Pseudo-typed Semliki Forest virus delivers EGFP into neurons.

Semliki Forest virus (SFV), a neurotropic virus, has been used to deliver heterologous genes into cells in vitro and in vivo. In this study, we constructed a reporter SFV4-FL-EGFP and found that it can deliver EGFP into neurons located at the injection site without disseminating throughout the brain. Lacking of the capsid gene of SFV4-FL-EGFP does not block its life cycle, while forming replication-competent virus-like particles (VLPs). These VLPs hold subviral genome by using the packaging sequence (PS) located within the nsP2 gene, and can transfer their genome into cells. In addition, we found that the G protein of vesicular stomatitis virus (VSVG) can package SFV subviral genome, which is consistent with the previous reports. The G protein of rabies virus (RVG) could also package SFV subviral genome. These pseudo-typed SFV can deliver EGFP gene into neurons. Taken together, these findings may be used to construct various SFV-based delivery systems for virological studies, gene therapy, and neural circuit labeling.

Co- and posttranslational modification of the alpha(1B)-adrenergic receptor: effects on receptor expression and function.

We have characterized the maturation, co- and posttranslational modifications, and functional properties of the alpha(1B)-adrenergic receptor (AR) expressed in different mammalian cells transfected using conventional approaches or the Semliki Forest virus system. We found that the alpha(1B)-AR undergoes N-linked glycosylation as demonstrated by its sensitivity to endoglycosidases and by the effect of tunicamycin on receptor maturation. Pulse-chase labeling experiments in BHK-21 cells demonstrate that the alpha(1B)-AR is synthesized as a 70 kDa core glycosylated precursor that is converted to the 90 kDa mature form of the receptor with a half-time of approximately 2 h. N-Linked glycosylation of the alpha(1B)-AR occurs at four asparagines on the N-terminus of the receptor. Mutations of the N-linked glycosylation sites did not have a significant effect on receptor function or expression. Surprisingly, receptor mutants lacking N-linked glycosylation migrated as heterogeneous bands in SDS-PAGE. Our findings demonstrate that N-linked glycosylation and phosphorylation, but not palmitoylation or O-linked glycosylation, contribute to the structural heterogeneity of the alpha(1B)-AR as it is observed in SDS-PAGE. The modifications found are similar in the different mammalian expression systems explored. Our findings indicate that the Semliki Forest virus system can provide large amounts of functional and fully glycosylated alpha(1B)-AR protein suitable for biochemical and structural studies. The results of this study contribute to elucidate the basic steps involved in the processing of G protein-coupled receptors as well as to optimize strategies for their overexpression.

Hypericum perforatum inhibits the binding of mu- and kappa-opioid receptor expressed with the Semliki Forest virus system.

The effects of Hypericum perforatum extracts on in vitro [3H]naloxone binding to the human mu- and rat kappa-opioid receptors were studied in chinese hamster ovary (CHO) cells using the Semliki Forest virus (SFV) expression system. Binding of [3H]naloxone to the mu- and kappa-opioid receptor was inhibited in the presence of Hypericum extracts showing IC50 values of approximately 25 and 90 micrograms/ml, respectively. In contrast, extracts of Valeriana officinalis did not inhibit binding to the mu-opioid receptor. Also, single constituents of H. perforatum like the flavonoids quercetin and kaempferol and the glycosilated flavonoid quercitrin did not inhibit [3H]naloxone binding to the mu-opioid receptor up to a concentration of 10 microM. The present in vitro data may suggest a new possible mechanism for the anti-depressant effect of H. perforatum.

Semliki Forest virus core protein fragmentation: its possible role in nucleocapsid disassembly.

Semliki Forest virus (SFV) envelope proteins function as proton pores under mildly acidic conditions and translocate protons across the viral membrane [Schlegel, A., Omar, A., Jentsch, P., Morell, A. and Kemp, F. C. (1991) Biosci. Rep. 11, 243-255]. As a consequence, during uptake of SFV by cells via receptor-mediated endocytosis the nucleocapsid is supposed to be exposed to protons. In this paper the effects of mildly acidic pH on SFV nucleocapsids were examined. A partial proteolytic fragmentation of core proteins was observed when nucleocapsids were exposed to mildly acidic pH. A similar proteolytic event was detected when intact SFV virions were exposed to identical conditions. Protease protection assays with exogenous bromelain provided evidence that the capsid protein degradation was due to an endogenous proteolytic activity and not to a proteolytic contamination. Detergent solubilization of virus particles containing degraded nucleocapsids followed by sucrose gradient centrifugation led to a separation of capsid protein fragments and remaining nucleocapsids. These data are discussed in terms of a putative biological significance, namely that the core protein fragmentation may play a role in nucleocapsid disassembly.

Semliki Forest virus envelope proteins function as proton channels.

It has been shown that isolated nucleocapsids of Semliki Forest virus (SFV) contract upon low pH exposure (Soederlund et al., 1972). This contraction of the nucleocapsids has been used as an indicator to demonstrate that the spike proteins of SFV can translocate protons into the interior of the virus particle upon low pH (5.8) exposure. Spikeless virus particles obtained after bromelain digestion, which were used as a control, did not translocate protons. This implies that the ectodomain of the spike plays a crucial role for the proton translocation.