ABSTRACT
Several genomic disorders are caused by an excessive number of DNA triplet repeats. We developed a DNA-templated reaction in which product formation occurs only when the number of repeats exceeds a threshold indicative for the outbreak of Chorea Huntington. The combined use of native chemical PNA ligation and auxiliary DNA probes enabled reactions on templates obtained from human genomic DNA.
ABSTRACT
The rescue of recombinant rabies virus (RV) from cloned cDNA is an inefficient process because it relies on the de novo formation within cells of functional ribonucleoprotein (RNP) complexes from plasmid-expressed viral-like antigenome RNAs and three helper proteins. In the standard RV reverse genetics systems, bacteriophage T7 RNA polymerase drives the transcription of virus antigenome-like RNAs containing three nonviral G residues at the 5'-end and a correct 3'-end generated by the autocatalytic activity of an 85 nucleotides long hepatitis delta virus antigenomic "core" ribozyme (HDVagrz). Here, we show that employing optimized ribozyme sequences significantly improves RV rescue. Substitution of the "core" HDVagrz by a ribozyme with an enhanced cleavage activity resulted in an approximately 10-fold higher number of rescue events and faster initiation of an infectious cycle. The alternative use of a hammerhead ribozyme for the generation of an exact 5'-end similarly enhanced rescue efficiency. Notably, RV cDNA clones containing the combination of optimized 3'- and 5'-ribozymes were rescued at an at least 100-fold increase. In addition to virus rescue, reporter gene expression from transfected minigenome cDNAs was significantly enhanced by the novel ribozymes. The improved RV reverse genetics system greatly facilitates recovery of strongly attenuated viruses and vectors for biomedical applications.
Subject(s)
DNA, Complementary/biosynthesis , Genetic Engineering/methods , Genome, Viral , Plasmids/genetics , RNA, Catalytic/genetics , RNA, Viral/biosynthesis , Rabies virus/genetics , Reverse Genetics/methods , Animals , Base Sequence , Cell Line , Cricetinae , DNA, Complementary/genetics , DNA-Directed RNA Polymerases/genetics , Genes, Reporter , Hepatitis Delta Virus/chemistry , Hepatitis Delta Virus/genetics , Molecular Sequence Data , Plasmids/chemistry , RNA, Catalytic/chemistry , RNA, Viral/genetics , Rabies/virology , Rabies virus/chemistry , Rabies virus/metabolism , Transcription, Genetic , Transfection , Viral Proteins/genetics , Virus Replication/geneticsABSTRACT
The coronavirus membrane protein (M) is the key player in the assembly of virions at intracellular membranes between endoplasmic-reticulum and Golgi-complex. Using a newly established human monoclonal anti-M antibody we detected glycosylated and nonglycosylated membrane-associated M in severe acute respiratory syndrome-associated coronavirus (SARS-CoV) infected cells and in purified virions. Further analyses revealed that M contained a single N-glycosylation site at asparagine 4. Recombinant M was transported to the plasma membrane and gained complex-type N-glycosylation. In SARS-CoV infected cells and in purified virions, however, N-glycosylation of M remained endoglycosidase H-sensitive suggesting that trimming of the N-linked sugar side chain is inhibited.
