pH-dependent entry of severe acute respiratory syndrome coronavirus is mediated by the spike glycoprotein and enhanced by dendritic cell transfer through DC-SIGN

The severe acute respiratory syndrome coronavirus (SARS-CoV) synthesizes several putative viral envelope proteins, including the spike (S), membrane (M), and small envelope (E) glycoproteins. Although these proteins likely are essential for viral replication, their specific roles in SARS-CoV entry have not been defined. In this report, we show that the SARS-CoV S glycoprotein mediates viral entry through pH-dependent endocytosis. Further, we define its cellular tropism and demonstrate that virus transmission occurs through cell-mediated transfer by dendritic cells. The S glycoprotein was used successfully to pseudotype replication-defective retroviral and lentiviral vectors that readily infected Vero cells as well as primary pulmonary and renal epithelial cells from human, nonhuman primate, and, to a lesser extent, feline species. The tropism of this reporter virus was similar to that of wild-type, replication-competent SARS-CoV, and binding of purified S to susceptible target cells was demonstrated by flow cytometry. Although myeloid dendritic cells were able to interact with S and to bind virus, these cells could not be infected by SARS-CoV. However, these cells were able to transfer the virus to susceptible target cells through a synapse-like structure. Both cell-mediated infection and direct infection were inhibited by anti-S antisera, indicating that strategies directed toward this gene product are likely to confer a therapeutic benefit for antiviral drugs or the development of a SARS vaccine.     

Rudhira/BCAS3 is a cytoskeletal protein that controls Cdc42 activation and directional cell migration during angiogenesis

Cell migration is a common cellular process in angiogenesis and tumor metastasis. Rudhira/BCAS3 (Breast Cancer Amplified Sequence 3) is a conserved protein expressed in the embryonic vasculature and malignant tumors. Here, we show for the first time that Rudhira plays an active role in directional cell migration. Rudhira depletion in endothelial cells inhibits Matrigel-induced tube formation and retards healing of wounded cell monolayers. We demonstrate that during wound healing, Rudhira rapidly re-localizes and promotes Cdc42 activation and recruitment to the leading edge of migrating cells. Rudhira deficient cells show impaired downstream signaling of Cdc42 leading to dramatic changes in actin organization and classic cell polarity defects such as loss of microtubule organizing center (MTOC) and Golgi re-orientation. Biochemical assays and co-localization studies show that Rudhira interacts with microtubules as well as intermediate filaments. Thus, Rudhira could control directional cell migration and angiogenesis by facilitating crosstalk between cytoskeletal elements.     

A novel group of negative-sense RNA viruses associated with epizootics in managed and free-ranging freshwater turtles in Florida,USA

Few aquatic animal negative-sense RNA viruses have been characterized, and their role in disease is poorly understood. Here, we describe a virus isolated from diseased freshwater turtles from a Florida farm in 2007 and from an ongoing epizootic among free-ranging populations of Florida softshell turtles (Apalone ferox), Florida red-bellied cooters (Pseudemys nelsoni), and peninsula cooters (Pseudemys peninsularis). Affected turtles presented with similar neurological signs, oral and genital ulceration, and secondary microbial infections. Microscopic lesions were most severe in the softshell turtles and included heterophilic/histiocytic meningoencephalitis, multi-organ vasculitis, and cytologic observation of leukocytic intracytoplasmic inclusions. The virus was isolated using Terrapene heart (TH-1) cells. Ultrastructurally, viral particles were round to pleomorphic and acquired an envelope with prominent surface projections by budding from the cell membrane. Viral genomes were sequenced from cDNA libraries of two nearly identical isolates and determined to be bi-segmented, with an ambisense coding arrangement. The larger segment encodes a predicted RNA-directed RNA polymerase (RdRP) and a putative zinc-binding matrix protein. The smaller segment encodes a putative nucleoprotein and an envelope glycoprotein precursor (GPC). Thus, the genome organization of this turtle virus resembles that of arenaviruses. Phylogenetic analysis shows that the RdRP of the turtle virus is highly diverged from the RdRPs of all known negative-sense RNA viruses and forms a deep branch within the phylum Negarnaviricota, that is not affiliated with any known group of viruses, even at the class level. In contrast, the GPC protein of the turtle virus is confidently affiliated with homologs from a distinct group of fish hantaviruses. Thus, the turtle virus is expected to become the founder of a new taxon of negative-sense RNA viruses, at least with a family rank, but likely, an order or even a class. These viruses probably evolved either by reassortment or by intrasegment recombination between a virus from a distinct branch of negarnaviruses distant from all known groups and a hanta-like aquatic virus. We suggest the provisional name Tosoviridae for the putative new family, with Turtle fraservirus 1 (TFV1) as the type species within the genus Fraservirus. A conventional RT-PCR assay, targeting the TFV1 RdRP, confirmed the presence of viral RNA in multiple tissues and exudates from diseased turtles. The systemic nature of the TFV1 infection was further supported by labeling of cells within lesions using in situ hybridization targeting the RNA of the TFV1 RdRP.     

Prenatal alcohol exposure and early postnatal changes in the developing nerve-muscle system

BACKGROUND: Extensive research on prenatal alcohol exposure has proven the potent teratogenicity of this substance of abuse. Children born to alcoholic mothers are often diagnosed with fetal alcohol syndrome (FAS). Those afflicted with FAS often have muscle weakness, muscle wasting, and atrophy. This study assessed the effects of prenatal alcohol exposure on the developing rat neuromuscular system. METHODS: Pregnant Sprague-Dawley rats were injected intraperitoneally with 1.0 ml of 20% ethyl alcohol/100 gm body weight. Unexposed rats served as controls. The offspring were killed 2, 3, 4, and 5 weeks after birth, and their body weights were recorded. The tibialis anterior (TA) and extensor digitorum longus (EDL) muscles were recovered and weighed. The TA muscles were histochemically stained by silver cholinesterase in order to study the pattern of innervation. The EDL muscles were processed and stained by hematoxylin-eosin. The number and size of the EDL muscle fibers was quantified. The sciatic nerve was also removed and stained by Swank and Davenport's method to demonstrate the myelin pattern. RESULTS: Assessment at the neuromuscular junction showed a higher proportion of endplates polyneuronally innervated in the alcohol-exposed rats. The muscle weights, as well as the number and size of the muscle fibers, were significantly reduced in these animals. A light-microscopy examination of the nerve sections revealed alterations in the connectivity of myelin. CONCLUSIONS: The finding that a higher proportion of endplates were polyneuronally innervated in the alcohol-exposed rats indicates that the maturation process of the neuromuscular system was delayed, thus confirming the deleterious effects of alcohol on growth and maturation of the nerve-muscle system.     

Dual role of MutS glutamate 38 in DNA mismatch discrimination and in the authorization of repair

MutS plays a critical role in DNA mismatch repair in Escherichia coli by binding to mismatches and initiating repair in an ATP-dependent manner. Mutational analysis of a highly conserved glutamate, Glu38, has revealed its role in mismatch recognition by enabling MutS to discriminate between homoduplex and mismatched DNA. Crystal structures of MutS have shown that Glu38 forms a hydrogen bond to one of the mismatched bases. In this study, we have analyzed the crystal structures, DNA binding and the response to ATP binding of three Glu38 mutants. While confirming the role of the negative charge in initial discrimination, we show that in vivo mismatch repair can proceed even when discrimination is low. We demonstrate that the formation of a hydrogen bond by residue 38 to the mismatched base authorizes repair by inducing intramolecular signaling, which results in the inhibition of rapid hydrolysis of distally bound ATP. This allows formation of the stable MutS-ATP-DNA clamp, a key intermediate in triggering downstream repair events.     

FTIR spectroscopic discrimination of Saccharomyces cerevisiae and Saccharomyces bayanus strains

In this study, we tested the potential of Fourier-transform infrared absorption spectroscopy to screen, on the one hand, Saccharomyces cerevisiae and non-S. cerevisiae strains and, on the other hand, to discriminate between S. cerevisiae and Saccharomyces bayanus strains. Principal components analysis (PCA), used to compare 20 S. cerevisiae and 21 non-Saccharomyces strains, showed only 2 misclassifications. The PCA model was then used to classify spectra from 14 Samos strains. All 14 Samos strains clustered together with the S. cerevisiae group. This result was confirmed by a routinely used electrophoretic pattern obtained by pulsed-field gel electrophoresis. The method was then tested to compare S. cerevisiae and S. bayanus strains. Our results indicate that identification at the strain level is possible. This first result shows that yeast classification and S. bayanus identification can be feasible in a single measurement.     

Suppression of experimental autoimmune myasthenia gravis by granulocyte-macrophage colony-stimulating factor is associated with an expansion of FoxP3+ regulatory T cells

Dendritic cells (DCs) have the potential to activate or tolerize T cells in an Ag-specific manner. Although the precise mechanism that determines whether DCs exhibit tolerogenic or immunogenic functions has not been precisely elucidated, growing evidence suggests that DC function is largely dependent on differentiation status, which can be manipulated using various growth factors. In this study, we investigated the effects of mobilization of specific DC subsets-using GM-CSF and fms-like tyrosine kinase receptor 3-ligand (Flt3-L)-on the susceptibility to induction of experimental autoimmune myasthenia gravis (EAMG). We administered GM-CSF or Flt3-L to C57BL/6 mice before immunization with acetylcholine receptor (AChR) and observed the effect on the frequency and severity of EAMG development. Compared with AChR-immunized controls, mice treated with Flt3-L before immunization developed EAMG at an accelerated pace initially, but disease frequency and severity was comparable at the end of the observation period. In contrast, GM-CSF administered before immunization exerted a sustained suppressive effect against the induction of EAMG. This suppression was associated with lowered serum autoantibody levels, reduced T cell proliferative responses to AChR, and an expansion in the population of FoxP3+ regulatory T cells. These results highlight the potential of manipulating DCs to expand regulatory T cells for the control of autoimmune diseases such as MG.