Alphaviruses, such as the Sindbis virus and the Chikungunya virus, are RNA viruses with a positive sense single-stranded RNA genome that infect various vertebrates, including humans. A conserved... Show moreAlphaviruses, such as the Sindbis virus and the Chikungunya virus, are RNA viruses with a positive sense single-stranded RNA genome that infect various vertebrates, including humans. A conserved sequence element (CSE) of similar to 19 nt in the 3 ' noncoding region is important for replication. Despite extensive mutational analysis of the CSE, no comprehensive model of this element exists to date. Here, it is shown that the CSE can form an RNA pseudoknot with part of the poly(A) tail and is similar to the human telomerase pseudoknot with which it shares 17 nt. Mutants that alter the stability of the pseudoknot were investigated in the context of a replicon of the Sindbis virus and by native gel electrophoresis. These studies reveal that the pseudoknot is required for virus replication and is stabilized by UAU base triples. The new model is discussed in relation to previous data on Sindbis virus mutants and revertants lacking (part of) the CSE. Show less
Pepino mosaic virus (PepMV) is a potexvirus of the family Alphaflexiviridae within the order of Tymovirales that threatens tomato production worldwide. PepMV possesses a positive-strand RNA genome... Show morePepino mosaic virus (PepMV) is a potexvirus of the family Alphaflexiviridae within the order of Tymovirales that threatens tomato production worldwide. PepMV possesses a positive-strand RNA genome with a 5 '-methylguanosine cap and a 3 '-polyA tail. Previously, using partially-purified viral RNA polymerase important secondary structures within the 3 '-untranslated region (UTR) of PepMV RNA were identified. Here we show that an RNA pseudoknot can be formed in the 3 '-UTR that includes part of the polyA tail. Using protoplasts, we demonstrate that the pseudoknot is required for replication of PepMV RNA. Mutational analysis and native gel electrophoresis further show that the pseudoknot is stabilized by UAU base triples, as is the human telomerase RNA pseudoknot. The presence of a pseudoknot in several other members of the Alpha- and Betaflexiviridae is supported by covariance analysis and native gel electrophoresis of other potexvirus, capillovirus and trichovirus RNAs. The ubiquitous presence of the pseudoknot in viruses of the Betaflexiviridae, suggests that the pseudoknot is a typical trait of the Betaflexiviridae that may have been adopted by many potexviruses during evolution. Show less
Magnetic-activated cell sorting (MACS) is an affinity-based technique used to separate cells according to the presence of specific markers. Current MACS systems generally require an antigen to be... Show moreMagnetic-activated cell sorting (MACS) is an affinity-based technique used to separate cells according to the presence of specific markers. Current MACS systems generally require an antigen to be expressed at the cell surface; these antigen-presenting cells subsequently interact with antibody-labeled magnetic particles, facilitating separation. Here, we present an alternative MACS method based on coiled-coil peptide interactions. We demonstrate that HeLa, CHO, and NIH3T3 cells can either incorporate a lipid-modified coiled-coil-forming peptide into their membrane, or that the cells can be transfected with a plasmid containing a gene encoding a coiled-coil-forming peptide. Iron oxide particles are functionalized with the complementary peptide and, upon incubation with the cells, labeled cells are facilely separated from nonlabeled populations. In addition, the resulting cells and particles can be treated with trypsin to facilitate detachment of the cells from the particles. Therefore, our new MACS method promotes efficient cell sorting of different cell lines, without the need for antigen presentation, and enables simple detachment of the magnetic particles from cells after the sorting process. Such a system can be applied to rapidly developing, sensitive research areas, such as the separation of genetically modified cells from their unmodified counterparts. Show less
