We describe four monoclonal antibodies (MAB) which specifically recognize double-stranded RNA (dsRNA) together with their use in new methods for detecting and characterizing dsRNA in unfractionated nucleic acid extracts.

The monoclonal anti-dsRNA antibody J2 binds double-stranded RNAs (dsRNA) in an apparently sequence-nonspecific way. The mAb only recognizes antigens with double-stranded regions of at least 40 bp and its affinity to poly(A) poly(U) and to dsRNAs with mixed base pair composition is about tenfold higher than to poly(I) poly(C). Because no specific binding site could be determined, the number, the exact dimensions, and other distinct features of the binding sites on a given antigen are difficult to evaluate by biochemical methods. We therefore employed scanning force microscopy (SFM) as a method to analyze antibody-dsRNA interaction and protein-RNA binding in general. Several in vitro-synthesized dsRNA substrates, generated from the Dictyostelium PSV-A gene, were used. In addition to the expected sequence-nonspecific binding, imaging of the complexes indicated preferential binding of antibodies to the ends of dsRNA molecules as well as to certain internal sites. Analysis of 2,000 bound antibodies suggested that the consensus sequence of a preferential internal binding site is A 2 N 9 A 3 N 9 A 2 , thus presenting A residues on one face of the helix. The site was verified by site-directed mutagenesis, which abolished preferential binding to this region. The data demonstrate that SFM can be efficiently used to identify and characterize binding sites for proteins with no or incomplete sequence specificity. This is especially the case for many proteins involved in RNA metabolism.

The intracellular localization of viroids has been investigated by viroid‐specific in situ hybridization and analysis by digital microscopy of the distribution of the fluorescent hybridization signals. Isolated nuclei from green leaf tissue of tomato plants infected with potato spindle tuber viroid (PSTVd) were bound to microscope slides, fixed with formaldehyde and hybridized with biotinylated transcripts of cloned PSTVd cDNA. The bound probe was detected with lissamine‐‐rhodamine conjugated streptavidin. Nucleoli were identified by immunofluorescence using the monoclonal antibody Bv96 and a secondary FITC‐conjugated antibody. In plants infected with either a lethal or an intermediate PSTVd strain, the highest intensity of fluorescence that arose from hybridization with the probe specific for the viroid (+)strand was found in the nucleoli, confirming results of previous fractionation studies. A similar distribution was found for (‐)strand replication intermediates of PSTVd using specific (+)strand transcripts as hybridization probes. In order to determine if viroids are located at the surface or in the interior of the nucleoli, the distribution of the fluorescence hybridization signals was studied with a confocal laser scanning microscope (CLSM). It was shown by three‐dimensional reconstruction that viroids are neither restricted to the surface of the nucleoli nor to a peripheral zone, but are instead homogeneously distributed throughout the nucleolus. The functional implications of the intranucleolar location of viroids and their replication intermediates are discussed with respect to proposed mechanisms of viroid replication and pathogenesis.

The glycoproteins of pseudorabies virus (PRV) Phylaxia were characterized with monoclonal antibodies as specific reagents. Three major structural glycoproteins with molecular weights of 155,000 (155K) (gC), 122K (gA), and 90K (gB) could be identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions. We investigated the processing of glycoproteins gA, gB, and gC by in vitro translation, pulse-chase experiments, and in the presence of the ionophore monensin which inhibits glycosylation. gA and gB were found to compose a single polypeptide, whereas gC was found to be a disulfide-linked glycoprotein complex. Immunoprecipitates formed with the aid of anti-gC monoclonal antibodies gave rise to three glycoprotein bands (gC0 [120K], gC1 [67K], and gC2 [58K]) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Limited proteolysis of gCo, gC1, and gC2 resulted in peptide maps of gCo related to those of both gC1 and gC2. No common peptide bands between gC1 and gC2, however, were seen. We suggest that (i) gC1 and gC2 arise by proteolytic cleavage from the same precursor molecule and stay joined via disulfide bridges and (ii) gCo is an uncleaved precursor.