Molecular and diagnostic aspects of the protein p41 of HHV-6 and silencing of the CD46 receptor by RNA interference

Detta är en avhandling från Stockholm : Karolinska Institutet, Microbiology and Tumor Biology Center (MTC)

Sammanfattning: Multiple sclerosis (MS) is one of the most common diseases of the central nervous system (CNS), leading to sensory and motor disability in young adults. There is no efficient treatment of the disease. Its etiology is unknown. Identification of an infectious cause of MS will be of importance to establish an efficient strategy in treating or preventing the disease. HHV-6 is a lymphotropic herpesvirus and one cause of exanthema subitum (ES). All HHV-6 isolates can be classified into two variants, HHV-6A and HHV-6B. CD46 has been found to be a cellular receptor for HHV-6 entry. A link between human herpesvirus 6 (HHV-6) and the pathogenesis of MS has been suggested on the basis of some laboratory findings. Presence of HHV-6 early protein (p41) in MS brains and an elevated serum IgM response to p41 in MS patients are the main evidence. In order to further examine a possible role of HHV-6 in MS, p41 genes (U27) from HHV-6A strain GS and HHV-6B strain Z29 were cloned and expressed in HEK-293 cells and as glutathione-S-transferase fusion proteins (GST-p41) in E. coli. The transfected cells and recombinant p41 proteins were detected with two monoclonal antibodies (mAb to p41/38 and mAb to p41). The mAb to p41/38 recognized the HHV-6A but not the HHV-6B. The epitope recognized by the antibody and the critical amino acid of the epitope were identified by peptide ELISA. Serum IgM and IgG responses to p41 were measured in MS patients and controls by ELISA using the recombinant GST-p41 proteins as the antigen. No significant difference was observed between the examined groups. Our data do not indicate a link between HHV-6 and MS, and suggest that p41 is not an ideal antigen for HHV-6 serology. RNA interference (RNAi) is a gene silencing generated by double-stranded RNAs. Small interfering RNAs (siRNA), composed of double-stranded RNAs of about 21-nucleotides, can induce specific RNAi and don t activate a nonspecific protein kinase mediated activity as long double-stranded RNAs do. RNAi is a promising method for probing gene functions and for treatment of human diseases. Some data suggests that siRNA acts by the antisense strand. It is unknown if there is an association between the antisense accessible sites and the siRNA accessible sites in the mRNAs. We tested 12 pairs of antisense DNAs and siRNAs against reporter luciferase genes in cultured mammalian cells. Both the antisense DNA and siRNA in one pair were directed towards the same site. Five siRNAs and two antisense DNAs turned out to be effective, but no overlapping was found among the sites targeted by those effective antisense DNAs or siRNAs. These findings suggest that effective antisense DNAs and siRNAs have different target sites in the mRNA. Targeting the exogenous luciferase and endogenous CD46, the duplex siRNAs were more potent than the antisense-stranded siRNAs as RNAi triggers. Antisense-stranded siRNAs had a shorter duration in the transfected cells than the duplex siRNAs, but it doesn t seem to be a main reason for the fact that antisense-stranded siRNAs have significantly lower efficiencies than duplex siRNAs in mammalian cells. Double-stranded siRNAs and antisense-stranded siRNAs may act in cells by different mechanisms. CD46 expression was efficiently blocked by the specific duplex siRNAs in human cells. This CD46-specific RNAi model could be used in study of the biological roles of CD46 in human diseases and the interaction between cells and microorganisms using the CD46 receptor.

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