Mutations in one of the duplicated survival of motor neuron (SMN) genes lead to the progressive loss of motor neurons and subsequent development of spinal muscular atrophy (SMA), a common, and usually fatal, hereditary disease. Homozygous absence of the telomeric copy (SMN1) correlates with development of SMA because differential splicing of the centromeric copy (SMN2) leads to exon 7 skipping and predominantly produces a biologically inactive protein isoform. To increase exon 7 inclusion of SMN2, we have designed a series of vectors that express modified U7 snRNAs containing antisense sequences complementary to the 3' splice site of SMN exon 8. Over 20 anti-SMN U7 snRNAs were tested for their ability to promote exon 7 inclusion in the SMN2 gene. Transient expression of anti-SMN U7 snRNAs in HeLa cells modulated SMN2 splicing to approximately 70% exon 7 inclusion in a sequence-specific and dose-dependent manner. Significantly, the administration of anti-SMN U7 snRNPs results in an increase in the concentration of SMN protein. These results suggest that modulation of SMN2 pre-mRNA splicing by modified U7 snRNAs provides a promising form of gene therapy for the treatment of SMA.
The genome of human herpes virus 8, which is associated with Kaposi’s sarcoma, encodes proteins with similarities to cytokines and chemokines including a homologue of IL-6. Although the function of these viral proteins is unclear, they might have the potential to modulate the immune system. For viral IL-6 (vIL-6), it has been demonstrated that it stimulates IL-6-dependent cells, indicating that the IL-6R system is used. IL-6 binds to IL-6R, and the IL-6/IL-6R complex associates with gp130 which dimerizes and initiates intracellular signaling. Cells that only express gp130 but no IL-6R cannot be stimulated by IL-6 unless a soluble form of the IL-6R is present. This type of signaling has been shown for hematopoietic progenitor cells, endothelial cells, and smooth muscle cells. In this paper we show that purified recombinant vIL-6 binds to gp130 and stimulates primary human smooth muscle cells. IL-6R fails to bind vIL-6 and is not involved in its signaling. A Fc fusion protein of gp130 turned out to be a potent inhibitor of vIL-6. Our data demonstrate that vIL-6 is the first cytokine which directly binds and activates gp130. This property points to a possible role of this viral cytokine in the pathophysiology of human herpes virus 8.
Persistent viral infections can render host cells resistant to superinfection with closely related viruses by largely uncharacterized mechanisms. We present evidence for superinfection exclusion in brains of Borna disease virus (BDV)-infected rats and in persistently infected Vero cells, and we suggest that acquired resistance to BDV is due to unbalanced intracellular levels of viral nucleocapsid components. We observed that expression of BDV protein P, N, or X rendered human cells resistant to subsequent challenge with BDV but not with other RNA viruses, indicating that incorrect stoichiometry of nucleocapsid components selectively blocked the polymerase activity of incoming viruses. Vero cells containing high levels of an untranslatable BDV-N transcript remained virus susceptible, demonstrating that viral protein rather than RNA mediated resistance. Transient overexpression of BDV-P in persistently infected Vero cells was also remarkably effective against BDV, indicating that the intracellular balance of viral nucleocapsid components could serve as a target for future therapeutic antiviral strategies.Borna disease virus (BDV) persistently infects neurons and other cells of the central nervous systems of a broad range of warm-blooded animals and possibly humans (7,21,32,34). Natural and experimental infections with BDV may result in immune system-mediated neurological disease and behavioral abnormalities (17,28). BDV is a newly classified nonsegmented negative-stranded RNA virus that, unlike other Mononegavirales, replicates in the nuclei of infected cells and employs the splicing machinery for the maturation of some of its transcripts. Its genome codes for at least six viral proteins, of which the phosphoprotein P, the nucleoprotein N, and the RNA polymerase complex-associated protein X are abundantly present in infected cells (19,31).It was recently suggested that the remarkable stability of the BDV genome might be a consequence of superinfection exclusion (11). It is well known that certain persistent viral infections can confer resistance to subsequent infection of host cells with the same virus or a closely related virus. This phenomenon, designated superinfection exclusion or homologous interference (1, 20), has been described for retroviruses (5, 9), hepatitis B virus (6), alphaviruses (1, 20), Rift valley fever virus (3), and pestiviruses (23). Superinfection exclusion by retroviruses is primarily due to down-regulation of host cell surface entry receptors (5, 9). A similar mechanism may account for superinfection exclusion in hepatitis B virus-infected duck hepatocytes (6). Poorly defined mechanisms seem to contribute to superinfection exclusion of alphaviruses; one of these is posttranscriptional gene silencing (2). RNA silencing also seems to play a role in the case of Rift valley fever virus, because superinfection exclusion in insect cells could be mimicked by expression of nontranslatable viral RNAs (3).We now present evidence that superinfection exclusion is operative in brain cells of BDV-infect...
Human herpes virus‐8 (HHV8) encodes a cytokine named viral interleukin‐6 (vIL‐6) that shares 25% amino‐acid identity with its human homologue. Human IL‐6 is known to be a growth and differentiation factor of lymphatic cells and plays a potential role in the pathophysiology of various lymphoproliferative diseases. vIL‐6 is expressed in HHV8‐associated‐diseases including Kaposi's sarcoma, Body‐cavity‐based‐lymphoma and Castleman's disease, suggesting a pathogenetic involvement in the malignant growth of B‐cell associated diseases and other malignant tumours. We expressed vIL‐6 in Escherichia coli as a fusion protein with recombinant periplasmic maltose binding protein. After cleavage from the maltose binding protein moiety and purification, vIL‐6 was shown to be correctly folded using circular dichroism spectroscopy. A rabbit antiserum was raised against the recombinant vIL‐6 protein. vIL‐6 turned out to be active on cells that expressed gp130 but no IL‐6 receptor (IL‐6‐R) suggesting that, in contrast to human IL‐6, vIL‐6 stimulated gp130 directly. Accordingly, vIL‐6 activity could be inhibited by a soluble gp130 Fc Fusion protein. vIL‐6 was shown to induce neuronal differentiation of rat pheochromocytoma cells and to stimulate colony formation of human hematopoietic progenitor cells. Thus, vIL‐6 exhibits biologic activity that has only been observed for the IL‐6/soluble IL‐6‐R complex but not for IL‐6 alone. These properties are important for the evaluation of the pathophysiological potential of vIL‐6.
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