We have constructed a series of MLV-based retroviral vectors and packaging components expressed from the CMV promoter and carried on plasmids containing SV40 origins of replication. These two features greatly enhanced retroviral gene expression when introduced into cell lines carrying the SV40 large T antigen. The two packaging components, gag-pol and env, were placed on separate plasmids to reduce helper virus formation. Using a highly transfectable human cell line and sodium butyrate to further increase expression of each component, we achieved helper-free viral stocks of approximately 10(7) infectious units/ml by 48 h after transient co-transfection with the three plasmid components. This system can be used both for the generation of high titer retroviral stocks for transduction and for the rapid screening of a large number of MLV gag-pol or env mutants.
Herpesvirus saimiri has characteristics that make it amenable to development as a gene therapy vector. The viral genome is thought to be capable of accommodating large quantities of heterologous DNA while the virus itself can infect many different cell types. Virus infection has been shown in many cases to be persistent by virtue of episomal maintenance in the target cell. In this article we examine the ability of nonselectable recombinant viruses expressing the beta-galactosidase gene product to infect a variety of human cells and demonstrate that this virus could be developed as an alternative hematopoietic stem cell gene therapy vector. In contrast to earlier observations, we demonstrate by a number of methods that the virus has the ability to replicate in many human cell types, suggesting the need for the development of a disabled virus for use as a gene therapy vector.
Objectives The dorsal vagal complex (DVC) senses insulin and controls glucose homeostasis, feeding behaviour and body weight. Three-days of high-fat diet (HFD) in rats are sufficient to induce insulin resistance in the DVC and impair its ability to regulate feeding behaviour. HFD-feeding is associated with increased dynamin-related protein 1 (Drp1)-dependent mitochondrial fission in the DVC. We investigated the effects that altered Drp1 activity in the DVC has on feeding behaviour. Additionally, we aimed to uncover the molecular events and the neuronal cell populations associated with DVC insulin sensing and resistance. Methods Eight-week-old male Sprague Dawley rats received DVC stereotactic surgery for brain infusion to facilitate the localised administration of insulin or viruses to express mutated forms of Drp1 or to knockdown inducible nitric oxide synthase (iNOS) in the NTS of the DVC. High-Fat diet feeding was used to cause insulin resistance and obesity. Results We showed that Drp1 activation in the DVC increases weight gain in rats and Drp1 inhibition in HFD-fed rats reduced food intake, weight gain and adipose tissue. Rats expressing active Drp1 in the DVC had higher levels of iNOS and knockdown of DVC iNOS in HFD-fed rats led to a reduction of food intake, weight gain and adipose tissue. Finally, inhibiting mitochondrial fission in DVC astrocytes was sufficient to protect rats from HFD-dependent insulin resistance, hyperphagia, weight gain and fat deposition. Conclusion We uncovered new molecular and cellular targets for brain regulation of whole-body metabolism, which could inform new strategies to combat obesity and diabetes.
The mRNA species encoding the herpesvirus saimiri (HVS) homolog of the Epstein-Barr virus R transcriptional activator (termed ORF50) have been identified and used to determine transcriptional start sites within the gene. The first transcript is spliced and starts from a promoter within ORF49 containing a single intron; the second is produced from a promoter within the second exon and is in the same reading frame. The spliced transcript is detected at early times during productive virus replication in OMK cells, whereas the nonspliced transcript is detected later. The spliced transcript is fivefold-more potent in activating the delayed-early ORF6 promoter; the function of the nonspliced transcript is unclear. Thus, the role of this protein in activating herpesvirus saimiri from the latent state may differ significantly from that of the Epstein-Barr virus R protein.Herpesvirus saimiri (HVS) establishes asymptomatic infections involving a subset of T lymphocytes in its natural host, the squirrel monkey (Saimiri sciureus), but causes fatal T-cell lymphomas and lymphoproliferative diseases in other species of New World primates (6). HVS has been classified as a gamma-2 herpesvirus and shares significant homology with Epstein-Barr virus (EBV) (1,2,9,10,16,24). The genomes of HVS and EBV are generally colinear, in that homologous genes are found in approximately equivalent locations and in the same relative orientations in the two viruses (22). As in many other herpesviruses, gene expression during lytic infection occurs in three main temporal phases: immediate-early (IE), delayed-early (DE), and late (17). The major IE transcript in HVS is encoded by the HindIII-G IE gene (ORF14) (2,(25)(26)(27). Analysis of the sequence shows that it does not exhibit homology with any EBV-encoded proteins (25), but it does contain local homology with a putative superantigen (29); however, its function remains to be established. The 52-kDa product of the HVS IE gene (ORF57) is homologous to the EBV transactivator encoded by BMLF1 (also known as Mta, M-IE, or IE-2) and to the 27-kDa or IE 63-kDa protein encoded by UL54 of herpes simplex virus 1 and RF4 of varicellazoster virus (4,14,21,22,24).The EcoRI-D or HVS R protein transactivator (ORF50) is homologous to the BRLF1 gene product (also known as R or Rta) (23). BRLF1 is translated from a bicistronic mRNA which also encodes another IE gene product, BZLF1 (12, 20). These two EBV gene products function in activating expression from a number of viral (3,5,8,13,15,18,28) and perhaps cellular (7) promoters, thereby initiating the lytic cascade. However, HVS R RNA is expressed as an early transcript in HVS-infected cells (23, 25). The HVS R gene is proposed to be spliced with more than one exon, although the initiation codon has not been mapped. Despite the likelihood of a spliced transcript, there is evidence that a DNA sequence within the identified exon is able to produce a functionally active protein capable of activating a DE ORF6 gene promoter, a component of the major DNA binding prot...
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