the importance of Class II MHC localization in membrane microdomains.     

AID-/-mus-/- mice are agammaglobulinemic and fail to maintain B220-CD138+ plasma cells

The terminal stage of B cell differentiation culminates in the formation of plasma cells (PC), which secrete large quantities of Igs. Despite recent progress in understanding the molecular aspect of PC differentiation and maintenance, the requirement for the synthesis of secretory Igs as a contributing factor has not been explored. To address this issue, we generated activation-induced cytidine deaminase (AID)/secretory mu-chain (mus) double-knockout mice, in which a normally diverse repertoire of B cell receptors is retained, yet B cells are unable to synthesize secretory Igs. These mice possess polyclonal B cells but have no serum Igs. Following immunization in vivo, PCs, identified by CD138 expression and loss of the B220 marker, were starkly reduced in number in spleen and bone marrow of AID(-/-)mus(-/-) agammaglobulinemic mice compared with wild-type mice. Upon mitogenic stimulation in vitro, AID(-/-)mus(-/-) B cells differentiated into plasmablasts to some extent, but showed reduced survival compared with wild-type B cells. We found no evidence that this reduced survival was attributable to accumulation of membrane IgM. Our results indicate that the synthesis of secretory Igs is a requirement for maintenance of B220(-)CD138(+) PCs.     

The mimivirus genome encodes a mitochondrial carrier that transports dATP and dTTP

Members of the mitochondrial carrier family have been reported in eukaryotes only, where they transport metabolites and cofactors across the mitochondrial inner membrane to link the metabolic pathways of the cytosol and the matrix. The genome of the giant virus Mimiviridae mimivirus encodes a member of the mitochondrial carrier family of transport proteins. This viral protein has been expressed in Lactococcus lactis and is shown to transport dATP and dTTP. As the 1.2-Mb double-stranded DNA mimivirus genome is rich in A and T residues, we speculate that the virus is using this protein to target the host mitochondria as a source of deoxynucleotides for its replication.