The capacity for self-renewal and differentiation of human embryonic stem (ES) cells makes them a potential source for generation of pancreatic beta cells for treating type I diabetes mellitus. Here, we report a newly developed and effective method, carried out in a serum-free system, which induced human ES cells to differentiate into insulin-producing cells. Activin A was used in the initial stage to induce definitive endoderm differentiation from human ES cells, as detected by the expression of the definitive endoderm markers Sox17 and Brachyury. Further, all-trans retinoic acid (RA) was used to promote pancreatic differentiation, as indicated by the expression of the early pancreatic transcription factors pdx1 and hlxb9. After maturation in DMEM/F12 serum-free medium with bFGF and nicotinamide, the differentiated cells expressed islet specific markers such as C-peptide, insulin, glucagon and glut2. The percentage of C-peptide-positive cells exceeded 15%. The secretion of insulin and C-peptide by these cells corresponded to the variations in glucose levels. When transplanted into renal capsules of Streptozotocin (STZ)-treated nude mice, these differentiated human ES cells survived and maintained the expression of beta cell marker genes, including C-peptide, pdx1, glucokinase, nkx6.1, IAPP, pax6 and Tcf1. Thirty percent of the transplanted nude mice exhibited apparent restoration of stable euglycemia; and the corrected phenotype was sustained for more than six weeks. Our new method provides a promising in vitro differentiation model for studying the mechanisms of human pancreas development and illustrates the potential of using human ES cells for the treatment of type I diabetes mellitus.

No abstract

While much of the genetic variation in RNA viruses arises because of the error-prone nature of their RNA-dependent RNA polymerases, much larger changes may occur as a result of recombination. An extreme example of genetic change is found in defective interfering (DI) viral particles, where large sections of the genome of a parental virus have been deleted and the residual sub-genome fragment is replicated by complementation by co-infecting functional viruses. While most reports of DI particles have referred to studies in vitro, there is some evidence for the presence of DI particles in chronic viral infections in vivo. In this study, short fragments of dengue virus (DENV) RNA containing only key regulatory elements at the 3′ and 5′ ends of the genome were recovered from the sera of patients infected with any of the four DENV serotypes. Identical RNA fragments were detected in the supernatant from cultures of Aedes mosquito cells that were infected by the addition of sera from dengue patients, suggesting that the sub-genomic RNA might be transmitted between human and mosquito hosts in defective interfering (DI) viral particles. In vitro transcribed sub-genomic RNA corresponding to that detected in vivo could be packaged in virus like particles in the presence of wild type virus and transmitted for at least three passages in cell culture. DENV preparations enriched for these putative DI particles reduced the yield of wild type dengue virus following co-infections of C6–36 cells. This is the first report of DI particles in an acute arboviral infection in nature. The internal genomic deletions described here are the most extensive defects observed in DENV and may be part of a much broader disease attenuating process that is mediated by defective viruses.

Cytoplasmic inclusions in respiratory syncytial virus-infected cells comprising viral nucleocapsid proteins (L,N Respiratory syncytial virus (RSV), a member of the familyParamyxoviridae, is one of the most important viral agents causing lower respiratory tract disease in infants, the elderly, and immunocompromised patients of all ages (3, 6, 21). The genome of RSV is a nonsegmented negative-strand RNA encoding 11 proteins (3). In RSV-infected cells, the viral RNA with the L (polymerase), N (nucleocapsid), P (phosphoprotein), and M2-1 proteins form the polymerase complex in which the transcription of messenger and genomic RNA takes place. As the cytoplasmic inclusions in RSV-infected cells have been shown to contain all elements of the polymerase complex and are capable of transcription in isolation (1, 7), it is presumed that they are major sites of viral transcription.Several studies have shown that the N protein is the major driver for the formation of these cytoplasmic inclusions. N associates with viral RNA, and N-RNA complexes are resistant to RNase treatment (18). Inclusion-like structures are formed when the N and P proteins are coexpressed in cells (7), and this association results from a specific protein-protein interaction between N and P, which can be disrupted by mutagenesis (8, 24). Garcia et al. (7) also showed that the M2-1 protein is present in cytoplasmic inclusions; subsequent investigations confirmed that the association of the M2-1 protein with inclusions resulted from its association with P (16).We previously reported that the RSV M protein is also found in cytoplasmic inclusions late during infection, in association with the N, P, and M2-1 proteins (11). Since we have also shown that the M protein inhibits virus transcription (11), the role of the M protein in cytoplasmic inclusions may be to inhibit viral transcription as a prelude to viral assembly and budding, driven by the M protein bringing cytoplasmic nucleocapsids into association with RSV envelope proteins (10). The concept is supported by data indicating specific interactions between M and the cytoplasmic domains of envelope glycoproteins (10).To date, it is not known how M becomes associated with the nucleocapsid complex. In the current study, we demonstrate that the N terminus of M can bind directly to M2-1 in a cell-free assay and that M colocalizes with M2-1 in the cytoplasm of cells either infected with RSV or expressing only M and M2-1 proteins. Using a cotransfection system, it was demonstrated that M associates with inclusion-like structures formed by N and P only in the presence of M2-1. MATERIALS AND METHODS Cells and virus.Human epithelial (HEp2) cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum (FCS) at 37°C and 5% CO 2 . RSV subgroup A strain A2 (a gift from Paul Young, University of Queensland, Brisbane, Australia) was grown in HEp2 cells as previously described (9). To prepare the virus stock, an 80% confluent cell monolayer was infected with RSV at a multiplicity of infect...

SUMMARY The prokaryotic translation elongation factors were identified as essential cofactors for RNA-dependent RNA polymerase activity of the bacteriophage Qβ more than 40 years ago. A growing body of evidence now shows that eukaryotic translation elongation factors (eEFs), predominantly eEF1A, acting in partially characterized complexes sometimes involving additional eEFs, facilitate virus replication. The functions of eEF1A as a protein chaperone and an RNA- and actin-binding protein enable its “moonlighting” roles as a virus replication cofactor. A diverse group of viruses, from human immunodeficiency type 1 and West Nile virus to tomato bushy stunt virus, have adapted to use eEFs as cofactors for viral transcription, translation, assembly, and pathogenesis. Here we review the mechanisms used by viral pathogens to usurp these abundant cellular proteins for their replication.