The Drosophila X virus contains a 1-μ M double-stranded RNA circularized by a 67-kd terminal protein: High-resolution denaturation mapping of its genome

Abstract

The effect of urea on Drosophila X virus (DXV) is presented and analyzed by electron microscopy. At 4 M urea a RNA-protein complex is liberated consisting of one segment of double-stranded (ds) RNA which is maintained in a circular form inside the virion by a protein of 67 kd. The RNA-protein complex was purified by chromatography and had a tendency to form very spectacular flower-like structures. By crosslinking the RNA inside the virus with psoralen and uv radiation it is shown that the RNA was double-stranded in situ. A new method for partial denaturation mapping of double-stranded nucleic acids by electron microscopy is described and was applied to DXV RNA. Under these conditions it is shown that native and denatured regions as small as 30 RNA base pairs (bp) can be visualized. In the case of DXV RNA it was observed that the RNA possessed at one extremity a GC-rich region of 30 by followed by an AU-rich region of 160 bp. These molecules were observed in a conventional transmission electron microscope and a scanning transmission electron microscope under dark-field illumination. Absolute electron microscope length measurements and molecular weight determinations by gel migration revealed that the DXV RNA is one segment of ds-RNA of 0.97 μm in the electron microscopic conditions used in this work, giving a molecular weight of 2.2 × 10 6 d which corresponds to 3170 RNA bp. An average melting temperature of 83.3° was obtained in 0.1 SSC. Viral protein analysis was performed on the virus and on the purified protein-RNA complex. The virus is made of six major proteins and two minor proteins which were revealed after in vitro iodination.