Novel circular forms of mengovirus-specific double-stranded RNA detected by electron microscopy

Abstract

Examination of mengovirus replicative form (RF) RNA by electron microscopy has revealed a substantial frequency of circular double-stranded RNAs which have an unusual projection on their circular contour. Circular structures comprised 14–16% of the genome-length double-stranded RNA in different preparations and have a contour length of 2.25 ± 0.08 μm corresponding to 8840 ± 310 nucleotide pairs in the A-form nucleic acid. The projection from the circular contour was typically found on more than 98% of the molecules and has an average heterogeneous length of 0.08 ± 0.02 μm (300 ± 80 nucleotide pairs). Linear duplex molecules with opposed ends were also detected at frequencies of 5–9% in different preparations and appear to be derived from dissociation of circular structures at the site of the projection. These linear duplexes had a length 2.32 ± 0.08 μm (9100 ± 310 nucleotide pairs) which is slightly greater than the contour length of circular structures excluding their projection. Linear dimers (3–6% of total), circular dimers (0.3%), and catenanes of two and three circular RF molecules, respectively, have been detected at low frequencies; such molecules usually contained a projection. The integrity of circular structures was destroyed by mild treatment with ribonucleases A, T1, or T2 or by incubation in 50% formamide or 40% formamide plus 2 M urea but was unaltered by treatment with DNase I or proteinase K. When examined by electron microscopy in the presence of formamide or formamide plus urea, approximately 14–17% of the linear duplex molecules were detected which contain one single-strand terminus typically less than 500 nucleotides in length. We suggest that circular structures are formed by noncovalent association of one single-strand terminus and one double-strand terminus of linear duplex molecules and that this association produces circular structures with the projection described at the site of association. A fixed fraction of linear RF RNA molecules in any given preparation contains termini suitable for association into circular structures that may have biological significance in the replication of picornaviruses.