Role of the Pepino mosaic virus 3'-untranslated region elements in negative-strand RNA synthesis in vitro.

Pepino mosaic virus (PepMV) is a mechanically-transmitted positive-strand RNA potexvirus, with a 6410 nt long single-stranded (ss) RNA genome flanked by a 5'-methylguanosine cap and a 3' poly-A tail. Computer-assisted folding of the 64 nt long PepMV 3'-untranslated region (UTR) resulted in the prediction of three stem-loop structures (hp1, hp2, and hp3 in the 3'-5' direction). The importance of these structures and/or sequences for promotion of negative-strand RNA synthesis and binding to the RNA dependent RNA polymerase (RdRp) was tested in vitro using a specific RdRp assay.

In vitro template-dependent synthesis of Pepino mosaic virus positive- and negative-strand RNA by its RNA-dependent RNA polymerase.

Pepino mosaic virus (PepMV)-infected tomato plants were used to develop an in vitro template-dependent system for the study of viral RNA synthesis. Differential sedimentation and sucrose-gradient purification of PepMV-infected tomato extracts resulted in fractions containing a transcriptionally active membrane-bound RNA-dependent RNA polymerase (RdRp). In the presence of Mg(2+) ions, (32)P-labelled UTP and unlabelled ATP, CTP, GTP, the PepMV RdRp catalysed the conversion of endogenous RNA templates into single- and double-stranded (ds) genomic RNAs and three 3'-co-terminal subgenomic dsRNAs.

In vitro synthesis of minus-strand RNA by an isolated cereal yellow dwarf virus RNA-dependent RNA polymerase requires VPg and a stem-loop structure at the 3' end of the virus RNA.

Cereal yellow dwarf virus (CYDV) RNA has a 5'-terminal genome-linked protein (VPg). We have expressed the VPg region of the CYDV genome in bacteria and used the purified protein (bVPg) to raise an antiserum which was able to detect free VPg in extracts of CYDV-infected oat plants. A template-dependent RNA-dependent RNA polymerase (RdRp) has been produced from a CYDV membrane-bound RNA polymerase by treatment with BAL 31 nuclease.

Molecular characterization of a partitivirus from Ophiostoma himal-ulmi.

The complete nucleotide sequences of two double-stranded (ds) RNA molecules, S1 (1,744 bp) and S2 (1,567 bp), isolated from an isolate HP62 of the Himalayan Dutch elm disease fungus, Ophiostoma himal-ulmi, were determined. RNA S1 had the potential to encode a protein, P1, of 539 amino acids (62.7 kDa), which contained sequence motifs characteristic of RNA-dependent RNA polymerases (RdRps).