Inhibition of RNAse A family enzymes prevents degradation and loss of silencing activity of siRNAs in serum.

Small interfering RNAs (siRNA), RNA duplexes of approximately 21 nucleotides, offer a promising approach to specifically degrade RNAs in target cells by a process termed RNA interference. Insufficient in vivo-stability is a major problem of a systemic application of siRNAs in humans. The present study demonstrated that RNAse A-like RNAses degraded siRNAs in serum. The susceptibility of siRNAs towards degradation in serum was strongly enhanced by local clustering of A/Us within the siRNA sequence, i.e. regions showing low thermal stability, most notably at the ends of the molecule, and by 3'-overhanging bases. Importantly, inhibition of RNAse A family enzymes prevented the degradation and loss of silencing activity of siRNAs in serum. Furthermore, the degradation of siRNAs was considerably faster in human than in mouse serum, suggesting that the degradation of siRNAs by RNAse A family enzymes might be a more challenging problem in a future therapeutic application of siRNAs in humans than in mouse models. Together, the present study indicates that siRNAs are degraded by RNAse A family enzymes in serum and that the kinetics of their degradation in serum depends on their sequence. These findings might be of great importance for a possible future human therapeutic application of siRNAs.

Epac activation converts cAMP from a proliferative into a differentiation signal in PC12 cells.

Elevation of the intracellular cAMP concentration ([cAMP]i) regulates metabolism, cell proliferation, and differentiation and plays roles in memory formation and neoplastic growth. cAMP mediates its effects mainly through activation of protein kinase A (PKA) as well as Epac1 and Epac2, exchange factors activating the small GTPases Rap1 and Rap2. However, how cAMP utilizes these effectors to induce distinct biological responses is unknown. We here studied the specific roles of PKA and Epac in neuroendocrine PC12 cells. In these cells, elevation of [cAMP]i activates extracellular signal-regulated kinase (ERK) 1/2 and induces low-degree neurite outgrowth. The present study showed that specific stimulation of PKA triggered ERK1/2 activation that was considerably more transient than that observed upon simultaneous activation of both PKA and Epac. Unexpectedly, the PKA-specific cAMP analog induced cell proliferation rather than neurite outgrowth. The proliferative signaling pathway activated by the PKA-specific cAMP analog involved activation of the epidermal growth factor receptor and ERK1/2. Activation of Epac appeared to extend the duration of PKA-dependent ERK1/2 activation and converted cAMP from a proliferative into an anti-proliferative, neurite outgrowth-promoting signal. Thus, the present study showed that the outcome of cAMP signaling can depend heavily on the set of cAMP effectors activated.

Maturation of Borna disease virus glycoprotein.

The maturation of Borna disease virus (BDV) glycoprotein GP was studied in regard to intracellular compartmentalization, compartmentalization signal-domains, proteolytic processing, and packaging into virus particles. Our data show that BDV-GP is (i) predominantly located in the endoplasmic reticulum (ER), (ii) partially exists in the ER already as cleaved subunits GP-N and GP-C, (iii) is directed to the ER/cis-Golgi region by its transmembrane and/or cytoplasmic domains in CD8-BDV-GP hybrid constructs and (iv) is incorporated in the virus particles as authentic BDV glycoprotein exclusively in the cleaved form decorated with N-glycans of the complex type. Downregulation of BDV-glycoproteins on the cell surface, their limited proteolytic processing, and protection of antigenic epitopes on the viral glycoproteins by host-identical N-glycans are different strategies for persistent virus infections.

Identification of the amino terminal subunit of the glycoprotein of Borna disease virus.

The only surface membrane glycoprotein of Borna disease virus (BDV) is synthesized as a polypeptide with a molecular mass of 57 kDa and N-glycosylated to a precursor glycoprotein (GP) of about 94 kDa. It is processed by the cellular protease furin into the C-terminal membrane-anchored subunit GP-C, also known as gp43, and a presumptive N-terminal subunit GP-N, that is highly glycosylated and has a molecular mass of about 51 kDa. However, up to now the latter remained undetected in BDV-infected material. We describe a novel approach to identify glycan masked linear antigenic epitopes. In the present study, GP-N was identified in BDV-infected cells by a combination of lectin precipitation, enzymatic deglycosylation on blot and immunochemistry using an N-terminal specific antiserum. The GP-N has an apparent molecular mass of 45-50 kDa in its glycosylated form and 27 kDa in its deglycosylated form. N-glycan analysis revealed that the precursor GP contains only mannose-rich N-glycans, whereas GP-N and GP-C contain mannose-rich and complex-type N-glycans.

Crystallization and preliminary X-ray analysis of the matrix protein of Borna disease virus.

The matrix protein M of Borna disease virus (BDV) is associated with the inner viral membrane and is thought to be a mediator between the nucleocapsid and the lipid-containing envelope in stabilizing the virus shape. The full-length BDV-M gene encoding a 16 kDa protein was expressed in Escherichia coli. M was purified to homogeneity and crystallized by the sitting-drop vapour-diffusion method. The crystals of M belong to the space group I432, with unit-cell parameters a = b = c = 144.6 A, and diffract to 3.1 A.